JP2000515761A - Whole cell analysis system - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for determining the presence of a particular type of cell in a mixed cell population. The method involves the identification and detection of markers of the target cells, the preparation of primers specific for the markers, and the technique of reverse transcription to amplify these markers. The method of the invention is sensitive enough to be able to detect the presence of as few as 100 target cells in a cell population. Specific examples of target cells include human chondrocytes and human keratinocytes.

Description

DETAILED DESCRIPTION OF THE INVENTION Whole Cell Analysis System Related Application This application is a provisional application No. 60 / 022,636 filed on July 26, 1996 and a provisional application No. 60 filed on January 23, 1997. Relates to and claims priority to / 035,415. BACKGROUND OF THE INVENTION The present invention relates to a rapid and sensitive assay system and method for confirming the presence of a particular mRNA species present in a cell suspension. The assay systems and methods of the present invention utilize whole cells as a starting material for reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Primers are designed and used to amplify a specific cDNA sequence that matches the mRNA. In this way, specific and positive identification of cell types present in the mixed cell population can be performed with minimal time and sample manipulation. Various strategies can be used to determine cell type with currently available techniques. One such strategy is to determine cell type by examining cell or tissue morphology. The disadvantage of this method is that it requires a considerable amount of training, and even then it is very subjective and always poorly reproducible. This can be improved by performing histological staining on the cells or tissues. In general, histological manipulations are time consuming and staining is not very specific. Also, staining and fixing cells or tissues often requires hazardous chemicals and facilities to work with and dispose of these substances. A second strategy for determining cell types is to look for protein markers that are known to be expressed only by specific cell types. For example, immunohistology or FACS technology. Although these methods are much more specific than examining morphology, they rely heavily on the availability of antibodies raised against the protein marker of interest. However, antibodies are often not readily available. Furthermore, each antigen-antibody reaction is unique and requires individual optimization of reaction conditions. A third strategy for typifying cells is to examine the mRNA transcribed by the cells. Although each cell type has unique mRNA properties, the method used to engineer the RNA does not depend on the particular mRNA being examined. The technology can be applied to a wide range of cell sources with very little improvement at best. Northern blotting and RNAse (ribonuclease) protection assays are two methods by which the presence of specific mRNA can be detected and quantified. However, both of these methods require significant amounts (1-20 μg) of RNA and generally require that it be first purified. Although in situ hybridization and in situ PCR can be performed with much less starting material, both of these methods are technically difficult, time consuming to perform, and difficult to analyze. In the last few years, several technical improvements have been made in eukaryotic cell gene expression studies regarding the conditions of sensitivity, specificity, and time for detection of target mRNA. RNAse protection assays are superior to Northern blot analysis because they are quantitative, require very little starting material, and can be performed directly on whole cells without prior RNA purification. (Strauss and Jacobowitz, Brain Res. Mol. Brain Res., 20, 229-239, (1993)). However, RNase protection assays still require a significant amount of time and manipulation. RT-PCR analysis systems allow the detection of dilute mRNA transcripts from very small amounts of RNA (Klebe et al., BioTechniqes 21 (6), 1094-1100 (1966). However, RNA isolation. Some steps are still required, such as first-strand cDNA synthesis, followed by PCR amplification, etc. These limitations make RNase protection assays or RT-PCR assays to screen cells for specific target mRNA. Therefore, it is difficult to use it as a rapid analysis system for rapid and accurate identification of target mRNA in cell culture with minimal sample manipulation and to overcome the disadvantages of the prior art. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an electrophoretic photograph showing the presence of aggrecan and GAPDH in passaged human chondrocytes and human dermal fibroblasts. Figure 2a shows the fractionation performed on total RNA isolated from chondrocytes and fibroblasts. Figure 2b is a photograph of an agarose gel showing the results of the analysis, Figure 2b is a photograph of a Southern blot showing the results of an analysis performed on total RNA isolated from chondrocytes and fibroblasts. Figure 4 is a schematic diagram of the chromosome and cDNA templates at positions, with the intervening sequence shown in the chromosomal DNA but absent in the cDNA and mRNA. Fig. 5 is a photograph of an agarose gel showing the results of an analysis performed on total RNA isolated with and without RNase Fig. 5 shows RNase and DNA degradation from chondrocytes and fibroblasts Figure 6 is a photograph of an agarose gel showing the results of an analysis performed on total RNA isolated with and without enzymes Figure 6 shows human chondrocytes from a chromosomal DNA template (250 bp) and an mRNA template (146 bp) And fiber Figure 7 is a photograph of a Southern blot showing the presence of GAPDH in blasts Figure 7 shows the presence of RNase A and RNase inhibitor for the detection of aggrecan and GAPDH from healthy human chondrocytes and fibroblasts. Fig. 8 is a photograph of an agarose gel comparing the effects Fig. 8 is a photograph of an agarose gel showing the presence of aggrecan and GAPDH derived from healthy human chondrocytes and fibroblasts Fig. 9 is a photograph of a fully human chondrocyte and fibroblast FIG. 2 is a photograph of a Southern blot showing the presence of cell-derived