KR20040047971A - Multiparameter analysis of comprehensive nucleic acids and morphological features on the same sample - Google Patents

Abstract

The present invention relates to a high sensitivity assay using a method for gene-specific priming and amplification of mRNA derived from rare cells and rare transcripts found in blood. This assay can detect rare mRNAs (10 copies / cell) derived from 1-10 cells isolated through immunomagnetic enrichment. This assay is an improvement over complex PCR, which enables efficient detection of rare coding sequences for circulating carcinoma cells in the blood.

The methods of the invention are useful for profiling cells isolated from tissues and body fluids, and also assist in the clinical diagnosis of various carcinomas, including early stage detection and classification of circulating tumor cells. By monitoring the nucleic acid and protein profiles of the cells in conventional or microarray formats, it is possible to facilitate the regulation of therapeutic intervention, including delaying treatment, monitoring response to treatment, confirming recurrence, and determining whether it is armored.

Description

MULTIPARAMETER ANALYSIS OF COMPREHENSIVE NUCLEIC ACIDS AND MORPHOLOGICAL FEATURES ON THE SAME SAMPLE}

Any given cell will express the gene only in a proportion relative to the total number of genes present in that genome. Some of the total number of expressed genes determines aspects of cellular function such as development and differentiation, homeostasis, cell cycle regulation, aging, apoptosis and the like. Modification of gene expression determines the nature of normal cell development processes and conditions, such as cancer. Expression of certain genes will have a significant impact on the specific properties of any cell. Thus, methods for analyzing gene expression, such as those provided by the present invention, are important for basic molecular biological research and tumor biology. The identification of specific genes, especially rare genes, can provide important diagnostic, prognostic and therapeutic measures for many diseases that reflect these expression levels [Levsky et al., Single-Cell Gene Expression Profiling, Science , 297: 836-840 (2002).

Differential gene expression is a commonly used method for evaluating intracellular gene expression. In particular, cDNA microarray assays compare levels of cDNA target sequences from cells or organs obtained from healthy individuals and affected individuals. These targets are then hybridized with a set of probe fragments anchored to the membrane. Differences in the resulting hybridization patterns are then identified and correlated with differences in gene expression of both sources (US Pat. No. 6,383,749). This method requires the analysis of hundreds of thousands of gene-specific probes slowly and over time. In addition, competitive phenomena, such as interactions between non-complementary target sequences with nonspecific binding between the target and the probe and secondary structures in the target sequence, prevent hybridization, resulting in signal-to-noise. Lowers.

Gene specific primer sets are described in differential expression assays (US Pat. Nos. 5,994,076 and 6,352,829). In this document, gene specific primer sets were used to specifically amplify mRNA library subsets in a complex library to raise cDNA array signals compared to total library labeled amplification. The point of this type of assay is to compare sample array expression profiles as part of gene discovery studies, but not to improve the practical cellular RNA assays used for diagnosis.

Thus, gene specific primer sets have been used to selectively amplify specific mRNA subsets derived from mRNA libraries, where they can be applied to rare cell detection for diagnostic methods involving amplification methods, ie both quantitative and qualitative analysis. In any possible amplification method, there is a need to reliably reduce the signal-to-noise ratio.

The presence of circulating tumor cells (CTCs) generally present in the blood of a patient provides an early stage detection system in assessing whether therapeutic intervention is required. High sensitivity assays to accurately count circulating carcinoma cells show that the load of peripheral blood tumor cells correlates with the pathology [Terstappen et al., Peripheral Blood Tumor Cell Load Reflects the Clinical Activity of the Disease in Patients with Carcinoma of the Breast, International J. of Oncology, 17: 573-578, 2000].

In addition, classifying cell types and origins will provide a broader platform for treatment. Therapies for some injured cancers such as diffuse diffuse B-cell lymphoma (DLBCL) are based on two types of different diseases correlated with clinical prognosis [Rosenwald et al., Use of Molecular Profiling to Predict Survival After Chemotherapy after Diffuse Large -B Cell Lymphoma, New England Journal of Medicine, 346: 1937-1947 (2002). In the DLBCL, tumors originating from embryonic central B cells are more likely to survive as susceptible to chemotherapy, whereas tumors derived from activated B cells tend to be more difficult to treat. Thus, the subtypes of these cells depend on the origin of the tumor cells.

Layer formation of this subtype depends on the tumor cell of origin. In a few cases, the differences between these subtypes can be measured by analyzing a single gene, while the entire array of genes combined is more critical. Charting gene expression patterns through microarray analysis of gene expression levels will be a desirable indicator of tumor character in other diseases such as lymphoma, acute leukemia, breast cancer, lung cancer and liver cancer. However, the application of this genetic information to diagnostic applications requires the identification of intrinsic critical signal-to-noise as disclosed in the art.

Therefore, attention is particularly focused on developing novel methods for analyzing gene expression, which are provided to achieve rapid hybridization, high specific binding of targets to complementary probes, and substantially improved signal-to-noise ratios. In addition, this method is also important when evaluating gene expression because it is associated with cancer and disease related symptoms [US Patent Application No. 10 / 079,939 and US Patent Application No. 09 / 904,472; Both documents are incorporated herein by reference.

This application is directed to U.S. Provisional Application Nos. 60 / 369,945 filed April 4, 2002 and 60 / 330,669 filed October 26, 2002, and PCT / US02 / 26867 (August 23, 2002). Claiming priority under 35 USC §119 (e).

Technical Field of the Invention

The present invention relates generally to gene specific amplification, analysis and profiling of cytoplasmic biological molecules useful in the field of oncology and diagnostic testing. The present invention is particularly useful for chemotherapy treatment or in such fields as screening, screening and monitoring of cancer for cancer recurrence. More specifically, the present invention provides methods, devices, and kits for facilitating extensive analysis of mRNA and DNA of tumor cells, or other rare cells derived from biological samples, while maintaining cell integrity for counting and morphological image analysis. To provide. To this end, the present invention also provides a method for analyzing soluble cytoplasmic biomolecules that can be released from cells, such as tumor cells, using permeabilization reagents to determine the expression profile of the released nucleic acid. The method maintains the morphological and antigenic properties of the cells used in subsequent or concurrent multiparameter flow cytometry, imaging and immunocytochemical analysis (see US Pat. No. 6,365,362). The present invention also provides a method capable of performing such a wide range of assays using samples stabilized with aldehyde and aldehyde-urea derivative based fixatives.

1 illustrates a number of performances and choices described herein in a multi-parameter assay for a single sample feasible by the present invention. Phenotypic and genotyping assays are performed on fixed or unfixed cells.

FIG. 2 shows 2% agarose after SYBR gold staining of about 1600 SKBR3 breast cancer cells treated with Imuniperm at various time points prior to isolation of total RNA from the supernatant produced via Trizol + pellet paint coprecipitation. Inverted (negative) images of denatured total RNA were analyzed by gel electrophoresis.

FIG. 3 shows ethidium comprising whole cells, immunopumin (saponin) -infiltrated cells derived from cell pellet fractions, and cells obtained from the supernatant fraction of immunopenim® impregnated SKBR3 breast cancer cells. 1% denatured total RNA agarose gel stained with bromide.

FIG. 4 shows a Northern blot phosphor image of the gel shown in FIG. 3, hybridized with polynucleotide kinase treated ³² P-labeled oligo (dT) (25 mer) probes. Radioactive signals indicate the presence of all poly (A) + mRNA transcripts of total RNA, derived from whole cells and two immunoperm (R) treated cell fractions obtained from the gel shown in FIG. 3.

FIG. 5 shows the northern blot results of FIG. 4, stripped through conventional digestion and removal of labeled oligo (dT) probes and reprobed with nuclear specific precursor rRNA probes.

FIG. 6 shows the Northern blot results obtained in FIG. 5, stripped and reprobed with mitochondrial specific 12s rRNA probes.

FIG. 7 shows cDNA array dot blot hybridization pattern comparison results when the mRNA used was an alpha- ³² P-nucleotide labeled during first strand oligo (dT) primed cDNA synthesis, used to obtain the gel image of FIG. 3. will be. The labeled first strand cDNA was then used as hybridization probe. As a result of the pattern comparison, the mRNA present in all three RNA cell fractions shows the same relative abundance.

FIG. 8 shows gel images of relative cytoplasmic total RNA obtained quantitatively and quantitatively after each of multiple aliquots containing about 770 PC-3 cells were treated with Immunep®.

FIG. 9 shows the results of analysis on preservation, recovery, and RNA integrity for 90-100% of the total RNA libraries, which were subjected to enzyme digestion after treatment with cytochrome® and other aldehyde-based fixatives. In the present experiment, initially, three different fixatives, ie, lanes (phosphate buffered saline, PBS), which do not possess cytostatics (trade name), Stabilsite (trade name) and Transfix (trade name), and those having Large standardized aliquots of 300,000 SKBR3 cell line cells from control lanes such as lanes were first spiked into 7.5 ml of freshly collected peripheral blood. After mixing this, it was incubated for 24 hours at room temperature (20-25 degreeC). SKBR3 cells were then amplified from blood via VU-1D9 (EpCAM) -magnetic fluid immunomagnetic screening. The amplified cells obtained in each treatment were divided into three homogenous aliquots and treated with proteinase K digestion (labeled “Post”) or without proteinase K digestion (for immediate RNA isolation, “ Pre ", where the lane marked" No "differs from Post in that the phosphotinase K component is added and all other operations are identical to Post). The resulting standardized RNA isolation was isolated with 1% denatured agarose stained with SYBR Gold and imaged with alpha image density meter, followed by Northern blotting, and finally total RNA and mRNA libraries recovered by oligo (dT) probing, respectively. Relative qualitative and quantitative analysis results for.

10A and 10B show cytotoxicity, Stabilsite®, transfix®, paraformaldehyde, formaldehyde, glutaraldehyde and glyoxal fixation after 1 hour, 2 hours and 4 hours It represents the relative ratio of. In this experiment, the relative ratio stiffness to cytosfex®, stabilsite®, transfix®, paraformaldehyde, formaldehyde, glutaraldehyde and glyoxal fixation was 1 hour, 2 hours and Evaluation was made after 4 hours. 7.5 ml of a blood sample was prepared from a single donor by the same method as shown in FIG. The only difference is that the samples were screened and processed for RNA isolation at 1, 2 and 4 hours. The resulting standardized RNA isolate was isolated using 1% denatured agarose, stained with SYBR Gold, imaged with an alpha image forming density meter, followed by Northern blotting, and finally oligo (dT) probed recovered. The relative qualitative and quantitative analysis of each total RNA and mRNA library is shown.

FIG. 10C shows the relative fixed ratio of cytoscopy to paraformaldehyde at 15, 30 and 45 minutes. In this experiment, the relative velocity over time results of cytotoxic® versus paraformaldehyde were evaluated at 15, 30 and 45 minutes, respectively. 7.5 ml of blood sample was prepared from a single donor by the same method as shown in FIG. 9. The only difference is that the samples are after 15, 30 and 45 minutes. Phosphorescence image quantitative results of the oligo (dT) probed blots indicate the relative quantitative and quantitative results of the mRNA library, with the following relative ratios: formaldehyde = paraformaldehyde (about 4 times)> transfix ( Trademark name) (approx. 2 times)> Stabilsite (trade name) (approx. 1.5 times)> Cytoxex (trade name) (approx. 1x) = glutaraldehyde and glyoxal are too slow to be evaluated. The resulting standardized RNA isolate was isolated using 1% denatured agarose, stained with SYBR Gold, imaged with an alpha image forming density meter, followed by Northern blotting, and finally oligo (dT) probing Relative quantitative and qualitative analysis results were shown for RNA and mRNA libraries, respectively.

FIG. 11 shows the effect of nucleophile and enzyme mutations for the qualitative and quantitative analysis of RNA recovery in cytospheric preservation, supporting the fact that improved treatment can be achieved by using multiple treatments in combination. . The resulting standardized RNA isolate was isolated using 1% denatured agarose, stained with SYBR Gold, imaged with an alpha image forming density meter, followed by Northern blotting, and finally oligo (dT) probing, recovered. The relative qualitative and quantitative analysis of each total RNA and mRNA library is shown.

FIG. 12A shows the reliability of a proven assay by detecting specific mRNAs derived from 10 SKBR3 cells / 7.5 mL blood in 24 hour cytostable stabilized blood by proteinase recovery and aRNA preliminary amplification. As used herein, the reliability of a diagnostic method is evidenced by the sensitivity and repeat detection of specific mRNAs derived from 10 or 20 SKBR3 cells spiked three times in 7.5 ml of peripheral blood. The spiked blood was immediately stabilized by cytotoxicity, resulting in stabilization of cellular RNA. After incubation for 1 day at room temperature (20-25 ° C.), stabilized cells were selectively amplified using VU-1D9 (EpCam) -magnetic fluid immunomagnetic screening. After amplification, RNA was isolated by proteinase K digestion, followed by silica binding RNA separation, followed by aRNA preamplification and gene specific quantitative RT-PCR for CK19 and EpCAM.

12B and 12C show the respective Q-PCR results for CK19 and EpCAM, obtained by reverse proteinase treatment after SKBR cell spikes, magnetic fluid screening and cytoscetics treatment. This experiment shows the results of quantitative RT-PCR quantified against the original mass of total RNA prior to graphing. Thus, the figures presented here are the same as for the original mRNA population contained in the original RNA isolate prior to aRNA amplification.

12D and 12E show the respective Q-PCR results for CD10 and EpCAM for aRNAs derived from SKBR cells treated with cell stabilization reagents. This experiment shows RNA derived from three different fixatives (shown as Cytox®, Stabilsite® and Transfix® in FIG. 9). Proteinase K recovered RNA samples herein were also subjected to the same mRNA template assay as in aRNA pre-amplification and gene specific quantitative RT-PCR for CK19 and EpCAM, respectively, shown in FIGS. 12B and 12C. The data presented herein were normalized to total RNA mass, which is equivalent to aRNA mass yield in this experiment. Thus, these fixative inducible relative RNA quantifications and quantitative comparisons were made for the two aRNAs (FIG. 12A), followed by the qualitative RT-PCR shown herein. The results of these two comparisons are the results of each fixative dependent RT template qualities after RNA was recovered using only the proteinase K method.

FIG. 13A shows the RT-PCR amplification efficiency of the CK19-cRNA standard containing a CK19 mRNA transcript sequence consisting of approximately 800 bases at the 3 'end. Two consecutive dilutions of the CK19-cRNA standard containing 200, 100, 50, 25, 12 and 5 copies were spiked three times to 2 ng of CK19 negative total RNA derived from leukocytes, The maximum coefficient of variation was 27%. Standard deviation bars are shown. The cRNA was diluted to less than one copy and no detectable signal was generated in the absence of template control.

Figure 13b shows the relative RT-PCR gene expression levels after agarose gel electrophoresis. CK19 cRNA was spiked to total RNA derived from leukocytes at concentrations of 25 copies, 250 copies, 2,500 copies and 25,000 copies in Panel 1, Panel 2, Panel 3 and Panel 4.

FIG. 13C shows the comparison of relative positions in the same mRNA library of unamplified and T7 promoter-based amplified mRNA transcripts. Eight different mRNA transcripts (PSA, PSM, MGB1, MGB2, CK8, CK19, PIP, EpCam) were observed by using the RT-PCR dynamics curve method to assess relative abundance.

FIG. 14A shows a scatter plot bar graph for gene vista suggesting the presence of circulating epithelial cells. Human blood samples, which were immunomagnetically amplified at their cell surface for cells expressing EpCAM antigen, were initially aRNA pre-amplified and then 25 ng of this was reverse transcribed. After the RT-PCR for the gene selection group was analyzed by agarose gel electrophoresis. Thirteen healthy donors (7 males and 6 females) were named N columns for each gene measured, and nine consecutively sampled HRPC patients containing circulating tumor cells (CTCs) were examined by flow cytometry. This was named P column for each gene measured. The horizontal line in each column represents a threshold above the numerical value counted as a real positive number.

FIG. 14B is a result of observation of a gene representing an integrity prostate tumor organ in the same manner as described in the description of FIG. 14A. FIG.

FIG. 14C shows that there is a target symptom to be treated in the same manner as described in the description of FIG. 14A.

15A, 15B and 15C show in column columns each HRPC patient monitoring CTC and RT-PCR multigene analysis before, during and after a new series of chemotherapy. Here, the x-axis represents the sampling time (week), and in the y-axis on the opposite side, the symbol ○ indicates the CTC level. The y-axis on the right shows the relative expression levels of mRNA for androgen receptor (AR) (□), heptin (HPN) (○) and multiple drug resistance (MDR1) (Δ). This figure shows the relative mRNA levels when only Lupron was treated (FIG. 15A) and two patients were treated in parallel with Taxotere administration and Estramustine chemotherapy (vertical on the X-axis of FIGS. 15B and 15C). Indicated by arrow (Tx / Ex)]. The bar at the top shows the case where the hormone removal treatment is in progress for a long time.

Summary of the invention

The present invention provides methods, devices, and kits for assessing gene expression in amplified mRNA isolated from circulating rare cells (see FIG. 1) that overcome the disadvantages of the prior art described above. The present invention provides a method for separating soluble or releasable cytoplasmic biomolecules from a single target cell or cell population while maintaining properties relating to the structural integrity or phenotype of the cell. Thus, the cell (s) are fresh or stabilized and, after immobilization with a crosslinker, are contacted with a pore-forming permeabilization, the nucleic acid is recovered. RNA obtained from stabilized cells (PCT / US02 / 26867) is a proteinase for amplification and subsequent quantitative and quantitative PCR assays or for quantitative analysis through gene specific subsets of reverse transcriptase (RT) primers fused with universal primers. And recovery using a nucleophiles reversal agent, followed by PCR amplification using the universal primer. Thus, one aspect of the present invention is to prepare cells for both cytoplasmic biomolecular assays and cell phenotyping assays by stabilizing and immobilizing cells prior to infiltration and then releasing nucleic acids from these stabilized cells.

The invention also provides a method of contacting a cell or cell population with a permeation composition and releasing / released or unreleased fractions into one or more components such as nuclear and / or mitochondrial DNA, total RNA, mRNA, soluble proteins and By independently analyzing other target materials, it relates to the isolation of DNA, RNA, proteins and other soluble components of targeted intracellular nuclei and / or mitochondria (US Patent Application No. 60 / 330,669).

The present invention is directed to contacting cell (s) with a permeation composition and analyzing cytoplasmic and surface biomolecules, respectively, to quasi-transform the transformed and normal cells, including features derived from genotypic and phenotypic cell properties. By obtaining a functional cell profile, it is cited to analyze cytoplasmic biomolecules and membrane or surface biomolecules derived from the same cell (s) or cell population.

The isolation and rare cell assays of the present invention provide methods and reagents that, in parallel, allow for extensive profiling of mRNA obtained from rare cells. For example, the population of cells that define circulating tumor cells (CTCs) is a rare cell of the type found in the peripheral blood and bone marrow of cancer patients. The mRNA is obtained through the cell preparation described in the present invention, but could also be combined with any protocol commonly used in the art.

After isolating and purifying mRNA from a sample containing the cells of interest, very rare cellular phenomena with a small number of mRNA copies may be genetically specific RT-PCR with or without T7 RNA polymerase (T7RNAP) based preamplification methods. It can be detected through the panel (US Patent Application No. 60 / 369,945). Preliminary amplification is terminated by first amplifying the entire mRNA library using a variation of the Eberwine a RNA method (Van Gelder et al., 1990). In a preferred embodiment, the preamplification of the antisense mRNA library (aRNA library) increases at least 1000 fold all the messages present in the original mRNA isolated from ferrofluid enriched circulating cells. The gene specific primers are then used to amplify only the panel of genes of interest. The primers are designed to amplify transcripts that exhibit known whirling phenomena such as circulating tumor cells. The number of target sequences may be on the order of two or may be of a size that may be associated with some feature exhibiting this rare phenomenon. This can occur as individual independent reactions or in a single reaction vial. Subsequent assays allow at least qualitative evaluation of the target sequence, which is also provided, for example, in two types of multiplexes, presented herein as, for example, gene specific primed (GSP) arrays and / or GSP set-RT (universal PCR). It is performed by one of the genetic assays, such as but not limited to.

Preliminary amplification of the mRNA library through universal PCR (PCR) may be performed, or a multigene assay may be performed on a sample of recovered mRNA in a single reaction tube. No preliminary amplification can make it possible to analyze only one panel of genes at a time. Preliminary amplification further provides the advantage of analyzing a single sample in up to 1000 different reactions, allowing multiple panels of different genes to be observed for different times. Note that other methods are useful, the universal PCR cocktail panel assay is performed by array or capillary gel electrophoresis (CGE). Thus, this system enables both quantitative and qualitative determination of one to thousands of mRNAs of each type simultaneously when measured in cDNA microarray format.

Therefore, the present invention utilizes small or large scale microarrays, capillary gel electrophoresis (CGE), HPLC, electrophoresis and other analytical techniques to understand and analyze a wide range of RNA analysis results, including permeation compositions, post-crosslinked RNA recovery, The isolation and profiling described above in connection with kits and protocols comprising some or all of the essential reagents, including magnetic microspheres and other gene specific magnetic microsphere-binding probes, wherein the oligo (dT) probes are covalently bound to the surface Include combinations of assays.

Detailed Description of the Preferred Embodiments

As indicated above, the broader and more practical types of cancer diagnostics have also been described in the same cell or cell population, as well as in the analysis of endothelial and extracellular membrane antigens, which so far are mutually exclusive methods (US Pat. No. 6,365,362). Cellular RNA content and DNA content analysis. The monopoly of this method was due to the fundamental inadequacy of the pre-analytical cell preparation method for structural intracellular antigens and the separation of cytoplasmic biomolecules, whose primary purpose is to maintain cell integrity. Alternatively, pre-analytical cell preparation may also be limited to soluble cytoplasmic RNA, total cellular RNA, total cellular DNA, and / or protein, wherein the primary purpose is to homogenize the cells to release soluble intracellular components. (US Pat. No. 6,329,179). In particular, conventional phenotypic characterization required fixing cell structures by exposing the cells to crosslinking agents such as paraformaldehyde, formaldehyde, glutaraldehyde and the like. Such vigorous immobilization conditions cause undesirable crosslinking and / or fragmentation of all separable RNA species. Recently, similar intracellular DNA-protein crosslinking has been reported [Quievryn and Zhitkovick, Loss of DNA-Protein Crosslinks from Formaldehyde-Exposed Cells Occurs Through Spontaneous Hydrolysis and an Active Repair Process Linked to Proteosome Function, Carcinogenesis, 21: 1573-1580 (2000). So-called non-formaldehyde or non-paraformaldehyde fixatives (e.g., Cyto-Chex ™ Streck Labs, Omaha, NE) are cell-stabilizing additives containing formaldehyde-urea derivative donor compounds to be. It is used as a preservative for blood circulating tumor cells upon shipment or storage as disclosed in co-pending patent application (PCT / US02 / 26867, incorporated herein by reference). However, studies conducted by the inventors have shown that even cytoscene® containing only trace amounts of free formaldehyde releases formaldehyde capable of crosslinking intracellular RNA with intracellular proteins. Such crosslinking is completely reversed by the method of the present invention to enable extensive RNA analysis. Therefore, cellular RNA and DNA assays are typically prepared for fresh cells that are not immobilized, or for cells that can be preserved with reagents that do not crosslink, or whose crosslinking can be reversed while mRNA is released from the cell. RNAlater ™ is a commercially available RNA stabilization solution that stabilizes RNA but cannot be immunomagnetic, immunochemical or image assayed on the same sample and is not useful for blood. PreAnalytiX provides a blood RNA stabilizer, but is only a guanidine isothiocyanate solution (GITC solution), a chaotropic agent in a Vacutainer ™ tube that enables common homogenization based on total RNA isolation. Do.

In general, mRNA recovered from immobilized cells is not quantitative and has an average size of about 1750 bases, which is about 10 bases larger than the average size of about 200 bases, compared to intact RNA. It is well known that the base has been partially degraded or fragmented, reducing its size, and also causing a number of complex chemical modifications. However, the overall effect on fixative-derived RNA is significantly compromised with mRNA analysis (Current Protocols in Molecular Biology, Wiley, (2002)). In combination with reverse-transcription polymerase chain reaction (RT-PCR) assays designed for amplicons less than 100 base pairs in length, the lengthy non-quantitative mRNA rescue technique, although performed in a qualitative rather than quantitative manner, [US Pat. No. 5,346,994]. In addition, this limited RNA assay of immobilized cells should be performed according to phenotypic analysis. Therefore, the two methods cannot be performed continuously on the same cell sample, because conventional RNA separation techniques destroy cell structure and further remove cellular DNA and RNA populations when complete cell digestion or homogenization is performed. This is because it is necessary to hybridize with each other to perform complex analyzes [Maniatis et al., Molecular Cloning-A Laboratory Manual, 2nd ed. Cold Spring Harbor Press (1989). Previous reports have stated that there is a need to improve the method of RNA recovery in tissues [Godfrey et al., Quantitative mRNA Expression Analysis from Formalin-Fixed, Paraffin-Embedded Tissues Using 5 'Nuclease Quantitative Reverse Transcription-Polymerase Chain Reaction, J. of Molecular Diagnostics, 2: 84-91 (2000). Using formaldehyde and urea-based fixatives to stabilize quantitative, high-field integrity total mRNA libraries recoverable from blood, it is the basis of one aspect of the present invention that enables extensive analysis to be performed.

Surprisingly, it was found that saponins used as penetrants are highly selective and efficient release agents for intracellular cytoplasmic RNA and other biomolecules, requiring no cell degradation or homogenization process. This novel use of saponins as RNA release agents is one of the particularly beneficial elements of the present invention. Surfactants such as saponins have been commonly used to observe the expression of intracellular antigens by penetration of cell membranes, which can incorporate staining reagents while maintaining cell integrity. For example, chromosomes by fluorescence in in situ hybridization (FISH) or in intracellular component staining such as staining of nuclear DNA using DAPI, or immunostaining of cytokeratin using specifically labeled antibodies. Or gene analysis plays a very important role. In general, release of cytoplasmic intracellular protein RNA or DNA can be achieved by lysing or completely dissolving the cells with a strong surfactant such as Triton X-100. However, saponins are used for the study of both the expression of soluble intracellular antigens, including RNA, and the phenotypic characterization of each cell or cell population in the same sample. Therefore, continuous phenotypic assays in the cytoplasm of the same cell sample and assays of integrity RNAs and soluble proteins are highly desirable and are also subject of the present invention.

Accordingly, the present invention provides methods, devices, and kits that are advantageous for rapid and effective RNA profiling of all cells found in biological samples, particularly targeted cells. The present invention provides a method capable of independently analyzing both phenotypes and genotypes. Phenotypes are identified and profiled through antibody antigen proteins and mass spectrometric profiling methods of integrity cytoplasmic RNA derived from cells or cell populations and extensive analysis. Genotyping of the sample, ie, genotype and mitochondrial DNA, can be independently profiled by any method known to the skilled person. Similar to the amplification of the mRNA library, each genotype and mitochondrial library is preamplified so that multiple assays can be performed without losing medical sensitivity, because multiple substitution amplification (MDA) techniques are the primary This is because it enables an effective whole genome amplification method. MDA is a fast and reliable way to generate unlimited DNA from a small number of cells.

The invention described herein can be used to effectively isolate and characterize cell phenotypes such as cell surface antigens, intercellular antigens and any type of RNA, and genotypes. Both phenotypic and genotyping assays can be performed continuously on exactly the same sample. For example, after cell surface assays and RNA collection, the remaining integrity nuclei and mitochondria are all standard RNA (mtRNA, hRNA), DNA and protein based assay techniques such as S1 nuclease, ribonuclease protection, RT- Downstream can be analyzed by PCR, SAGE, DD-RT-PCR, microarray cDNA hybridization, ISH, FISH, SNP, all RNA and all genome-based PCR techniques and any protein analysis system.

One of the many uses of this type of cell assay is to diagnose cancer. Many clinicians believe that cancer is an organ specific disease that is limited in its early stages. This disease occurs systemically over time and was first detected using current methods. Thus, evidence suggesting the presence of tumor cells in the blood stream suggests that it will provide a first line of detection mechanism that will replace, or work in conjunction with, other tests such as mammography or measurement of prostate specific antigens. do. By analyzing cell phenotypes (proteins and RNA) and genotypes through markers specific to the cells, organs of these cells such as breast, prostate, colon, lung, ovary or other non-hematopoietic cancers can be readily measured. Can be. Therefore, if proteins, RNA and genomes can be analyzed, especially if no clinical signs of the carp are present, specific tumors and organs of origin will be identified. Because this profile defines the function of the cell, it also indicates what type of treatment method and treatment route is most appropriate when used for cancer cell observation. In addition, post-surgical or other continuous treatment, when monitored in the absence of detectable evidence for circulating tumor cells, further clinical studies may determine whether additional treatment is needed.

In order to allow for a broader and earlier diagnosis, one embodiment of the present invention separates cytoplasmic biological molecules from cells or cell populations while maintaining cell integrity for subsequent phenotypic and morphological analysis. And contacting the cell or cell population with a permeabilization compound, and separating the desired cellular biological molecule from the cell.

By targeted rare event in the present invention is meant expressing any biological molecule that at least partially indicates a known rare phenomenon. Thus, hormones, proteins, peptides, lectins, oligonucleotides, drugs, chemicals, nucleic acid molecules (eg RNA and / or DNA) and biological particles such as cells, apoptotic bodies, cell fragments, nuclei, mitochondria, Viruses, bacteria, and the like will be included in embodiments of the present invention.

Fluid samples include, but are not limited to, cell-containing body fluids, peripheral blood, bone marrow, urine, saliva, sputum, semen, tissue homogenates, milk effluents, and any other rare cell source available from human subjects. no.

A "cytoplasmic biological molecule" includes a target cellular target molecule, examples of which include, but are not limited to, proteins, polypeptides, glycoproteins, oligosaccharides, lipids, electrolytes, RNA, DNA, etc., located in the cytoplasmic fraction of cells. It doesn't happen. When separating cells after contacting the cells with the permeable compound, cytoplasmic biological molecules are present in the supernatant for downstream analysis. All soluble cytoplasmic biomolecules, such as whole cytoplasmic RNA libraries or target components capable of passing through membrane pores, can be isolated and analyzed. Preferred embodiments focus on the analysis of transcribed mRNA and translated proteins in CTCs, for example, as indicators of carcinogenic metastasis aimed at performing cancer diagnosis and treatment.

“Membrane biomolecule” includes any desired extracellular, intercellular, or intracellular domain molecule that is associated with or embedded in a cell membrane, such as an outer cell membrane, nuclear membrane, mitochondria, and other organelle membranes. Include. When infiltrated with the penetrating compound of the present invention, the targeted membrane biomolecule is usually not dissolved or removed from the membrane. That is, the membrane biomolecules remain bound to the invasive cells. Membrane biomolecules bind to cell membranes, including those present on the outer or extracellular surface of the outer membrane, as well as those present on the inner surface of the outer membrane, and proteins associated with nuclear membranes, mitochondrial membranes and all other intracellular organ membranes. Proteins, glycoproteins, lipids, carbohydrates, nucleic acids, and combinations thereof. Membrane biomolecules also include cytoskeletal proteins.

By "genotype" or "genotyping" is meant a method of identifying an intracellular genetic material, such as DNA, that possesses encoded genetic indications that form and control all aspects such as cell survival and death. By "phenotype" or "phenotyping" is meant to classify cells based on observable external structural elements and their products (ie, including intermediate RNAs). These include taxonomic, morphological and other surface area characteristics, all of which can be obtained from information about internally encoded genotypes and can be used in combination with the methods of the present invention. In contrast, cell structure and integrity ensure that at least all cell constructs, except the nucleus and mitochondria, are completely degraded in the presence of NP-40, usually by disrupting all cell structures during chaotropic salt treatment and / or mitochondrial cell homogenization. It is not maintained in the performance of conventional RNA isolation techniques associated with this. .

Morphological analysis or morphology of the cell structure may be used to determine the anatomical aspects and surface properties of cells and nuclei such as intracellular or surface markers, epitopes or detectably labeled binding partners such as antibodies that can be stained with histochemical reagents. It is used as commonly defined in relation to the anatomy and surface properties of cells and nuclei, including their interaction with. In addition to morphology, it covers the entire field of “morphometry”, defined as follows: Quantitative measurement of chromatin distribution in the nucleus.

The terms genotype and proteomics are used as commonly defined. As used herein, the term "functional" is an adjective that refers to a biological feature or characteristic of an experimentally detectable cell, for example, the term "functional celomic" refers to genomic and proteomic and other target categories. The broader meaning encompasses all, but includes, but is not limited to, "glyconomic" for carbohydrates and "lipidomic" for cell lipids. The resulting cellular characteristics provide a profile that can differentiate normal cells from transformed cells.

"Contact" means contacting a compound or reagent directly or indirectly with a physical contiguous portion of a cell. Cells and / or compounds are present in any number of buffers, salts, solutions, and the like. Contacting made to obtain the cell (s) includes, for example, placing the reagent solution into a tube, microtiter plate, microarray, cell culture flask, or the like. Microtiter plates and microarray formats for simultaneous analysis of multiple cellular target compounds or components, including but not limited to nucleic acids and proteins, make it possible to perform additional complex assays.

An “invasive compound, reagent or composition” refers to the formation of pores in cell membranes, including lipid-cholesterol bilayers, while maintaining the membrane, cytoplasm and nuclear structure sufficient to perform subsequent phenotyping assays on the infiltrated cell (s). Means any reagent. For example, saponins complex with cell membrane components to form multiple transmembrane pores of about 8 nm in size on the cell wall or cell membrane, thereby allowing small soluble cell substrate components such as enzymes, proteins, glycoproteins, globulins, electrolytes, etc. Is a known “pore-forming” compound that diffuses outward and is in internal equilibrium with extracellular reagent components such as electrolytes.

“Immunomagnetic beads” are substantially covalently bound to binding reagents (eg, antibodies) that retain selective affinity for cellular surface markers or epitopes, and thus are used in magnetic fields such as high gradient magnetic separation systems (HGMS). Magnetically labeled nanoparticles or microparticles that can selectively capture magnetically labeled cells when exposed to a magnetic field being formed. Other terms used herein for methods, reagents, and instruments are as commonly defined and known to those skilled in the art.

Preferred gene expression targets (mRNAs and proteins) for identification, diagnosis, prognosis, therapeutic target characterization and monitoring of tissues of origin include, for example, mammoglobin 1 (MGB1), mammoglobin 2 (MGB2), prolactin induction. Sex protein (PIP), carcinoembryonic antigen (CEA), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), onboard kallikrein 2 (hK2), androgen receptor (AR), prosta God, Hepsin (HPN), DD3, Her-2 / Neu, BCL2, Epidermal Growth Factor Receptor (EGFR), Tyrosine Kinase Receptor (HER2), Thymidylate Synthetase (TS), Vascular Endothelial Growth Factor VEGF, Pancreas Mucin (Muc1), guanylyl cyclase C (GC-C), phosphatidylinositol 3 kinase (PIK3CG), protein kinase B gamma (AKT), incision repair protein (ERCC1), alpha-1 globin (F6), macrophages Inhibitory cytokin-1 (G6), dihydropyrimidine dehydrogenase (DPYD), insulin growth factor receptor (IGF2), alpha and beta Estrogen receptor (ER), progesterone receptor (PR), aromatase (cyp19), telomerase (TERT), systemic epithelial tissue specific genes, cytokeratin 19 (CK19), cytokeratin 5 (CK5), cytokeratin 8 ( CK8), cytokeratin 10 (CK10), cytokeratin 20 (CK20), epithelial cell adhesion molecule (EpCAM), mucins such as mucin 1 (MUC1), topoisomerase, urokinase plasminogen activator (uPA), urokinase Detection of marker expression sensitization or resistance genes such as plasminogen activator receptor (uPAR), matrix metalloproteinase (MMP), systemic leukocyte specific mRNA, alpha-1-globin, CD16, CD45, and CD31, and Cells derived from breast, prostate, lung, colon, ovary, kidney, bladder, and the like for monitoring include, but are not limited to. The above list is intended to illustrate the general diversity of mRNA-specific gene arrays that can be incorporated to differentiate cells from various origins, types and diseases, but this is not all.

Stabilization, release and recovery

Circulating epithelial cells were compared to leukocytes using the methods disclosed in the above-described invention (US Pat. No. 6,365,362 and US Patent Application No. 10 / 079,939, co-assigned herein; all of which are incorporated herein by reference). It can be amplified by 2,500 to about 10,000 times. Following immunomagnetic screening of circulating epithelial cells in blood, nucleotide assays specified in the present invention are performed. Such amplification is an example of one of a number of methods known in the art for selecting specific cell populations for use in embodiments of the present invention.

The method of releasing integrity cytoplasmic total RNA and mRNA from these cells, isolating and purifying them is performed by staining and immunostaining, both cytoplasmic RNA and intracellular antigen phenotyping and DNA genotyping for the same cell, cell population or sample. In the case of prior to performing continuous or simultaneous assays, they were usually developed when infiltrating cells with saponins, which was surprising and groundbreaking.

Permeabilization can be carried out under the criteria of the present invention using one of three types of common surfactants or detergents: pore forming reagents such as saponin, or saponin aliquots such as QS-21, esin, digionine , Cardenolide etc. All of these agents increase the porosity of the membrane to release small soluble intracellular components. Another group of formulations are surfactants. These agents maintain a relatively high hydrophilic-lipophilic balance and do not degrade and penetrate the membrane. In addition, more soluble surfactants with lower hydrophilic-lipophilic balances also tend to release RNA but dissolve the membrane. Examples of such surfactants include t-octyl phenoxy ethoxy ethanol or SDS, such as polyoxyethylene sorbitan (commercially available as Tween 20, 40 or 80), nonylphenoxy polyethoxy ethanol (NP-40), It is not limited to this.

Subsequent analysis of cytoplasmic RNA (and other RNAs such as mtRNA and hnRNA), cell surface and soluble intracellular antigens, organelles such as mitochondria and the rest of the indexed nuclei is down by all standard RNA, DNA and protein based assay techniques. Stream can be analyzed. Such techniques include protein microarrays for all types of cDNA, RNA and profile analysis, mass spectrometry, fluorescent in situ hybridization (FISH), single nucleotide polymorphism (SNP), all genome-based amplification techniques such as PCR, etc., microsatellite ) Assays, restriction fragment length polymorphism (RFLP, ALFP), SAGE, DD-RT-PCR, and the like.

Such assays can be performed on 1-10 RNA molecules for each of any of the RNA sequence types, but are preferably performed on tens of millions to millions of target copies to provide cellular translation or transcription profiles as indicators of the condition in clinical conditions. It is possible to detect whether this is a slight deformation. Other functional cell profiles for releasable and non-releasable cellular components such as proteins, glycoproteins, lipoproteins, oligoglycosides, etc. can be applied to conventional microarrays, HPLC, electrophoresis such as high resolution 2D electrophoresis or antibody array profiling. By analysis in two classes of methods.

The penetrating compounds of the present invention include saponins, cholesterol-like aglycones or ginines (triterpenes or steroids containing no carbohydrates) and one or more carbohydrates (which are readily dispersed in water to form spherical micelles), namely And natural products consisting of species active in pore formation. Those mentioned above as suitable pore formers and others, such as polyoxyethylene sorbitan (commercially available as Tween 20, 40 or 80), nonylphenoxy polyethoxy ethanol (NP-40) and t-octyl phenoxy Toxic ethanol has a high HLB (hydrophilic-lipophilic balance) number, which should be used to a degree sufficient to minimize solubilization and membrane degradation of undesirable cellular components even at low concentrations. The concentration of the permeating compound is about 0.01-0.5% (w / v) when using saponin containing about 10% saponenin. Preferred penetrating compounds include saponins (Sigma Catalog Number S-7900). Saponins derived from other sources and saponins of high purity may also be used, such as saponin (Sigma S-4521), which is about 20-25% of saponin, and QS-21 (about 99%, which is high purity, saponin). , Aquila Biopharmaceuticals, Framingham, Mass.). Other useful compounds include alpha-essin and beta-essin (Sigma E-1378), all of which are derived from horse chestnut. Permeation compounds such as phosphate buffer solutions may be present in the composition, such as compositions that also include antimicrobial agents such as sodium azide, Proclin 300 (Rohm & Hass, Philadelphia, PA) and the like. Another preferred penetrant is Immunperm ™, which itself releases about 50% of cellular RNA (85% of all RNA in the cell) that does not affect the nuclear or mitochondrial nucleotide pool. The remaining 50% of total cellular RNA in immobilized cells and all DNA can be released in a release cocktail comprising SDS, protease and formaldehyde scavenging agent, wherein the composition is one of the invention A specific example is achieved. Although the exact mode of action of each cocktail component is unknown, the SDS dissolves intracellular RNA and DNA cross-linked to structural intracellular proteins, making proteolysis and formaldehyde cross-linked nucleic acid release more efficient. . Examples of novel formaldehyde scavenging reagents include, but are not limited to, hydroxylamine, carboxymethoxylamine, hydrazine, acethydrazine and other hydrazide or hydrazine derivatives, and amines such as tris, And to increase yield, it is believed to increase the amount and “quality” of nucleic acid released. The two classes and release cocktails released into the immunoperme can be analyzed or pooled, respectively.

Accordingly, the invention also stores and maintains any surfactant or protease (or combination thereof) with or without formaldehyde scavenger, which can release cellular nucleotides, in a form suitable for simultaneous analysis. It is included in the range of.

Unlike current cell fixation and RNA recovery protocols that tend to fragment large amounts of cellular RNA, the present invention provides for integrity derived from cells treated with penetrants such as saponins that penetrate the cell membrane while maintaining cell integrity. More than 90% of the cytoplasmic total RNA and mRNA can be extracted and separated. mRNA isolation is also suitable for immunomagnetic cell amplification and immunofluorescent cell labeling methods. Comprehensive RNA expression profile analysis of cells identified and characterized by a cell assay platform such as T7, SP6, or T3 promoters, flow cytometry, microarrays, and Cell Spotter® or CellTracks systems (all of which are Immunicon, Pennsylvania) RNA polymerase promoter-based primary amplification method using (manufactured by Corp.) can directly validate, supplement, and expand expression profiles to enhance information obtained therefrom.

Although not limited to a specific fixative, the permeated cells are treated with a crosslinking agent to maintain morphology, antigen and nucleotide integrity as described above. Three types of commercially available stabilizers are Cytox®, Stabilcyte® and Transfix®, which are useful for stabilizing blood cells in blood samples for long periods of time. These stabilizers are mostly optimized to maintain cell size (predominantly by minimizing contraction) and to preserve antigen on the cell surface, as measured by flow cytometry. The present application generally includes direct assays and does not require extensive sample manipulation or amplification of specific cell populations. In contrast, circulating tumor cells or other rare target cells isolated and detected in the present invention include and are defined as abnormal pathogenic cells or rare cells which are present at very low frequencies, and for this purpose, significant amplification before detection. I need to.

As demonstrated in the practice of the present invention, cytostatic® stabilizers can be used as cell stabilizers, which form macromolecular complexes with intracellular proteins as aldehyde-releasing fixatives of intracellular RNA. . Applicants have unexpectedly found that immobilization, preferably immobilization with formaldehyde donors (e.g., cytotoxic®), is essential for retaining and protecting the RNA in subsequent sample processing, and also for the full functionality of RNA It has been found that release and optimal release require saponin in conjunction with the release cocktail described above.

An ideal “stabilizer” or “preservative” (used herein interchangeably) is defined as a composition capable of rapidly preserving target cells present in a biological sample, which in some way isolates, detects, and counts target cells. And minimizes the formation of coherent aggregates and / or cell debris in the biological sample, which may interfere with distinction from non-target cells. In other words, when used with anticoagulants, stabilizers should not interfere with the performance of the anticoagulant. Conversely, the anticoagulant should not interfere with the performance of the stabilizer. In addition, the disclosed stabilizers may also have a third function of stabilizing by immobilizing penetrated cells [wherein "penetrated" or "penetrating" and "fixed", "fixed" or "fixed" Expression is used as commonly defined in the field of cell biology. As used herein, the term stabilizer refers to the use of such agents in suitable concentrations or amounts, as long as the concentration or amount is effective to stabilize without damaging the target cell (which will be readily understood to those skilled in the field of cell biology). ) Is included. The use of the compositions, methods and devices of the present invention to preserve rare cells is not intended to be used in a manner that damages or destroys such rare cells, and therefore selects an appropriate appropriate concentration or amount. For example, the formaldehyde donor imidazolidinyl urea is preferably effective at a concentration of 0.1 to 10% by volume, more preferably at a concentration of 0.5 to 5% by volume and most preferably at a concentration of 1 to 3% by volume. It is figured out. Additional agents, such as polyethylene glycol, are also understood to be effective for stabilizing cells when preferably added at a concentration of about 0.1-5%. The use of such formulations is described in PCT / US02 / 26867, which is incorporated herein by reference.

A surprising aspect of the present invention is that intracellular RNA, as part of the macromolecular complex, can be amplifiably recovered, mostly quantitatively, from cells previously treated with cell stabilizers and fixatives. Complete release of the crosslinked RNA requires saponins with enzymatic degradation in the presence of soluble detergents and formaldehyde scavengers. For example, as a result of pronase digestion of proteinase K, V8 proteinase, cytoscene-treated cells, RNA could be recovered completely or full-length RNA could be comprehensively analyzed. As disclosed herein, the presence of formaldehyde scavenger is believed to further improve RNA recovery.

In one embodiment of the invention, target cells such as circulating cancer cells or fetal cells effectively separate these cells from other non-target cells, purify their nucleic acids, and then target the target of interest for microarray analysis. It can be evaluated by amplifying (s).

Therefore, the separation of cytoplasmic biological molecules is performed by first separating the infiltrated cells from the infiltrating compounds through centrifugation or immunomagnetic bead enrichment. The cytoplasmic biomolecular mixture is then present in the supernatant. Separation of cytoplasmic biological molecules can be accomplished by capturing magnetic beads. For example, if the cytoplasmic biomolecule is mRNA, oligos (dT) immobilized on magnetic beads or nonmagnetic supports can be used to capture as such, thus allowing mRNA to be removed from the cells with or without centrifugation. Can be separated. If the cytoplasmic biological molecule is a protein, an antibody capable of binding to a specific protein can be used, in which case the antibody can be attached to a magnetic bead or a nonmagnetic support. Other separation techniques are well known to those skilled in the art, and examples include standard protein and RNA chemical extraction, electrophoresis, chromatography, immunoassay, and affinity techniques. Immunomagnetic amplification reagents and devices for isolating cells and biomolecules have been produced by several manufacturers, including Immunicon Corp. (Huntingdon Valley, PA), Dynal (New Hyde Park, NY), and Miltenyi Biotec Inc. (Auburn, CA). Including but not limited to those prepared. The cell may be a prokaryotic cell such as a bacterial cell or eukaryotic cell such as a mammalian cell, the most preferred cell being a cell of human origin. In a preferred embodiment, the cells are carcinogenic cells or tumor cells. Preferred target carcinomas include, but are not limited to, carcinomas derived from breast, prostate, lung, colon and ovarian tissues, such as those found in tissue sections or body fluids such as blood and bone marrow.

A method for preparing a cell for cytoplasmic and / or whole cell biomolecule analysis and membrane biomolecule analysis performed continuously on the same sample is described [Functionally, they are functional genomics or functional proteomics for the analysis of nucleic acids or proteins, respectively. Called]. As mentioned above, this assay can be performed on the same cell (s) before carrying out the method of the present invention. The cells are contacted with the permeating compound to release the cellular biological molecules as described above without modifying the structural and membrane biological molecules.

Thus, as disclosed herein, methods for analyzing cytoplasmic biomolecules obtained from a cell sample and methods for analyzing membrane biomolecules obtained from the same cell sample are stabilized by contacting the cells with the infiltrating compound as described above. And then after the cellular biological molecules have been recovered. Cytoplasmic biological molecules can be separated and analyzed simultaneously or sequentially with biological molecules bound thereto.

The invention also provides reagents and kits for separating cytoplasmic or whole-cell RNA, specifically mRNA. The kit may comprise permeabilization compounds for RNA isolation and detection and RNA extraction reagents, or hybridization probes such as oligo (dT) or gene specific sequences or random (degenerate) oligonucleotides of various lengths. The kit may also include an antibody that binds to a protein bound to the cell, such as an antibody that binds to a membrane biomolecule. The antibodies and probes can be detected by enzymatic labeling, fluorescent labeling or radiolabelled. The antibodies and probes may also be attached to, for example, magnetic beads or the like to facilitate separation.

analysis

Cytoplasmic biomolecule analysis includes any type of assay or assay involving biological molecules isolated from the cytoplasm of the cell. Cytoplasmic biomolecular assays include functional genomic expression profiling such as mRNA profiling, protein expression profiling, reverse transcriptase polymerase chain reaction, northern blotting, western blotting, nucleotide or amino acid sequencing, and continuous analysis of gene expression (SAGE ), Competitive genomic hybridization (CGH), electrophoresis, 2-D electrophoresis, mass spectrometry by MALDI or SELDI, gas chromatography, liquid chromatography, nuclear magnetic resonance, infrared, atomic adsorption, etc. But is not limited to such. Sequence analysis at the nucleotide or amino acid level can direct and identify the presence of mutations in protein, DNA / cDNA, or mRNA sequences. For example, original gene or protein profile assays can direct the presence of oncogenes in variant cells or tumor cells. Analysis after appropriate cancer therapy can further determine whether the tumor burden is reduced or the tumor recurs upon alleviation of the disease by further mutating the oncogene and its appearance of drug resistance or more aggressive tumor cells.

Membrane biomolecular assays include any type of assay or assay involving biological molecules, such as extracellular and intracellular biomolecules or markers, that are bound to or incorporated in the cell membrane present in the cell. Suitable assay methods include, but are not limited to, flow cytometry, enzyme-linked immunosorbent assay, morphological staining, cell sorting, and the like. Infiltrated cells can be sorted by, for example, fluorescent activated cell sorting (FACS) techniques based on the expression of specific detectable proteins. Cell sorting techniques are well known to those skilled in the art and have been used, for example, to simply count detectably labeled cells in the diagnosis of cancer. Infiltrated cells such as CD4 or CD8 cells may also be sorted based on the expression of a particular protein. Membrane biomolecular assays can also be performed on downstream membrane sorts and then via assays including, but not limited to, protein expression profiling. Western blotting, amino acid sequencing, mass spectrometry, gas chromatography, liquid chromatography, nuclear magnetic resonance, infrared, atomic adsorption, surface plasma resonance (SPR) and any other technique are suitable for the analysis of membrane components. .

Functional genomic assays or assays can be performed on genetic material suspended in infiltrated cells. For example, genomic DNA, nucleus (hnRNA), mitochondria (mtRNA), and any other RNA or DNA possessed by an organ (RNA or DNA bound or immobilized within a cell as it penetrates the cell) can be assessed. Can be. Therefore, assays for cytoplasmic biomolecules of the type described above include in situ hybridization, polymerase chain reaction, differential display PCR, random priming PCR, microsatellite analysis, single nucleotide polymorphism (SNP), competitive genomic hybridization ( CGH), restriction fragment length polymorphism assays, nuclear and mitochondrial transcription progression assays, and in vitro protein translation assays, including, but not limited to, genomic DNA, hnRNA, and mtRNA. . However, in order to obtain genomic DNA, nuclear hnRNA and mtRNA, the infiltrated cells must be completely lysed by exposure to the release cocktail of the present invention or further fractionated by conventional means well known to those skilled in the art. In stabilized cells, the combination of proteinases and nucleophiles allows the macromolecular complex containing the desired nucleic acid to be reversed and removed to release RNA and DNA nucleic acid components. In addition, the organelles remaining upon subsequent infiltration can be further fractionated and separated for metabolic functional analysis, such as, for example, mitochondria.

Accordingly, another embodiment of the present invention is to provide a method for separating nuclear or mitochondrial genetic material from cell substrate RNA. Cells containing the nuclear or mitochondrial genetic material and cytoplasmic RNA are contacted with the permeating compound as described above. Nuclear or mitochondrial genetic material can be separated, for example, by further appropriate subcell fractionation, followed by complete cell / organ lysis of this fractionated cellular material. The resulting organ specific components (DNA, RNA, proteins, lipid carbohydrates, etc.) can be extracted from homogenates or analyzed separately. Isolation may also be performed using organ-specific immunomagnetic beads, as described above.

Several substantially important automation benefits can be obtained from the present invention. For example, after removing the permeation solution from the cells, mRNA can be captured with oligo (dT) -magnetic beads, i.e. beads that are ideally suited for microarray-like automated downstream manipulation and a wide range of assays. In addition, in order to obtain both protein and mRNA profiles, it is necessary to make small changes to the mRNA analysis protocol of the present invention to reduce the time required and the amount of reagent. In addition, since the integrity cellular genomic DNA of each of the nucleus and mitochondria is still present in the infiltrated cells, it can be obtained in such infiltrated cells, and also in conventional methods for DNA, RNA and protein such as FISH, SNP, SAGE , DD-PCR, PCR, RFLP, RT-PCR, CGH, cDNA microarrays, mass spectroscopy, protein arrays and the like can also be analyzed downstream. Thus, simultaneous multicomponent analysis of DNA, RNA, proteins, lipids, carbohydrates (and their precursors, metabolites and cofactors) on large microarrays can be widely applied to any eukaryotic cell, tissue sample or body fluid. . This type of cell expression profiling in combination with multicellular component or multibody (eg microarray) assays is the ultimate goal to be achieved through techniques ranging from high throughput screening of drug candidates to disease diagnosis and management.

The present invention also provides reagents and kits for interstitial or whole cell RNA, in particular mRNA. The kit may comprise permeation compounds for RNA isolation and detection and RNA extraction reagents or hybridization probes such as oligo dT or gene-specific sequences or random (degenerate) oligonucleotides of various lengths. The kit may also include an antibody that binds to a protein incorporated into the cell, such as an antibody that binds to a membrane biomolecule. The antibodies and probes can be detected by enzymatic labeling, fluorescent labeling, or radiolabeling. Antibodies and probes may also be attached to, for example, magnetic beads or the like to facilitate separation.

The integrity library is then examined for the presence of any message related to confirming the presence of epithelial cells and / or confirming that tissue of epithelial cell origin exists. For this purpose, all mRNAs present in the sample must be analyzed for the specific gene of interest (each with the same sensitivity / selectivity as the other, capable of recognizing all of the target mRNAs at once).

Under the aforementioned criteria, overall gene expression analysis by microarray will be insensitive to rare phenomena. In particular, the signal-to-noise ratio in the sample is unrealistically low because, for example, problems such as contamination occur when performing leukocyte immunomagnetism in any given amplified sample. For example, in a fluid sample amplified for a particular target cell population by immunomagnetic selection, a target population of 1 to 10 cells could potentially contaminate about 10,000 white blood cells. The target cell (s) express the desired rare phenomenon and will be masked by nucleotides found in white blood cells. Excess leukocyte cells derived from background RNA noise due to the level of extremely rare copies of target mRNAs generate potential signals that cannot be detected.

In order to solve the above problems, the total RNA (or purified mRNA) is pre-amplified using any one of SP6, T3 or T7 RNA polymerase promoter-based in vitro primary preamplification methods. Typical examples include T7 RNA polymerase (T7RNAP), promoter (T7RNAPP) and enzyme amplification systems, but any equivalent system may be replaced with a system apparent to those skilled in the art. The primary preliminary amplification of all messages results in a minimum deviation of the relative abundance of each mRNA sequence in the RNA community, increasing the number of original mRNA libraries by more than 1000 times. The same preliminary amplification method may also be known as transcript amplification, continuous amplification or in vitro amplification. Thus, a feature of one embodiment of the invention is that the number of mRNA copies carrying a polyA tail in the entire library is 1000. The first preliminary amplification method is to increase fold. A single standard mRNA is one that is annealed to the polyA tail region of the oligo (dT) portion of the T7 promoter containing oligonucleotides. RNA polymerase produces an antisense copy (aRNA) of the entire mRNA library. Therefore, in general, the number of copies of mRNA carrying polyA tail in the entire library will increase by more than 1000 times, and in this regard the sensitivity of any particular mRNA sequence type will be increased by 1000 times.

For example, the T7 promoter oligonucleotide primer used as the first strand RT primer and subsequent T7RNAP amplification primers has a 3 'oligo (dT) moiety containing a 5' T7 RNA polymerase promoter sequence having the following base pair configuration: It consists of 67 bases.

The preamplification reaction is terminated by performing a randomly primed DNA polymerase dependent second strand synthesis after the reverse transcription reaction and finally incubating with T7RNAP overnight. PCR reaction analysis is then carried out using some of the total reaction mixtures, whereby a specific single band amplicon is obtained by a suitably designed gene specific primer (GSP) or any other suitable RNA analysis method of interest.

The design and synthesis of gene specific primers depends on the particular target sequence to be amplified, and it will be appreciated by those skilled in the art that it can be designed by any means known in the art and will also be recognized in the art. For example, gene specific primers are designed using the National Center for Biotechnology Information (NCBI) BLAST® Basic Local Alignment Search Tool software and the GenBank human cDNA sequence database. Primers are optimized for annealing temperatures of about 55-65 ° C., showing DNA-free, RT-PCR dependent single bands from complex mRNA libraries known to be positive for specific mRNAs. The complex mRNA library is often extracted from normal and cancerous human tissues and in vitro cell lines. The designed primers all produce the desired target sequence specific PCR bands electrophoresed on agarose gel, as a result of which the molecular weight predicted by the design can be compared with known standards. Computation is done using the R _f value measured on gel analysis software. Amplicon sequences can be further sequenced by direct sequencing, blot probing, restriction enzyme mapping, and the like.

In order to avoid the limitations due to signal-to-noise (S / N) inherent in cDNA array assays as described above, novel variations of RNA polymerase promoter-induced primary amplification techniques have been developed. Alternatively, a single tube, multi-gene RT-PCR analysis system based on universal PCR amplification of multiple gene-specific reverse transcription of cDNA in a single reaction tube substantially reduces background noise. These two signal-to-noise improvement methods are specific elements embodied in the present invention.

The second standard synthesis of the pre-amplified library is within the selected region, contains 1-1000 target independent regions for a single sample, and also maintains 100% sensitivity compared to the original library. The second strand synthesis is terminated by selectively amplifying the genes of interest. Thus, gene specific primers (GSPs) are designed for second strand synthesis to include only the region of interest. Such regions include, but are not limited to, for example, prostate specific antigen (PSA), PSM, CK19, EpCam, AR, HPN, F6, mammoglobin and / or all cytokeratin. GSPs are designed to include universal primers at their tail ends.

In contrast to the prior art, when the first strand synthesis is performed with a gene specific primer set, some of the novel aspects of the present invention can be achieved without using CAPswitch.TM. Oligonucleotides (US Pat. No. 6,352,829). One is to use gene specific primers for two strand synthesis only. The prior art teaches that a gene specific primer is designed to contain a random anchor sequence comprising a CAPswitch oligonucleotide at its 5 'end. Surprisingly, according to the invention disclosed herein, the universal portion of the primer does not include the CAPswitch portion.

The length of a gene specific primer will be about 15-30 nucleotides, while the length of the universal primer portion will typically be about 15 nucleotides.

Reverse transcription of a small portion of the T7 amplified antisense RNA (aRNA) library is performed using cycle conditions known in the art. All RT-PCR results are first analyzed on a 2% agarose gel containing ethidium bromide, according to the methods known in the present invention.

After amplifying a selected portion of the amplified aRNA library, the product is analyzed in an array format known in the art, or in any electrophoretic format.

In addition to amplifying the second strand after preliminary amplification as described above, a universal PCR multiple gene amplification method is used, a set of gene specific primers (P1) for simultaneous reverse transcriptase, and an opposite primer for simultaneous second strand synthesis. Incorporation with a suitable set of (P2) can be performed in a single tube. In addition, the primers define two terminal ends (alpha and beta) to form a complete set of gene specific amplicons equivalent to the desired GSP multiple gene panel. GSP1 and GSP2 priming for gene specific first and second strand synthesis is performed under primer-target annealing high specific conditions using suitable enzymes known to those skilled in the art. More sophisticated methods of achieving appropriate primer specificity can be performed using proteins such as recA from natural recombinant cell repair mechanisms. Appropriate use of such in vitro repair systems yields excellent, even complete primer template formation specificities. Standard templates include mRNA or mRNA: cDNA heterozygotes, or double stranded duplexes cDNA. In addition, the innovative idea of utilizing the natural repair mechanisms of the cells, as described herein, is another GSPs-RT sub that alters signal-to-noise that enables other gene-specific primer methods such as cDNA array analysis for rare cellular phenomena below. It can be used in the method described below for the set. P1 and P2 primers present in any of the GSP multiple gene panel sets of PCR primers each have a universal primer sequence at the 5 'end (in an independent universal sequence common to all gene-specific P1 and P2 (or P1 and all P2)). Consensus sequence]. In order to control the competitive reactions with the undesirable side after the second strand synthesis, all GSP1 and 2 should be removed from the set of target double strand cDNA amplicon panels to eliminate their nonspecific effects on their downstream methods. Those skilled in the art will appreciate various techniques such as molecular size exclusion (provided by Sephadex and Centricon et al.), Chromatography, solid support selective attachment, single strand specific DNase (Mung Bean, S1, etc.) primer sequence specific techniques such as Instead of thymidine (T), a combination of DNA oligonucleotide primer synthesized with deoxyuridine (U) and uracil-N-glycosylase (UNG) can be used. Alternatively, RNA-DNA oligo-primer hybrids may be used in place of DNA-uracil, and similarly above, may be removed by DNase RNase treatment after the first and / or second strand synthesis. With the ease of PCR integration for UNG degradation, the availability of uracil containing DNA primers provides an efficient way to eliminate undesirable complex primer interactions. This UNG digestion technique will produce smaller oligomers than oligomers that can be annealed under selected PCR annealing temperatures. After UNG treatment, the cDNA template mixture can also benefit from the treatment of DNase RNase-free, eliminating all undesirable side reactions that can be caused by highly complex RNA. After performing the UNG treatment (a method of removing all RNA by random RNase treatment), the only remaining nucleic acid hybridizes to the first and second complementary strands to form a dsDNA duplex that constitutes a PCR template obtainable from the sample. do. Then, a non-UMP containing universal primer (1 or 2) is added to perform subsequent PCR. The overall effect is to capture any desired set of any desired mRNA (or DNA-RT) sequences with one or two PCR compatible high efficiency primers, wherein the primers quantify RT-PCR multiple genes in a single tube and It can be amplified and analyzed simultaneously. Since the primers are universal, they can prime each GSP amplicon with exactly the same efficiency, thus avoiding complexity due to problems with multiplex GSP primer performance. Each GSP defines an amplicon with a panel, or sets of amplicons, each GSP sequence is based on size such as vertical and horizontal PAGE and agarose gel electrophoresis, capillary gel electrophoresis, SELDI, MALDI, cDNA array, etc. Certain different fragment sizes can be retained so that they can be analyzed and identified by unique R _f values in one analysis system. Therefore, a rapid multi-gene RNA / DNA panel can quickly examine multiple samples for various diagnostic treatment sets and monitoring. By this method multiple genes derived from each sample of mRNA can be analyzed in a single reaction tube with or without mRNA library preamplification. Preliminary amplification cannot make it possible to analyze only one panel of genes in a single assay. As preliminary amplification provides the advantage of analyzing a single sample in up to 1000 different assays, multiple panels of genes can be examined at different times in one sample. While not intending to be limited to any particular method, universal PCR panel analysis via cDNA array or capillary gel electrophoresis (CGE) is the preferred method.

Therefore, an important feature in distinguishing the present invention from the conventional techniques of the prior art is that it improves the signal to noise by selectively amplifying rare target mRNA species, thereby further improving the present invention over existing multivariate mRNA assays. Known multivariate analysis systems, such as complex RT-PCR, can substantially transform the signal into noise, but attempts to design and optimize useful complex systems may be directed to such systems, particularly for two or more target subsets in a reaction vessel. Generally made impractical.

The present invention also utilizes highly improving signal to noise to select individual transcripts, as well as amplifying the entire set of target sequence (s) to be detected in one reaction vial.

Thus, the present invention can be used to generate target gene subset (s) found in known conditions using each gene specific primer set (s), without being limited to the following specific uses. An exemplary set will include two or more different target genes that are indicative of the desired condition. For each reaction vial, the number of gene specific primer sets will be determined by known conditions that will define the condition and conditions.

The following examples illustrate the usefulness of the disclosed invention and demonstrate the effect on diagnostic techniques. The following examples are not intended to limit the scope of the invention. In addition, the disclosures of each patent, patent application, and publication cited or described herein are incorporated herein by reference in their entirety. Through the following examples, molecular cloning reactions and other standard recombinant molecular biology techniques are described in Maniatis et al., Molecular Cloning-A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989) (hereinafter "Maniatis et al. And Current Protocols in Molecular Biology, Wiley, 2002, commercially available reagents were used unless otherwise noted.

Example 1

Isolation of Cytoplasmic RNA

The supernatant obtained from magnetic fluid-selected unfixed cells infiltrated with Immunep, a phosphate buffer containing 0.05% saponin and 0.1% sodium azide, was found to contain at least 80% of the total cellular RNA remaining in the cytoplasm of the cell. Turned out. RNA isolated from this supernatant showed no evidence of degradation as determined by natural and modified agarose gel electrophoresis and ethidium bromide staining. These supernatants are usually discarded after intracellular staining of ferrofluid selected cells, where the supernatants contain mRNAs of the same cells that are used for morphological analysis, including RNA in the integrity or intact full-length form. The result was 2 shows that total RNA release occurs in less than 1 minute, and about 95% of cytoplasmic total RNA can be easily and repeatedly isolated.

In contrast, cells of the breast cancer cell line SKBR3 using conventional methods, ie, isolation methods by commercially available phenolic RNA lysis buffer Trizol® reagent (Gibco BRL, Gettersburg, MD, Cat # 10296). RNA isolated from complete lysis and homogenization of all cellular constructs, thereby releasing genomic and mitochondrial DNA and cytoplasm, mitochondrial and nuclear RNA. Examination of cell pellets derived from immunopump (saponin) treated SKBR3 cells using EpCAM magnetic fluid (see Example 2 for detailed procedures) revealed that nearly 100% of genomic and mitochondrial DNA was present. Nuclear and mitochondrial RNA, which accounts for about 15-20% of all cellular total RNA, is expected to be obtained from the same number of whole cells that have not been infiltrated. About 95% of the predicted cytoplasmic RNA is recovered to integrity from the immunoperm supernatant layer.

As shown in FIG. 3, two tubes containing about 250,000 cells of breast cancer cell line SKBR3 were first immunomagnetically concentrated, followed by the presence (+ IP) and absence (+ IP) of immunofem at room temperature for 15 minutes. Incubated under). Immune perm treatment Infiltrated cells were centrifuged for 5 min at 800 × g RCF as infiltrated visible cell pellet. The immunoperm supernatant fractions containing all cytosolic soluble components were transferred to a second tube. Total RNA isolated from untreated whole cells, immunotreated permeated cell pellets and immunotreated treated cell supernatant fractions were subjected to RNeasy® (Kiagen Incorporated, Valencia, CA) silica binding. Isolate using the method. This total RNA fraction was treated with DNase to remove trace DNA. Spectroscopic quantification of the DNase treated RNA fraction showed that the average yield of total RNA / cells for all cells was 20 pg (= 100%) and from the pellet fraction 4 pg (20%) of total RNA / infiltrating cells, And 16 pg (80%) of total RNA / cell was obtained from the supernatant fraction. As shown in FIG. 3, 2.5 × 10 ⁴ cell equivalents from DNase treated total RNA fractions were subjected to formamide denaturation and stained with ethidium bromide after electrophoresis through 1% agarose gel. Quantification by agarose gel density meter was consistent with the spectroscopic recovery value. In addition, the gel image of FIG. 3 shows full length and high integrity, as evidenced by the relative ratio of 4.4 kb ssRNA co-migrating with the 28S rRNA band to the 2 kb ssRNA marker co-migrating with the 18S rRNA band. Could. In general, the fact that the measure of the relative ratio of 28S rRNA to 18S rRNA is about 2, is further demonstrated by Northern blotting of this gel and probing it with oligo (dT) as shown in FIG. 4. Excellent indicator of unity. The literature figures for the percentage of total RNA resulting from the nucleus are 15-25%. Therefore, the immunoperm® treated cell fraction containing 20% RNA is consistent with the published nuclear contribution.

In conclusion, immunopeptide-based infiltration unexpectedly demonstrates complete isolation of nuclear and cytoplasmic total RNA that retains nearly 100% of totally recoverable cytoplasmic total RNA in the supernatants of immunotreated permeated cells. It became. In addition, the nuclear fraction of total RNA has been surprisingly found to remain integrity in the infiltrating cell structure generated after immunoprep treatment.

Using poly-A tail hybridization, the denatured RNA from the gel shown in FIG. 3 to the positively charged nylon filter was transferred to a Northern blot to isolate the mRNA portion of the RNA derived from the two Immunep® cell fractions. The whole cell was analyzed. Oligo (dT) 25-mer probes were labeled with ³² P and polynucleotide kinase and then hybridized to Northern blot. A size ladder of the size of the short-chain RNA containing the poly-A tail was used as a marker capable of forming a molecular weight band ladder for relative quantitative size fractionation of mRNA populations. The oligo (dT) hybridization results of FIG. 4 showed that there was no significant difference in the size range for the mRNA library between these three samples.

Comparative quantitative phosphor image analysis of whole mRNA-blotting regions (i.e., full dT hybrid signals) from whole cell mRNA libraries was performed from immunoperme treated infiltrating cell pellets (nuclear fractions) and immunoperm induced cytoplasmic fractions. It was almost the same as the sum. In addition, the cell fraction ratio of mRNA obtained from the dT-probe signal measured by phosphor imaging was equal to the 28S / 18S rRNA ratio measured from agarose gel image density meter analysis. These data demonstrate that all of the immunoferment-derived cytoplasmic total RNA and its mRNA components are quantitatively separated, which is large in integrity and full length. Since nearly 100% of the cytoplasmic mRNA is recoverable from the immunopereum supernatant, and the integrity of rRNA 28S / 18S indicates complete mRNA integrity, release of the immunoperim-derived mRNA is transcript size It is not limited by.

The northern blot shown in FIG. 4 was stripped and reprobed with nuclear specific precursor rRNA probes. According to Figure 5, it can be seen that if the cells are treated with the immunonuclear and cytoplasmic total RNA population can be completely separated. Thus, the nuclear membrane structure remains intact during the immunopreparation process, and the nucleus retains all of its soluble components.

The northern blot of FIG. 5 was stripped and reprobed with mitochondrial-specific 12s rRNA probes. The results shown in FIG. 6 demonstrate that immunization of cells can completely separate mitochondrial and cytoplasmic RNA populations. Thus, the mitochondrial membrane structure is maintained intact during immunological processing.

The same total RNA stock solution used to obtain the images of FIGS. 3-6 was used to obtain a ³² P-labeled first strand cDNA library with the same mass and specific activity. Hybridization of these three labeled first strand library probes yields an identical but generated cDNA array dot blot as shown in FIG. 7. The present application aims to assess the relative abundances present in each of the immunoperim derived cell fractions by comparing the relative proportions of each cDNA hybrid signal pattern for each image forming filter. Randomly selected cDNA gene array identity on the template is shown in FIG. 7. The uppermost dot is the house represented by the abbreviation: 23 kd = 23 kD protein, a-tub = α-tubulin, b-act = βactin, b2mic = β-2-microglobulin, phos = phospholipase A2 It is a set of keeping genes. The second column is labeled f6 = α-1-globin, CD16 = cluster determinants 16, CD12, CD38, CD45 and CD31. The column below shows g6 = macrophage inhibitory cytokine 1, CK8 = cytokeratin 8, CK18 = cytokeratin 18, CK19 = cytokeratin 19, EpCam = epithelial cell adhesion molecule, uPA = general epithelial specificity expressed as urokinase plasminogen activator It consists of enemy genes. Qualitative and quantitative phosphorescent imaging comparisons of each fraction showed no significant difference in the relative abundance of genes on the array. Interestingly, these data show that the relative abundance of mRNA from whole cells is approximately equal to its cytoplasmic fraction and also to the nuclear fraction, where the total RNA mass is from about 20% (nucleus) to about 80% ( Cytoplasmic). The length of each of the transcripts in this cDNA array is 1-5 kb, which further improves the Northern blot results of unbiased size in mRNA release from immuno-treated cells. In addition, the same relative presentation pattern of FIG. 7 demonstrates that the mRNA derived mRNA is as effective as the reverse transcriptase template for first strand synthesis as whole cell mRNA derived by conventional methods.

Overall data show that immunofibre-derived cytoplasmic RNA, which accounts for> 95% of all cytoplasmic total RNA, is a full-length form that accounts for about 80% of all cellular total RNA mass in total homogenized cells, It was surprising that silica extraction method had the same reverse transcription efficiency as total RNA isolated. Therefore, the immunoperm-derived cytoplasmic total RNA and its accompanying heterologous nuclear component are equally effective templates in all conventional downstream RNA assays when compared to conventional whole cell high difficulty RNA isolation methods.

FIG. 8 shows a gel image of cell substrate RNA obtained by treatment of about 770 PC-3 cells with an immunoperm. According to these data, the immunophim-derived cell substrate mRNA obtained from a small number of cells yielded the same proportion of RNA as shown in FIGS. 3, 4, 5, 6 and 7. Therefore, immunonime-mediated cell release of cell substrate RNA is independent of cell number.

Example 2

Isolation of Circulating Tumor Cells Derived from Peripheral Blood

After isolation of circulating tumor cells from peripheral blood, cell analysis by flow cytometry and gene expression analysis by RT-PCR can be performed as follows: EDTA-anticoagulated blood (7.5 mL) 15 Transfer to a ml conical tube and add 6.5 ml of system buffer (0.05% sodium azide, PBS with Cat # 7001, Immunicon Corporation, Huntingdon Valley, Pa.). The tube was capped and mixed upside down several times. The blood-buffer mixture was centrifuged at 800 x g for 10 minutes at room temperature. The supernatant was aspirated carefully to avoid mixing the pale yellow brown coating layer. Some supernatant may remain in the tube. Aspirated supernatant was followed. AB buffer (system buffer containing streptavidin as the reversible aggregation reagent, Immunicon Corporation, Huntingdon Valley, Pa.) Was added to the tube to a final volume of 10 ml. The tube was capped and mixed upside down several times. Inverted vial several times, inverted VU / desthiobiotin EpCAM magnetic fluid particles (immunomagnetic nanoparticles bound to anti-EpCAM monoclonal antibodies conjugated to desthiobiotin for biotin-reversible aggregation with streptavidin, Pennsylvania, USA Immunicon Corporation, Huntingdon Valley). 100 μl of VU / desthiobiotin EpCAM magnetic fluid was added to the samples in AB buffer and the tubes were mixed upside down several times. Shake the tube to prevent foaming. The tube was immediately inserted into a QMS17 (Cat. # AS017, Immunicon Corporation, Huntingdon Valley, Pennsylvania) magnetic separator and allowed to stand for 10 minutes. The tube was removed from the separator and the tube was inverted upside down several times to resuspend its contents. The tube was inserted back into the QMS17 magnetic separator and allowed to stand for 10 minutes. The tube was removed from the separator and the tube was inverted upside down several times to resuspend its contents. The stopper was removed and the tube was placed in the QMS17 separator for another 20 minutes. The cell-buffer mixture was carefully aspirated using a Pasteur pipette in a tube inserted into QMS17, and then the aspirated supernatant was discarded. Immediately the tube was removed from the separator and 3 ml of system buffer was added thereto. Self-collected cells were resuspended by weak vortex. During vortexing, the liquid must rise to the tube level in order to allow the upper cells to be washed down. The unsealed tube was put back into the QMS17 separator for 10 minutes and the supernatant was aspirated with a Pasteur pipette. Aspirated supernatant was discarded. Self-collected cells were placed in 200 μl of immunoperm / RNase inhibitor (infiltration reagent, Huntingdon Valley, Pennsylvania, USA) containing RNase inhibitor (RNase OUT, Cat. # 10777019, Invitrogen, Rockville, MD). Resuspended by vortexing at Immunicon Corporation). The liquid must rise to the tube level during the vortexing process so that all cells are washed off. 25 μl volume of antibodies, such as monoclonal anti-cytokeratin antibodies (C11-PE, 0.25 μg) (recognizing cytokeratins 4,5, 6, 8, 10, 13, 18 conjugated to R-phycoerythrin) Cocktail of antibodies; Immunicon Corporation, Huntingdon Valley, Pennsylvania) and 10 μl of CD45 PerCP (Pan anti-white blood cell marker, Cat. # 347464, Beckton Dickinson, San Jose, CA) or any other Appropriate antibody was added and vortexed and mixed. After 15 minutes of incubation, the sample was gently shaken by tapping the bottom of the tube. The tube was put back into QMS17 for 5 minutes. The supernatant was aspirated gently and the immunoperm-RNA fractions were transferred to appropriately labeled tubes.

Example 3

Cellular Analysis of Circulating Tumor Cells Derived from Peripheral Blood

The cells obtained from Example 2 were resuspended in 200 μl of CellFix (PBS based buffer containing biotin as a deagglomerating reagent and cell preservation component, Immunicon Corporation, Huntingdon Valley, Pa.) And incubated for 5 minutes. Treated. The sample was transferred to a 12 × 75 mm flow tube, combined with 300 μl of PB, followed by the nucleic acid dye thioflavin T (Sigma # T3516, 10 μl), and about 10 μl of fluorescent beads (10,000 beads; Flow-Set Fluorospheres). , Cat. # 6607007, Coulter, Miami, Florida, USA. Samples were mixed by vortexing. The fluorescent bead tubes are preferably swirled and mixed before pipetting the beads. Samples were analyzed with a flow cytometer.

Example 4

Gene Expression Analysis of Circulating Tumor Cells Derived from Peripheral Blood

Poly (A) + mRNAs are self- oligo (dT) labeled beads [Dynabeads® mRNA Direct® Micro Kit, Dynal, Prod. # 610.21, New Hyde Park, NY, USA. Alternatively, total RNA can be isolated using any other means suitable to those skilled in the art, such as silica bonds, polymer bonds, and more conventional phenol extractions such as Trizol® (GibcoBRL, Cat # 10296). Genomic DNA was removed by treatment with a DNase enzyme such as DNase I (GibcoBRL). With 2: 1 10 × DNase I (1 U /: 1), 1: 1 RNase inhibitor (cloned), 5: 1 dH ₂ 0 and 10: 1 RNA or control (250 ng, genomic DNA) An enzyme mixture was produced. The enzyme mixture was incubated at 37 ° C. for 20 minutes. DNase treated RNA was rearranged by magnetic oligo (dT) labeled beads or Trizol® separation and resuspended in 10: 1 RNase-free water. The activity of DNase enzyme was confirmed by developing control genomic DNA (+/- DNase treatment) on 2% agarose gel using ethidium bromide staining.

Specific mRNA sequences rTth; it can be amplified using a (servo filler servo Phyllis Thermos thermophilis) RT-PCR. 10: 1 5 × EZ buffer, 1.5: 1 dATP, 1.5: 1 dCTP, 1.5: 1 dGTP, 1.25: 1 dUTP, 5: 1 Mn ++ (25 mM), 2: 1 rTth (2.5 The master mix consisting of U /: 1), UNG (1U /: 1) of 0.5: 1, dH ₂ 0 of 12.25: 1, sense primer of 2.25: 1 and antisense primer of 2.25: 1 It can be prepared for reverse transcription of enemy mRNA species. A 40: 1 volume of main mixture was added to the corresponding negative control tube containing 10: 1 DNase treated RNA and 10: 1 H ₂ O. PCR thermal cycling was performed at 50 ° C. for 2 minutes (pre-PCR), 62 ° C./65° C. for 30 minutes (pre-PCR), 95 ° C. 1 minute (pre-PCR), 94 ° C. for 15 seconds (PCR), 62 ° C./65° C. 40 cycles were performed, such as 30 seconds (PCR) and 62 ° C./65° C. for 7 minutes (post-PCR). Immediately after completion of thermal cycling, the sample tube was placed in a block at -20 ° C for 2 minutes. After this was completed, the tube was placed in a block at 4 ° C. until gel analysis was performed. A volume of 20: 1 was developed on a 2% agarose gel with ethidium bromide staining. Qualitative and quantitative gene expression measurements of specific mRNA transcripts were performed by observing gel images for the presence of amplicons with expected molecular weights using alpha imagers and UV permeators.

In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Also, such modifications are intended to be included within the scope of the appended claims.

Example 5

Isolation and Analysis of Proteins from Circulating Tumor Cells in Peripheral Blood

The supernatant obtained from about 1 million magnetic fluid selected SKBR3 cells infiltrated with Immunepim, a phosphate buffer containing 0.05% saponin and 0.1% sodium azide, was used in the cytoplasm of the cells, in addition to the nucleic acid component analyzed in Examples 1-4. Contains soluble cytoplasmic protein released. Soluble proteins in these supernatants, and insoluble proteins remaining on or in the cell's surface, provide a means for measuring cell morphology as well as the total protein expression profile or proteomic profile of the cell.

First, the total cytoplasmic soluble protein fraction released from the cytoplasm due to the immunoperim treatment was released from two cellular preparations treated with NP-40 surfactant, which is the preferred formulation for the total cytoplasmic protein derived from the cell. The total amount of was measured. Both treated cell preparations were separated from the membrane debris by centrifugation or magnetic separation before observing total soluble protein by conventional methods such as spectroscopy Lowry and Bradford methods.

Secondly, aliquots of two sample preparations were subjected to electrophoresis on a 4-20% gradient SDS polyacrylamide gel (a) to determine molecular weight determinants for immuno-derived cell substrate proteins, (b) The band formation pattern and relative content of protein per band in dog formulations were compared.

Third, the aliquots are further analyzed by 2D electrophoresis and are typically stained or labeled so as to be detectable so that the protein in the two fractions is based on the qualitative and quantitative dot formation patterns of the identifiable and unidentified components. "Fingerprint" information was provided for size and isoelectric point. The derived information yields proteomic expression profiles of relative and absolute protein expression patterns in cell substrates and total protein fractions of normal and modified cell populations.

Example 6

Gene-specific primed (GSP) RNA polymerase systems of a subset of mRNA libraries that allow for diagnostic formats using unique signal-to-noise (S / N) limits, such as rare cell phenomena and cDNA array analysis of rare transcripts Amplification

After RNA isolation, several RNA assays were applied. Although conventional RT-PCR or more preferred quantitative modifications have been applied, these samples are generally considered undesirable for use in each sample because they contain very small amounts of starting material. As a result, clinical sensitivity was complemented for multigene analysis. Thus, an unamplified mRNA / cDNA library can only be analyzed once for one gene without compensating for clinical (and maximal technical) sensitivity. Since each sample is small, several high throughput methods have been developed.

Applicants have found that it is highly desirable to be able to measure the expression levels of multiple genes (2 to 1000) simultaneously through high throughput formats, as in all microarrays obtained from only one reaction tube. This is achieved without compromising the sensitivity without significantly reducing the workload. A major obstacle to rare cell phenomena and single tube cDNA microarray analysis of their rare mRNA samples is the undesirable inherent S / N ratio in the starting mRNA sample.

For radiolabeled cDNA arrays, this limit is labeled non-specific (background noise), which can be hybridized to a nylon filter array system at a time without increasing the background filter (solid support) noise component of the S / N ratio. Detectable in solution phase (about 5 × 10 ⁵ ) when one specific known target is spiked with the maximum amount (b) of target (20 ng = 2 × 10 ¹¹ , a library of randomly labeled target molecules) It is obtained from the lower limit (a) of the target copy number.

For immunomagnetically enriched samples, significant background noise mRNA is due to the production of WBCs which inevitably occurred during the enrichment process. One solution is to perform a second round of RNA polymerase amplification (RNAPA), which selects only a subset of a given gene, thereby changing the S / N ratio to 1000 times or less to obtain the desired rare mRNA from rare cells. .

Performed in this Example using a model system that reflects the typical WBC mRNA copy number ratio found in clinical samples (10 ng total RNA in about 5000 WBCs) / CTC (0.5 ng total RNA in about 50 CTCs) This GSP subset RNAPA selection method is reduced. In an equivalent aliquot of the starting sample stock consisting of 50 CTC, real-time quantitative RT-PCR counts are shown in Table 1 below, prostate specific antigen (PSA = 2650), prostate specific membrane antigen (PSM = 1750), All mRNAs detectable at the androgen receptor (AR = 100) and epithelial cell adhesion molecule (EpCAM = 1163) were measured. The starting WBC mRNA total copy number relative to non-specific background noise is about 10 ^{8-10 9} . In this embodiment, the starting total RNA / mRNA was treated with a first round of amplification that increased in proportion to all mRNA species nearly equivalent to those measured by real time quantitative RT-PCR (Table 1). 25 ng aliquots of aRNA amplified in the first round were then treated with a second round of GSP subset RNAPA and altered the signal-to-noise of the four GSP targets, as described below. One).

In the second round GSP RNAPA, an important screening step takes place during a single RT reaction which simultaneously forms the first strand only for a given mRNA library subset that contains gene specific RT primers. In this example the subset of GSP RT primers is for the four mRNAs (PSA, PSM, AR, EpCAM). After GSP-RT selective first strand synthesis, the synthesis of complementary second strand was performed using oligo (dT) primers carrying the T7RNAP promoter and appropriate DNA polymerase to prepare an optional set for double stranded DNA template T7RNAP.

Thus, RNAPA possible target template subsets were selected through GSP first and second strand synthesis. At this time, all remaining RNA is degraded by exposing the second strand reaction mixture to a cocktail of RNase without DNase. Alternatively, any remaining single-stranded RNA and any exogenous (non-poly U / poly A-dependent) single-stranded cDNAs formed during dT dependent second strand synthesis are single-chain specific nucleases such as mung bean nucleases. Can be removed by agent. Substrates cDNA template subsets are then purified by phenol extraction and / or silica bonding. The set of templates for the selected RNAPA was amplified overnight for RNAP, resulting in only about four orders of magnitude increase in S / N fluctuations for other possible templates such as F6 (α1 globin sequence) exhibiting WBC mRNA induced noise. It was. Table 1 shows the results of real-time quantitative RT-PCR for four target genes via methods including subsequent GSP-second round S / N fluctuations, where F6 is present at a very high rate in WBC but epithelial cells Is defined as the α1 globin sequence found in this system. These results clearly show that the four selected GSP targets increased by an average of 844 times, whereas the non-targeted F6 WBC noise increased by only 5.9 times. Thus, dividing the GSP target signal increment by F6 WBC noise improves the final signal versus noise for each GSP target. It is important to note that further improvements can be made by using modifications such as the mung bean nuclease and GSP-RT primer sequence selection / optimization described above.

gene rRNA Start Copy Total number of aRNA copies after the first round Total copy after round 2 of GSP First round: Second round GSPS / N (F6) PSA 2650 2.10 × 10 ⁷ 3.00 × 10 ¹⁰ 1238 510 PSM 1750 7.80 × 10 ⁶ 4.03 × 10 ⁹ 517 87 AR 100 64200 2.90 × 10 ⁷ 458 78 EpCAM 375 5.20 × 10 ⁶ 6.05 × 10 ⁹ 1163 197 F6 NA 1.40 × 10 ¹⁰ 8.30 × 10 ¹⁰ 5.9 One

Example 7

Proteinase and nucleophile recovery of cellular RNA from immobilized samples yields high quality RNA templates for downstream RT dependent analysis.

Surprising to samples exposed to aldehyde and urea stabilizers or fixatives, cytotoxics and other formaldehyde and formaldehyde-urea derivative fixers are found in whole blood compared to the corresponding unfixed control. Stabilizing nearly 100% of full-length total RNA, mRNA and other nucleic acids in all cells. Stabilized integrity RNAs as macromolecular complexes change the RNA's chemical properties, and RNA isolation methods based on current traditional cell lysis and chaotropic salts, such as phenol extraction, silica binding and oligo (dT) hybridization Not affected by

These macromolecular complexes (both covalent and non-covalent) are dissociated and reverse reactions occur through a combination of enzymatic degradation and / or chemical nucleophile incubation. The result is that the nucleic acid can be freed to separate the original DNA and RNA libraries with almost 100% integrity. This fixative derived RNA library provides a high quality template for reverse transcriptase (RT) dependent formation of the first strand cDNA.

RNA recovered by this fixative is combined with the aRNA preamplification or universal PCR methods described herein for comprehensive analysis downstream or general functional competence of total RNA libraries and mRNA libraries.

9 shows that Cytox® performs a similar function to other aldehyde-based fixatives. After 24 hours exposure to fixatives, less than 1% of mRNA and an unbalanced amount of 18S-rRNA can be recovered (about 10%). Extreme chaotropic salt denaturation chemistry (ie, GITC and phenol, silica or (dT) hybridization, BRL Trizol reagent, kiagen RNA mini silica bonds, and dynal Dynabeads mRNA direct oligo (dT) poly (A) ⁺ kits) Even when this is applied, the recovery rate is extremely low.

Surprisingly, treatment with nucleophiles such as proteinases such as proteinase K, and tris bases removes most of the proteins and polypeptides covalently bound to other macromolecules, including nucleic acids, and are completely intact. Enough nucleic acid chemistry to restore over 90% of total RNA and mRNA. 25 ng of aRNA aliquots (FIG. 12A) derived from the mass normalized aliquots of total RNA shown in FIG. 9 were applied to quantify RT-PCR comparative assays for both CK 19 and EpCAM. This comparison showed that the number of copies from fixative derived RNA was 3.8 and 3.9 times lower than unfixed RNA. This is understood as the current proteinase K treatment to recover at least 25% of the maximum RT-template activity for RT-PCR analysis.

Recovery of up to 25% of maximal RT-template activity through this fixed-recovery system allowed Percoll to specific mRNA (alpha globin) via quantitative RT-PCR for different operators performing the same procedure and for non-fixed corresponding samples. Analysis of derived white blood cells is reproducible. In addition, the Transfix® formulations used herein are known to bring the final concentration of paraformaldehyde fixative to 0.1% per unit volume of blood.

The loss and recovery behavior of RNA exposed to cytoscene® is the same as that of Transfix® and other formaldehydes below, but the formaldehyde urea observed in the preparations of cytoscece® and Stabilsite® The formaldehyde donor component of the derivative components is likely to be involved in the covalent binding of the nucleic acid to the protein.

Formaldehyde-urea derivatives in the presence of many macromolecular nucleophiles (ie proteins and nucleic acids) observed in biological systems increase the dissociation rate of these derivatives. Dissociation occurs very close to the biological nucleophile complex, possibly the regulatory protein specifically associated with RNA resulting in covalent bonds. Subsequent treatment of proteinases and more potent nucleophiles then removes and reverses such binding and association. The fact that the known crosslinker, Transfix®, yields a full-length mRNA library with high integrity derived from 24-hour stabilized whole blood cells, demonstrates that all aldehyde-based stabilizers yield similar high quality nucleic acids. This allows for reproducible calculation of nucleic acids after preservation and recovery.

Analysis of 90-100% of total RNA and their corresponding mRNA libraries is possible using the above and most other aldehyde and / urea derivative fixatives as further shown in Figures 10A, 10B and 10C below. . In addition, most of these types of immobilized nucleic acids can be recovered by a combination of a proteinase and a nucleophile cocktail heated at a high temperature such as 60 ° C. as shown in FIG. 11.

These results show that heating the immobilized RNA in buffer only for 1 hour at high temperature yields a portion of the mRNA library. The effect of this hot recovery has been previously disclosed for formalin fixation and paraffin embedded tissue RNA recovery, but these results have not been reported anywhere for whole blood. In addition, the quality and amount of mRNA libraries recovered herein have not been obtained in such reports using formalin fixed, paraffin embedded tissue RNA recovery.

Comparing this mRNA library with mRNA libraries recovered using other nucleophiles (Tris, acetic acid hydrazide and hydroxylamine), the ratio of mRNA transcript size distribution for each nucleophile shows that the sample shows no RNA degradation. Despite being different. This suggests that different types of mRNA sequences can be recovered by specific nucleophiles and incubation conditions (ie, different types of formaldehyde modifications are reversed). The various enzymes used also show that different ratios were recovered (FIG. 11, bottom of the gel).

The combination of the fact that proteinase K degradation alone recovers 25% of the maximum RT-template activity and the fact that different fixative reversals yield different proportions of the mRNA library are clearly largely achieved using a combination of nucleophiles and enzymes. It strongly suggests that improved recovery can be achieved.

In particular, to demonstrate the potential of mRNA diagnostic use for cancer using a relatively non-invasive peripheral blood model, FIG. 12A shows a single T7RNAP of total RNA / mRNA isolated from triple samples of 10 and 20 SKBR3 cells. The final relative quality and amount of aRNA from the preamplification shows that it is spiked into 7.5 ml peripheral blood after stabilizing with Cytox® for 24 hours at room temperature. Each of these multiple samples was then immunomagnetically concentrated from the cell lysate treated with proteinase inversion conditions followed by silica bound total RNA isolation. Normalized equivalent amounts of aRNA were used for the quantitative RT-PCR response for two specific genes CK19 and EpCAM. The results are shown in FIGS. 12B and 12C. As can be seen in FIGS. 12B and 12C, all spikes showed strong positives as compared to donor corresponding triple samples (non-spike controls) as measured by CK19. Similarly, EpCAM showed the same out-of-regular (endogenous) observed in this donor triple sample non-spike control and showed the same 100% positive value for all spikes despite extremely low expression rates. In fact, this low level of EpCAM mRNA detection by RT-PCR is generally observed for this sequence and not due to false positive PCR contamination.

12D and 12E show that the quality of mRNA RT-templates derived from three different aldehyde-based fixatives, namely Cytox®, Stabilsite® and Transfix®, are varied. As shown, Transfix® yields an mRNA template that may have slightly lower RT quality than Cytox® or Stabilsite®. In addition, these data were normalized to the number of spiked cells and compared to unmodified cells at time = 0 in exactly the same flask of SKBR3 cells, the difference between fixed CK19 and EpCAM mRNA and unfixed CK19 and EpCAM mRNA Only 4 times (RNA data derived from FIG. 9 is not shown). This is interpreted to mean that combining the 24 hour stabilization process with proteinase K recovery alone and aRNA amplification yields 25% of possible template quality for RT. In addition, the reason why the RT quality is lower than 100% may be due to an aldehyde modification that cannot be removed by proteinase K alone. Thus the combination of proteinase and nucleophile cocktail will significantly improve the RT-quality of these templates by 25% or more, as demonstrated in this experiment. For comparison with related literature under similar conditions, experiments evaluating the same parameters of RT RNA template quality from formalin fixed paraformaldehyde embedded tissues resulted in a 13-60 fold reduction in RT-template quality compared to unfixed tissue. (Godfrey et al., Quantitative mRNA Expression Analysis form Formalin-Fixed, Paraffin-Embedded Tissues Using 5 'Nuclease Quantitative Reverse Transcription-Polymerase Chain Reaction, J. of Molecular Diagnostics, 2: 84-91 (2000)). The reduction provides a significant improvement over the prior art.

12A, 12B, 12C, 12D and 12E confirm reproducible and product viable procedures for blood RNA sample preservation and a comprehensive analysis of epithelial cells likely to be circulating epithelial cells, cancer cells. High levels of mRNA retention are quantitative assays capable of detecting the presence of a single cell spiked with 7.5 ml of blood for any mRNA present in the cell with at least 50 mRNA molecules / cells (ie, only 50 copies / sample). Can be applied to

In summary, the rate and type of covalent fixation in whole blood depends on the type of fixative. Likewise, the rate and type of covalent fixation reversal and recovery depends on the type and combination of proteinases and nucleophiles used. Fixed rate is an important issue for applications where the half-life of the mRNA of interest is faster than the fixed rate. Both forward fixation and reverse recovery reactions can be optimized to further increase the quality and amount of RNA. However, the current quality and amount of stabilized and recovered RNA proves to be much better than anything previously disclosed in the blood.

Example 8

Concentration and analysis of mRNA from CTC in fresh unfixed blood

Human blood was isolated from nine patients with advanced hormone-resistant prostate cancer (HRPC) and 13 healthy volunteers and evaluated for gene expression mRNA specific for circulating epithelial cells.

patient

Blood from nine patients with advanced hormone-resistant prostate cancer (HRPC) and 13 healthy volunteers (7 males (ages 24-73, average 45 years) and 6 women (ages 27-61 years, 39 years old) Samples were taken and placed in 10 ml EDTA Vacutainer ^™ tubes (Becton-Dickinson, NJ). Of the nine HRPC patients, two had five longitudinal blood samples, one with four samples, three with two samples, and three at one sample time point. Patients ranged in age from 60 to 81 years (mean 74 years) and their initial diagnosis was made 2 to 10 years before the start of the study. Serial samples from three patients receiving treatment with Taxol / Estramusstin and / or Loopron were prepared and analyzed as long-term serials. Patients and healthy volunteers were informed and signed an informed consent form.

Target cell isolation

Unless otherwise noted, blood samples were collected at room temperature after treatment and treated within 2 to 3 hours. 15 ml of blood was divided into 7.5 ml aliquots and transferred to a disposable tube (Fisher Scientific) with an internal diameter of 17 mm and centrifuged at 800 g for 10 minutes without stopping. Phosphate buffered saline (PBS) containing bovine serum albumin (BSA) was added to make the volume up to 10 ml and the samples were inverted and mixed. Mab VU-1D9, which recognizes epithelial cell adhesion molecule (EpCAM), is broadly reactive with tissue derived from epithelial cells and is bound to magnetic nanoparticles (magnetic fluid, Immunicon, Huntington Valley, Pennsylvania).

EpCAM labeling as described in applications 09 / 351,515 and 09 / 702,188, incorporated herein by reference, in order to increase the self loading of EpCAM positive cells and to reduce the variation in capture efficiency due to differences in EpCAM density on the cell surface. Desthiobiotin was bound to the magnetic nanoparticles formed to form CA-EpCAM. CA-EpCAM ferrofluid and streptavidin containing buffer were then added to the sample to achieve this increase in self labeling of cells. Desthiobiotin on the CA-EpCAM magnetic fluid is then substituted with biotin contained in the permeation buffer described below. This reverses the crosslinking between the CA-EpCAM magnetic fluid particles. The sample is immediately placed in a quadrupole magnetic separator for 10 minutes (QMS17, Immunicon). After 10 minutes, remove the tube from the separator, turn it upside down five times, shake it, and put it back into the magnetic separator for 10 more minutes. This step was repeated one more time and the tube was placed back in the separator for 20 minutes. Supernatant was aspirated and discarded after separation. The tube was removed from the magnetic separator and resuspended in 3 ml of bovine serum albumin (BSA) containing phosphate buffered saline (PBS) and the fractions collected from the walls of the vessel.

Two 7.5 mL aliquots from each sample were treated separately. One aliquot was prepared and analyzed by flow cytometry (Example 12), and RNA from the other aliquot was analyzed as described above.

Nucleotide Purification and Amplification

One preferred method of using the present invention in a preferred embodiment is to first purify the nucleotide sample. Total RNA or mRNA is then isolated from the enriched cell population. Isolation can be performed by any method known in the art that can maintain mRNA intact and prevent degradation. For example, concentrated circulating tumor cells from duplicate blood samples are dissolved in 100 μl Trizol Reagent (BRL) or 100 μl RNA Extraction Buffer (ZYMO Research), and vortex homogenized samples are used when RNA is used. Store at -80 ° C until. Homogenates were used to separate total RNA according to the manufacturer's instructions. In summary, total RNA was treated with DNase I. DNase activity was verified by producing detectable genomic DNA on ethidium bromide gel after DNase treatment. DNase treated DNA was purified by repeated trizol separation procedure. One-tenth of the total RNA obtained was electrophoresed on a 1% agarose gel with total RNA mass and size standards, followed by northern blots and hybridized using a mixture of equimolar amounts of ribosomal 18S and 28S oligos. The hybrid blot formed was labeled with ³² P, imaged with phosphor (Packard cyclone) and analyzed to confirm RNA integrity and mass. The remaining total RNA mass value (90%) from each sample was then expressed as 7.5 ml blood donor equivalent of the sample of total RNA, of which 1.5% was calculated to be mRNA.

Example 9

Flow cytometry analysis in patients after immunomagnetic screening

Flow cytometry analysis of leukocytes taken from human blood was evaluated for gene expression in circulating epithelial cells. The isolated cells were prepared as described above and resuspended in 200 μl of permeate buffer containing biotin (immunicon corporation) to which monoclonal antibody (Mab) -fluorescent dye conjugate was added in saturation conditions. Monoclonal antibodies include phycoerythrin (PE) conjugated anti-cytokeratin monoclonal antibodies (MAb C11) and ferridinine chlorohill proteins that recognize cytokeratins 4,6,8,10,13 and 18 (immunicons). (PerCP) labeled anti-CD45 (Hle-1, BDIS, San Jose, CA). After incubating the cells and Mab for 15 minutes, 2 ml of cell buffer (PBS, 1% BSA, 50 mM EDTA) was added and the cell suspension was separated by magnetism for 10 minutes. After discarding the non-isolated suspension, the collected cells were resuspended in 0.5 ml PBS with the addition of the nucleic acid dye (Procount, BDIS) used in the Procount System ^™ . In addition, 10,000 fluorescence measurement beads were added to this suspension to confirm the sample volume analyzed (Flow-Set Fluorospheres, Coulter, Miami, FL).

Samples were analyzed on a FACSCalibur flow cytometer equipped with a 488 nm argon ion laser (BDIS). Data collection was performed using CellQuest ^™ (BDIS) using limit values for fluorescence of nucleic acid dyes. Data collection was stopped after 80% of 8000 beads or samples were analyzed. Multi-parameter data analysis was performed on the list mode data (Paint-A-Gate ^™ , BDIS). The analysis criteria included the size defined by the forward light scatterer and the granularity defined by the orthogonal light scatterer. Positive staining with nucleic acid dyes and staining with PE-labeled Pan anticytokeratin Mab C11 (CK 4, 5, 6, 8, 10, 13 and 18) and PerCP-labeled anti-CD45 Mab, together with differential CTC / Used for WBC fluorescence staining and analysis. The CTC's was confirmed by the presence of nucleic acid dyes and cytokeratin antigens in association with the absence of CD45 staining. For each sample, the sample volume that was not analyzed by flow cytometry was described by multiplying the number of events present in that region, usually epithelial cells, by 1.25.

In healthy non-cancer donor samples, leukocytes had immunomagnetic screening in the range of 655-5,560 (median 4,350; average 1,759). In the HRPC patient samples, leukocytes ranged from 813 to 92,000 (median 4,350; average 12,300). Blood samples from 7 men and 6 women in the healthy non-cancer control group did not show CTC, whereas blood samples from HRPC showed CTCs in the range of 4-283 in 7.5 ml of blood.

Example 10

Quantification of mRNA Transcripts from Amplified Libraries

Normalization of mRNA / aRNA mass was first determined by quantifying total RNA mass isolated from each immunomagnetically concentrated 7.5 ml blood sample volume. This was done by performing Northern Blotting of 10% of the total RNA of each sample, followed by hybridization using 28S + 18S radiolabeled oligo probes and known total RNA mass cell line standards side by side. This process was followed by phosphor image quantification (Cyclone, Packard Instruments). The total RNA mass obtained was defined as 1 donor sample equivalent of mRNA = 1 donor sample equivalent of aRNA:

[(Total RNA mass) x (1.5% mRNA)] / 3 ^* = 1 donor sample equivalent aRNA

^* (average molecular weight of aRNA library was found to be 3 times lower than unamplified mRNA molecular weight)

The relative gene expression levels of 0, 1, 2, 3, and 4 were determined according to the agarose gel dynamic curve band intensities of amplified products comprising CK19 in vitro transcription RNA construct (CK19-cRNA) standards of known copy number. Based on the assignment to the unknown. This CK19-cRNA standard contained 800 bases on the 3 'side of the CK19 wild type mRNA sequence. Standard CK19-cRNA curve covering 1000-fold dynamic range was 20,000; 2,000; 200; 100; 50; Progressed in triplicate samples at 25 and 12.5 copies, each sample spiked with 2 ng of total RNA isolated from WBC from Percoll. Standard dynamic curve progression for 40 cycles showed a linear signal response floating band intensity for the number of RNA copies between 13-200 copy CK19-cRNA transcripts (see FIGS. 13A and 13B). External standard curves showed a maximum CV of 27% for any standard analyzed in triplicate. CK19 external standard curves were constructed for multivariate gene analysis and relative gene expression levels 0-4 were assigned:

0-not detectable,

1-CK19 of about 25-50 copies,

2-CK19 of about 250 to 500 copies,

3-CK19 of about 2,500 to 5,000 copies, and

CK19 exceeding 4-25,000 copies.

FIG. 13B shows typical band intensities corresponding to the approximate number of copies of CK19 cRNA on a standard curve, which was used to assign relative gene expression levels 1-4.

CTC calculations and gene transcript expression profiles were measured using 23 different PCR amplification products from Ep-CAM immunomagnetically enriched blood samples from 13 healthy donors and 9 HRPC patients. Microarrays are not effective for analyzing these types of samples due to non-compatibility noise signals derived from nonspecifically retained WBCs during immunomagnetic enrichment. The ratio of CTC specific signal to WBC retention noise in these samples ranges from ¹ to 1000 CTC per 10 ³ to 10 ⁴ WBC. This microarray limit was overcome by including a 10,000-fold preamplification step using 90% of the total mRNA library from each immunomagnetically enriched blood sample, followed by multigene RT-PCR analysis instead of arrays. . This innovation provides enough starting material to allow hundreds of individual PCR reactions to be performed with each 7.5 ml of blood sample. Thus, for each mRNA member of each CTC mRNA library, individual CTC multivariate RT-PCR profile analysis can be performed without compromising assay sensitivity or clinical sensitivity.

After performing the volume normalization procedure described above, the remaining 90% total RNA from each sample was reverse transcribed (RT) using the SMART PCR cDNA synthesis kit, using the 67 base oligo (dT) primer described above.

The reaction was carried out by incubation at 42 ° C. for 90 minutes. Transfer all 10 μl RT to 50 μl PCR reaction using Advantage cDNA PCR Kit (Clontech), use P1-SMART primer and P2-T7 18 base primer (5'-TCTAGTCGACGGCCAGTGAATT-3 '), PE- 9600 and a thermal cycling program (1 minute at 95 ° C., 10 cycles for 15 seconds at 95 ° C., 1 second at 65 ° C., 6 minutes at 68 ° C. to 20 minutes at 72 ° C.). The entire PCR reaction volume was loaded onto a Sephadex G-50 Quick Spin (TE) column (Roche Diagnotics) and the eluate was produced according to the manufacturer's instructions.

The eluate was concentrated in vacuo to 3 μl at 60 ° C. on Vacufuge (Eppendorf) for about 30 minutes. T7 RNA polymerase transcript amplification reactions resulting in representative libraries of aRNAs were assembled in 20 μl volumes using AmpliScribe kit (Epicenter Technologies) and incubated at 37 ° C. for 6-12 hours. The trizol separation procedure was repeated to further purify the RNA transcription reactions.

1/10 of the RNA size standard, RNA mass standard, and transcription reaction product from each sample were denatured formaldehyde at 65 ° C. for 15 minutes and loaded onto a 2% agarose gel and run at 5 volts / cm for 15 minutes. After 1 hour post-staining with SYBR Gold ^™ (Molecular Probes), the gels were imaged with a density meter using AlphaImager ^™ (Alpha Innotek Corporation). The mass of each transcript library was measured.

Gene specific primers were designed as described above. All primer sets were designed to amplify specific gene target cDNA within 500 bases (average 344 bp and length range 266-513 bp) of the 3 'side of each specific gene target. All RNA samples were treated with DNase as described above to avoid amplification of genomic DNA. Table 1 shows the primer pairs for each amplicon analyzed by relative RT-PCR. Forward primer P1 is shown as the top sequence in each primer pair. The bottom sequence is the reverse primer. All sequences are described as 5 '-> 3'.

(▼ primer pair)

Reverse transcription (RT) was performed on 25 ng of T7 preamplified aRNA library using random 9mer 50 ng, 1 μl Superscript ™ (BRL) according to the manufacturer's instructions. RT was incubated at 25 ° C. for 10 minutes → 37 ° C. for 10 minutes → 42 ° C. for 20 minutes → 50 ° C. for 60 minutes. 10 equivalents of donor (50-1300 pg) per aRNA sample were used in 50 μl of each subsequent PCR reaction containing 1 unit of platinum taq (BRL). Individual PCR curves were drawn from each single PCR reaction tube by aliquoting 15 μl in 31, 35 and 40 cycles during the following high temperature cycling program: 1 min at 95 ° C. and 31, 35 and The 40th cycle was for 1 second at 95 ° C., 1 second at 65 ° C., 1 minute at 72 ° C. for 20 minutes at 72 ° C. (using PE-9600 thermal cycle system (or hot cycle system)). Each amplicon in each PCR batch included a cell line cDNA amplification positive control and a main mixed PCR reagent amplification negative control containing all components except cDNA samples. All RT-PCR results were analyzed on a 2% agarose gel containing ethidium bromide with BRL low mass molecular weight and point densitometric analysis (using AlphaEase ™ software version 5.04).

The purpose of the genetic investigation was to identify mRNA expression profiles in CTCs with the highest clinical specificity and sensitivity to detect epithelial cells. First, genes found in WBCs were amplified in healthy donors to assess the expression levels of these genes. Candidate genes were selected based on known literature data showing a wide range of epithelial specific expression levels. CTCs are profiled by identifying candidates that are not present in the WBC to characterize three basic anatomical dimensions: epithelial origin (FIG. 14A), origin tumor / organ (FIG. 14B), and tumor treatment target (FIG. 14C). On the basis of this, profiling for classification / characterization was possible.

RT-PCR profiling was performed according to the amplicon set of FIGS. 14A, 14B and 14C. 14A shows that when CK19 positive in 13 healthy donors was CK19 positive compared to 18 in 23 healthy donors (78%), the epithelial marker CK19 was 100% specific in the system of the present invention for HRPC. Indicates. HRPC patient samples were scored CK19 positive. CD5 and TROP2 were also 100% specific. However, their sensitivity was poor at 1/23 (4%) and 2/23 (9%), respectively, and could not provide enough profile values to justify their use. Of the non-100% specific genes (EpCAM, Muc-1 and MIC-1), the WBC background threshold was in the middle of levels 1 and 2, where all patients could be considered positive mistakes. It showed a greater signal. When combining CK19, EpCAM and Muc-1 sensitivity in profiling maximum epithelial sensitivity using this gene set, 23 of 23 (100%) patient samples were scored epithelial-positive. WBC specific genes (F6 = alpha 1-globin gene) were used as whole system positive controls for RNA processing and amplification because all blood samples retained WBC due to the low nonspecific binding derived from the magnetic fluid amplification method It was. All 36 samples were scored at level 4 or higher (see FIG. 14A).

Example 11

Reproducibility evaluation at lower limit of preamplification sensitivity

To assess the reproducible minimum threshold sensitivity performance in modified T7 transcription preprocessing amplification, a model system was constructed with clinical RNA samples on which all manipulations were performed. Trizol Reagent (Life Technologies, Inc.) was used to isolate total RNA from the breast and prostate cancer cell lines SKBR-3 and LNCaP (Schools of Caterpillar Culture, Manner, VA) according to the manufacturer's instructions. . Dilution serially prior to first strand synthesis yielded 2, 0.2, 0.02 SKBR-3 cell equivalents, where each dilution was spiked into 2 ng of total RNA derived from Percoll-induced WBC. The RT-PCR external CK19 standard curve was prepared parallel to the SKBR-3 dilution curve, indicating that there are about 1000 wild type CK19 mRNA copies per SKBR-3 cell equivalent. The CK19 characterized SKBR-3 dilution curve was used as either type of specific transcript, to model minimum sensitivity and reproducibility throughout the entire process of the T7 based mRNA library amplification system. The second transcript was an exogenous lambda DNA based construct (Walker Biotech Publication) that produced a 1.2 kb polyA (30) transcript. Curves for WBC spiked with SKBR-3 cells were assayed three times [2000, 200 and 20 CK19 mRNA copy levels]. Lambda curves were spiked three times with 2 ng of total RNA derived from Percoll-induced WBC at 500, 50 and 5 copy levels. In the final analysis, reproducible RT-PCR amplification was performed at levels above 50 copies (N = 12), with full T7-transcript preamplification performed on each of the samples, resulting in a measurable signal. In addition, the minimum limit of detection (starting with 50 mRNA transcripts of any one sequence) was reproduced in subsequent cell line spike models, in which case all six types of sequences (PSA, PSM, AR, HPN, CK19 and EpCAM) Serial dilution prior to aRNA analysis and quantitative RT-PCR analysis to dilute to known radiation levels (Example 6).

After RT-PCR, aliquots obtained from the kinematic curves of cycles 31, 35 and 40 were subjected to molecular weight and spot density determination gel analysis using Alphalmager. The CV calculated from 11 of the 12 spike levels (92%) was 19.25%. Only one of the 200 copy samples (8%) was 14 times smaller in intensity than expected, but this sample was still qualitatively detectable and named Level 1.

In a separate study, the effect of the T7 preamplification method of the present invention on mRNA abundance was modeled by comparing it with an identically prepared unamplified library. As shown in FIG. 13C, initially, 1000 WBC total RNA (2 ng) equivalents were spiked with 15 cell equivalents of prostate cancer cell line LNCaP and 15 equivalents of breast cancer cell line SKBR-3, followed by T7 preliminary When the amplification method and subsequent multi-gene RT-PCR dynamic curve analysis were performed, the difference in the intensity ratio of the bands for the N = 8 genes (PSA, PSM, MGB1, MGB2, PIP, CK8, CK19 and EpCAM) was Not much confirmed. The cDNA template negative amplification control group present with each batch did not detect any signal during the study.

The total RNA library was proportionally amplified with the first round of the modified T7 method of the present invention to generate an aRNA library with an average increase of at least 10,000 times the original mRNA mass. This is based on measurements of the original mRNA level, which is 1.5% of the given total RNA mass. The transcript amplification method resulted in a library with an intermediate transcript length of 600 bases (between 550 and 800 bases). The size of each transcript present in each library was 300-3000 bases. Each aRNA library was randomly primed for RT, from which multiple gene panels of each PCR reaction were performed using 10 donor equivalents of aRNA / cDNA.

The total amount of RNA derived from retained WBCs in healthy non-cancerous samples was 0.8-11.12 ng (average 3.5 ng). The total amount of RNA derived from HRPC patient samples was 0.8-35.12 ng (average 7.2 ng). All total RNA samples then produced an aRNA library with a mass primarily proportional to the starting total RNA level.

10% of the Northern blots for the total RNA of each sample were hybridized with 28S + 18S radiolabeled oligo probes with total RNA having a known mass derived from cell line standards, followed by phospho to measure the quantity and character. Imaged. If the SKBR-3 cell line standard for each amplified sample was on average 1.55 (range: 1.50 to 1.64), the quality was assessed at the ratio of 28S to the amount of 18S; the average of 1.36 (range) for 13 healthy donors : 1.16-1.60) and HRPC averaged 1.10 (range: 0.57-1.80).

In addition, the inventors found that 80% of the total RNA mass of the magnetic fluid-enriched CTC / WBC samples derived from HRPC patients was 6 times smaller than expected (1.5-15 fold). This projection is based on the fact that the WBC average total RNA mass = 2 pg per cell and the average epithelial cell total RNA mass = 20 pg per cell. This is particularly meaningful in evaluating the diagnosis and treatment status of an individual during treatment.

Example 12

Characterization of CTC Tumor-Derived Tissues Using Tissue-Specific Genes and Characterization of Patient-Specific Treatment Profiles

Further profiling was performed on 36 samples with amplicons with N = 8 to determine optimal specificity and sensitivity expression profiles for identifying the organs of origin of circulating epithelial tumor cells. For prostate specific identification, we evaluated PSA, PSM, HK2, HPN, PSGR, DD3, MGB1 and MGB2 (see FIG. 14B). None of the amplicons showed a signal from the WBC group, except for one female exogenous individual with hepsin (HPN). As shown in FIG. 14B, PSA was the highest sensitivity for the prostate group, with 20/23 (89%) samples scored +, followed by PSM 17/23 (74%), HPM 13 / 23 (57%), hK2 7/23 (30%), PSGR 2/23 (9%) and DD3 1/23 (4%). Unexpectedly, the "breast specific" genes mammoglobin 1 and 2 were both scored with strong signal levels in the prostate (+), MBG1 1/23 (4%) and MBG2 2/23 (9%), but healthy donors Not so with clusters. When the two high-sensitivity markers, PSA and PSM, were combined, the sensitivity was 20/23 (87%). As a result of adding HPN, the sensitivity was 21/23 (91%). Since hepsin and PSM are both tissue specific and transmembrane in which specific cell targeted delivery techniques can be implemented, they also play a central role in therapeutic techniques.

Characterization of each patient for potential treatment profiles based on CTC RNA profiling was performed and the results are shown in FIG. 14C. Here, each of the specificity scores of the samples represented by AR, NKX3A, EGFR and ER was 100%. MDR1, MRP and Topo2a all had significant background signals due to the WBC group. The sensitivity of AR to detect HRPC CTC was 16/23 (70%), followed by NKX3A 4/23 (17%), Topo2a 5/23 (22%), MDR1 5/23 (22%), EGFR 4/23 (17%) and ER 1/23 (4%). Unexpectedly, MDR and MRP did not seem useful to strategy the two groups.

Long-term samples were taken from three patients over 18-26 weeks. One patient was treated with loopon only and two patients received treatment with loopon and Taxane / Estramustine together. The series of samples showed a change in the expression of the therapeutic sensitivity / resistant genes, while there was no change in the remaining samples. As shown in Figures 15A, 15B and 15C, this change was independent of the number of CTCs and white blood cells. In patients treated with loopon alone (four long-term samples collected at 0, 5, 10, and 18 weeks), MDR1 was not detected (see FIG. 15A), and AR was relatively constant despite changes in hepcine levels. Maintained. 15B and 15C show that further MDR1 mRNA levels steadily decrease compared to healthy donors during TX / ES treatment. As shown in FIG. 15B, AR expression levels, which play an important role in HRPC progression, were not identified at all upon TX / ES treatment. In contrast, FIG. 15C shows that AR is relatively unaffected during a similar course of treatment. From the high expression level for the untreated group, the detection results for hepsin mRNA changed radically, indicating that there was no need for TX / ES treatment in both patients of FIGS. 15B and 15C.

Example 13

Profiling of the bed using plasma derived (non-CTC) RNA obtained from the same samples providing CTC-complementarity data

Hereinafter, a method for analyzing mRNA derived from CTC amplified from a blood sample is not described. An important step in the method is the T7 preamplification step, ie only a few transcripts can be analyzed with up to 1000 different individual gene specific RT-PCR responses. T7 preliminary amplification of each mRNA library effectively removes major limitations of limited sample mRNA mass. This same preliminary amplification can be performed for non-CTC RNA. Indeed, there are a number of RNA sources in a given blood sample, and some of these non-CTC RNA transcripts will provide a variety of information.

Confirmation of the presence of CTCs, determination of tissues of tumor origin, and comprehensive characterization of disease mechanisms can be performed with RNA derived from plasma blood fractions obtained during the magnetic fluid enrichment process. Preferably, this may be in parallel with the T7 preamplification method of the embodiment described above for concentrated CTC. The magnetic fluid concentration method first separates the plasma fraction of each sample. Typically this fraction is discarded when CTC is amplified.

Recently, however, plasma and serum derived mRNAs and DNA have been known in the literature to provide various cancer expression (phenotype) and genotype (DNA analysis) data. Plasma derived mRNA and DNA are isolated by conventional molecular biological methods for downstream analysis. Since mRNA is readily available from plasma and has been found to provide a variety of RT-PCR data, such identical RNA transcripts can be more extensively profiled as described herein. Therefore, CTC-independent and / or CTC-complementary mRNA expression profiles can be obtained by the same profiling method for CTC using RNA derived from the plasma-derived fraction of each sample.

In addition, the T7-based expression profiling methodology can be applied to the above-described enrichment method to enable analysis of the CTC-deficient fraction, which differentiates the contribution to the WBC expression profile retained nonspecifically during amplification. , Thereby contributing to the CTC specific profile. This can be done through differential pattern comparisons and subsequent deductions, providing additional mechanisms for accurately identifying CTCs during analysis. In addition, the CTC-deficient profile itself will provide a variety of patient-specific information regarding responsiveness and sensitivity to specific therapies.

Those skilled in the art to which the present invention pertains are not specifically limited to the description and examination results of the preferred embodiment described herein, but the method specifically disclosed herein without departing from the spirit and scope of the invention as defined only by the appended claims below. It will be understood that various modifications and variations can be made.

Claims (76)

a. Obtaining a sample from the blood of a test subject suspected of having cancer comprising a mixed cell population suspected of containing cancer cells of epithelial origin; b. Mixing said sample with immunomagnetic particles that specifically bind said cancer cells until other sample components are substantially excluded; c. Applying a high gradient magnetic field to the sample-immunomagnetic particle mixture to prepare individual cell fractions in which magnetic particle-bound cancer cells are concentrated; And d. Assaying the concentrated fraction for the presence of cancer cells, wherein the presence of the cancer cells in the sample is indicative of cancer A method of diagnosing the severity of a disease of a test subject, comprising. The method of claim 1, wherein the test subject is for assessing the presence of circulating cancer cells. The method of claim 1, wherein the responsiveness to the method for removing cancer of the test subject is assessed by the presence of the circulating cancer cells. The method according to claim 1, wherein the test subject is prostate cancer, breast cancer, colon cancer afudoma, isolated species, new species, malignant carcinoid syndrome, carcinoid heart disease, carcinoma such as Walker, basal cell, basal Squamous Cells, Brown-Pearce, Conduit, Erlich Tumor, In situ, Krebs 2, Merkel Cells, Mucin Productivity, Non-Small Cell Lungs, Oat Cells, Papillary, Hard Cancer, Bronchus, Bronchus Squamous Cells, Transient Cell Retinopathy, Melanoma, Chondroma, Chondroma, Chondroma, Fibrosarcoma, Fibrosarcoma, Giant Cell Tumor, Histicocytoma, Lipoma, Liposarcoma, Mesothelioma, Myxoma, Myxarcoma , Osteosarcoma, osteosarcoma, Ewing sarcoma, synovial tumor, adenoma, adenocarcinoma, carcinosarcoma, chordoma, pericarcinoma, mesothelioma, myoma, enamel blastoma, cementomas, dental carcinoma, teratoma, tropoblast tumor ( thropoblastic tumor) Tumor, adenoma, cholangiocarcinoma, cholesteroma, columnar, cyst adenocarcinoma, cyst adenoma, granulocyte cell tumor, glioblastoma, hepatocellular carcinoma, adenoma, islet cell tumor, Leidihi cell tumor, papilloma, sertoli cell tumor, follicle Membrane Cell Tumor, Leiomyoma, Leiomyosarcoma, Myoblastoma, Myoma, Myoma, Rhabdomyosarcoma, Rhabdomyosarcoma, Epithelial Cell Tumor, Ganglion Cell Tumor, Glioma Tumor Celloma, Meningioma, Neurofibromatosis, Neuroblastoma, Neuroepithelioma, Neurofibroma, Neuroma , Adrenal ganglia, Non-Chrome-affinity, Antikeratoma, Curable angioma, Multiple hemangioma, Glomerular angioma, Hemangioendothelioma, Hemangioma, Peripheral hemangioma, Peripheral cell sarcoma, Lymphangioma, Lymphangioma, Lymphangioma, Pineal somatoma , Carcinosarcoma, chondrosarcoma, filamentous sarcoma, fibrosarcoma, angiosarcoma, leiomyarcoma, leukemia, liposarcoma, lymphangiosarcoma, myoma, myxarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma (kaposi and obesity) Cells), neoplastic tumors (e.g., bone, digestive system, liver, pancreas, thyroid gland, testicles, orbits, head and neck, central nervous system, hearing, pelvis, airway and urogenital), neurobromatosis and cervical dysplasia Is diagnosed with a cancer selected from the group consisting of: The method of claim 1, wherein the assaying of the concentrated fraction for the presence of cancer cells consists of the following steps: a. Releasing cytoplasmic biomolecules from the cancer cells; b. Separating the cytoplasmic biomolecules; And c. Analyzing the cellular biomolecules. The method of claim 1, wherein said obtaining a fluid sample is performed by cell fractionation. The method of claim 5, wherein the release step is performed by adding a permeabilizing agent. 8. The method of claim 7, wherein said penetrant is selected from the group consisting of detergents, surfactants, and combinations thereof. 8. The method of claim 7, wherein said penetrant is selected from the group consisting of saponin, Imuniperm, and combinations thereof. 8. The method of claim 7, wherein the penetrant is an immunoperm. The method of claim 1, wherein obtaining the fluid sample is performed by cell stabilizer treatment. The method of claim 11, wherein the stabilizer is selected from the group consisting of aldehydes, ureas, and combinations thereof. The method of claim 12, wherein the aldehyde is selected from the group consisting of formaldehyde, paraformaldehyde and combinations thereof. The method of claim 11, wherein the stabilizer is dialdehyde. The method of claim 14, wherein the dialdehyde is selected from the group consisting of glutaraldehyde, glyoxal and combinations thereof. The method of claim 11, wherein the stabilizing agent is selected from the group consisting of Cyto-Chex®, Stabilocyte®, and Transfix®. 12. The method of claim 11, wherein the step of releasing cytoplasmic biomolecules involves macromolecular complexes formed by exposure to the stabilizer. The method of claim 17, wherein the step of releasing the cytoplasmic biomolecule from the cell is performed by enzymatic digestion. The method of claim 18, wherein the enzymatic digestion step is performed in the group consisting of proteinases, nucleophiles, and combinations thereof. The method of claim 19, wherein the proteinase is selected from the group consisting of proteinase K degradation, V8 proteinase degradation, pronase degradation, and combinations thereof. The method of claim 19, wherein the nucleophilic agent is selected from the group consisting of phosphate-based buffers, tris-based buffers, hydrazide acetates, hydroxylamines, and combinations thereof. The method of claim 5, wherein said cytoplasmic biomolecule is a protein. The method of claim 22, wherein the separation of the protein is performed by a method selected from the group consisting of chemical extraction, electrophoresis, chromatography, immunosparation, and affinity technique. The method of claim 5, wherein said cytoplasmic biomolecule is a nucleic acid. The method of claim 24, wherein the nucleic acid is selected from the group consisting of cytoplasmic RNA, nuclear and mitochondrial RNA, nuclear and mitochondrial DNA, and combinations thereof. The method of claim 24, wherein the nucleic acid is cytoplasmic mRNA. The method of claim 24, wherein the separation of the nucleic acid is performed by a method selected from the group consisting of RNA or DNA chemical extraction, electrophoresis, chromatography, immunoisolation and affinity techniques. The method of claim 24, wherein the separation of the nucleic acid is carried out by capture by magnetic beads attached to the oligo (dT). The method of claim 5, wherein said target cell is assessed for expression of the phenotype in addition to being assessed using said cytoplasmic biomolecules after obtaining said fluid sample. The method of claim 29, wherein the expression of the phenotype is selected from the group consisting of morphological observation, cell component staining, immunoassay, and a combination thereof. The method of claim 1, wherein the assay is from the group consisting of multi-parameter flow cytometry, immunofluorescence microscopy, laser injection cytometry, bright field imaging, capillary volumetric, spectral imaging, manual cell assay, and automated cell assay. The method performed by the selected method. The method of claim 5, wherein the analysis of cytoplasmic biomolecules is performed by functional proteomics. 33. The method of claim 32, wherein the functional proteomics is characterized by protein expression profiles, western blots, amino acid sequencing, electrophoresis, 2-D electrophoresis, mass spectrometry, gas chromatography, liquid chromatography nuclear magnetic resonance, infrared, atomic adsorption And a method of combining them. The method of claim 5, wherein the analysis of cytoplasmic biomolecules is performed by functional genomics. 35. The method of claim 34, wherein said functional genomics is characterized by mRNA profile analysis, protein expression profile analysis, polymerase chain reaction, northern blot, western blot, nucleotide or amino acid sequencing, gene expression on microarray, electrophoresis, 2- D electrophoresis, mass spectrometry, gas chromatography, liquid chromatography nuclear magnetic resonance, infrared and atomic adsorption methods. The method of claim 5, wherein the assay of cytoplasmic RNA for the presence of two or more genetic markers is performed by multiple gene RT-PCR. The method of claim 5, wherein said genetic marker identifies the tissue of origin of said cancer cell. The method of claim 5, wherein the genetic marker is to diagnose a known cancer. The method of claim 5, wherein the genetic marker designates a cancer selected from the group consisting of breast, prostate, lung, colon, ovary, kidney and bladder. The method according to claim 5, wherein the genetic marker is MGB1, MGB2, PIP, CEA, PSA, PSMA, hK2, AR, DD3, Her- / Neu, BCL2, EGFR, TS, DPYD, IGF2, ER, PR, cyp19, TERT , Systemic epithelial tissue specific genes, CK19, CK8, CK20, EpCAM, MUC1, topoisomerase, uPA, uPAR, MMP, systemic leukocyte specific mRNA, alpha-1-globin, CD16, CD45, and CD31 Selecting a cancer. The method of claim 5, wherein analyzing the cytoplasmic RNA for the genetic marker comprises the following steps: a. Reverse transcription of said genetic marker with one or more sets of gene specific primers having a universal primer extension at the 5 'end; b. Removing the gene specific primer; c. Amplifying the gene specific amplicon through PCR amplification using a universal primer extension; And d. Identifying the gene specific amplicon. 42. The method of claim 41, wherein the removal of said gene specific primers comprises uracil-N- using molecular size exclusion, solid support selective attachment, single strand specific DNase methods, and DNA oligonucleotide primers synthesized with deoxyuridine. The method is carried out using a method of the group consisting of incorporation of glycosylate. 42. The method of claim 41, wherein the removal of said gene specific primers is performed by uracil-N-glycosylase treatment of DNA oligonucleotide primers synthesized with deoxyuridine. 44. The method of claim 43, wherein DNase-RNase is incorporated after the uracil-N-glycosylase treatment. The method of claim 41, wherein the gene specific primer is used under high specific primer-target annealing conditions. 46. The method of claim 45, wherein said high specific primer-target annealing is performed by a protein derived from a native recombinant cell repair mechanism. 47. The method of claim 46, wherein said high specific primer-target annealing is performed by recA. 42. The method of claim 41, wherein identifying the gene specific amplicons is performed by measuring Rf values inherent to the analysis system itself based on size. 49. The method of claim 48, wherein said size based assay system is selected from the group consisting of PAGE, agarose gel electrophoresis, capillary gel electrophoresis, SELDI, MALDI, and cDNA arrays. The method of claim 5, wherein the analyzing step comprises the following steps: a. Preamplifying the nucleic acid extracted by primary amplification, wherein the preamplification results in an increase of at least 1000-fold in all library transcripts in the form of aRNA; b. Synthesizing at least 1000 independent selection genes of interest for the second strand derived from the aRNA; And c. Recognizing the amplified product using a method of the group consisting of array analysis, electrophoresis and a combination thereof. 51. The method of claim 50, wherein said preamplification step is performed by a promoter of the group consisting of an SP6 RNA polymerase promoter, a T3 RNA polymerase promoter, and a T7 RNA polymerase promoter. 51. The method of claim 50, wherein said preamplification step is performed with RNA polymerase using random primers. 51. The method of claim 50, wherein said step of synthesizing said second strand is performed under high specific primer-target annealing conditions. The method of claim 53, wherein the high specific primer-target annealing is performed with a protein derived from a native recombinant cell repair mechanism. 55. The method of claim 54, wherein said high specific primer-target annealing is performed by recA. 51. The method of claim 50, wherein the recognition step is performed by pretreatment with DNase-RNase free. The method of claim 56, wherein the pretreatment step is selected from the group consisting of phenol extraction, silica bonding, and combinations thereof. 51. The method of claim 50, wherein said recognition step is performed by amplifying all double stranded products. 51. The method of claim 50, wherein said recognition step is selected from the group consisting of array analysis, electrophoresis, and combinations thereof. a. Immunomagnetic particles that specifically bind to cancer cells; b. Cell penetrant; c. Cell stabilizer; d. Nucleic acid release agents; e. Preparations for nucleic acid amplification; f. Tablet formulations for selecting double stranded products; g. Nucleotide specific detection agents capable of identifying double-stranded products; And h. Protocol for implementing the method of claim 1 A test kit for screening a patient sample for the presence of circulating cancer cells. 61. The kit of claim 60, further comprising immunomagnetic particles retaining binding specificity for said cancer cells. 61. The kit of claim 60, wherein said cell penetrant is selected from the group consisting of saponins, detergents, surfactants, and combinations thereof. 61. The kit of claim 60, wherein said penetrant is an immunoperm. 61. The kit of claim 60, wherein the stabilizer is selected from the group consisting of aldehydes, dialdehydes, ureas, and combinations thereof. 61. The method of claim 60, wherein said stabilizing agent is cytostatics. The kit is selected from the group consisting of Stabilites® and Transfix®. 61. The kit of claim 60, wherein said release agent is selected from the group consisting of proteinases, nucleophiles, and combinations thereof. 67. The kit of claim 66, wherein said proteinase is selected from the group consisting of proteinase K, V8 proteinase, pronase and combinations thereof. 67. The kit of claim 66, wherein said nucleophilic agent is selected from the group consisting of phosphate-based buffers, tris-based buffers, hydrazide acetates, hydroxylamines, and combinations thereof. 61. The kit of claim 60, wherein said preparative formulation is an immunomagnetic particle that retains binding specificity for said nucleic acid. 61. The kit according to claim 60, wherein the preparation for purification is selected from the group consisting of RNA or DNA chemical extraction preparations, electrophoretic preparations, chromatography preparations, immunoassay preparations and nucleotide affinity preparations. 61. The method of claim 60, wherein the amplification agent is selected from the group consisting of SP6 RNA polymerase / promoter, T3 RNA polymerase / promoter, and T7 RNA polymerase / promoter, wherein the amplification agent increases the nucleic acid content by 1000 times. Kit. 61. The kit of claim 60, wherein the amplification agent is an RNA polymerase / random primer, which increases the nucleic acid content by 1000 times. 61. The kit of claim 60, wherein said amplification agent is a polymerase / primer that is highly specific primer-target annealed. 61. The kit of claim 60, wherein said tablet formulation is DNase-RNase free. 61. The kit of claim 60, wherein said tablet formulation is selected from the group consisting of phenolic extraction formulations and silica binding formulations. 61. The kit of claim 60, wherein the nucleotide specific detection agent is selected from the group consisting of a double stranded nucleotide amplification agent, an array detection agent, and an electrophoretic agent.