aggrecan and GAPDH. FIG. 10 is a photograph of an agarose gel comparing the effect of the presence of RNase A and RNase inhibitor on the detection of aggrecan and GAPDH from healthy human chondrocytes and fibroblasts. SUMMARY OF THE INVENTION The present invention relates to a rapid and accurate method for the determination of specific types of cells, such as chondrocytes, in a mixed cell population. The method of the invention uses whole cells as starting material. Markers specific to the target cells are identified and primers for the markers are assembled. It is desirable to assemble primers to make a nucleic acid product from a reverse transcribed RNA template that is smaller than the DNA amplified from the corresponding chromosomal template. In carrying out the method of the present invention, cells are separated from the remaining components of the liquid sample by a simple means such as centrifugation. Preferably, the cells are pelleted. Prepare the reaction by mixing the pellet, RT-PCR media, cell lysing or detergent such as Tween 20, and, optionally, RNase inhibitors to minimize mRNA degradation . By keeping the reaction solution at a sufficiently high temperature (about 60 to 90 ° C.) to temporarily inactivate the RNase, it suppresses mRNA degradation and erroneous priming. The method of the present invention is particularly useful for analyzing a human cartilage tissue sample that is surgically removed from a patient during cartilage repair surgery. After removal from the patient, the sample is analyzed for the presence of aggrecan mRNA specific for chondrocytes. If sufficient cells are present in the sample, as determined by the procedure of the present invention, the cells may be cultured to form a patch for repairing cartilage defects. It will be readily appreciated that a similar approach can be taken to replace keratinocytes. DETAILED DESCRIPTION OF THE INVENTION The methods of the present invention utilize molecular markers that are known to be specific for certain cell types. It is desirable to perform a modified single tube RT-PCR analysis to amplify the signal for these markers. Such techniques are known methods and are commercially available. Any known amplification technique can be used so long as it achieves the purpose of the method and analytical system. The method and assay can be used for any procedure that requires confirming the presence of a particular cell type in a mixed cell population. More generally, whenever a particular RNA is expected to be transcribed by some or all cells, it can be used. Transcribed only in chondrocytes (aggrecan mRNA), keratinocytes (keratin mRNA), viral or bacterial RNA, cells expressing mRNA isoforms with deletion or insertion mutations, and cancer alone, to name a few. There is RNA that is performed. The ability to use whole cells as a starting material without having to first purify the mRNA or total RNA from the cells saves a great deal of time and samples required for gene expression analysis procedures. The entire operation can be performed in a single tube using a single buffer, with little manipulation of the sample. In many situations, currently available methods for cell sorting are inappropriate because they require too much time, manipulation, or are too subjective. Primers with a relatively high melting point are chosen to amplify a short portion of the mRNA where the intervening sequence is present in the corresponding genomic sequence. Such primers allow a distinction between signal generated from chromosomal DNA templates present in all cells and cell type-specific reverse transcribed mRNA. High primer annealing temperatures allow for very specific interactions between the primer and the template. It also allows the use of a two-step PCR amplification with a combined annealing and extension step. The short distance between the primers, combined with the high operability of the enzyme and two-step PCR amplification, greatly reduces the number of cycles required to generate a signal from a specific mRNA. The speed and simplicity of this assay is optimized by having a mixture of enzymes or enzymes having thermostable reverse transcriptase and DNA polymerase activities in a single buffer. If this bi-activity / single buffer is possible, the analysis can be completed in a single tube without any manipulation required during the analysis. Thermostable polymerase rTth is a thermostable enzyme that has both reverse transcriptase and DNA polymerase activities, and both activities are obtained in a buffer system such as the PCR reaction buffer available from Perkin Elmer Cetus. . The assays of the present invention can be used to determine whether a particular gene is transcribed in a given cell population by choosing a primer pair that distinguishes the RNA signal from the chromosomal DNA signal. The method used in the present invention can generate a signal directly from whole cells, indicating that it is a specific, simple, rapid, and highly sensitive analysis requiring a minimum handling time and operation. This method can be used to confirm the presence of a particular target mRNA in a mixed cell population. The method of the present invention is very sensitive in detecting specific low concentrations of mRNA from less than about 1000 and as few as 100 cells. Further, amplified DNA fragments are not made from chromosomal DNA, but from transcribed genes. This procedure considerably simplifies qualitative gene expression analysis, since all cells are used as templates in a single tube, single buffer, one enzyme RT-PCR reaction. Accurate quantitation of RNA using RT-PCR has been made possible by recent innovations that limit small variations in sample-to-sample conditions during the reverse transcription process and the amplification step to introduce artificial errors. Assays that allow for efficient and quantitative screening of cells for specific target mRNAs will have many potential applications. For example, you can analyze conditions that enhance the transcription of specific mRNAs by cells in vitro, examine heterogeneous cell populations for abundance of specific cell types, and determine the transcription level of plasmid transfection. Could be measured and optimized. Various features and aspects of the present invention are illustrated in the following examples, which should not be considered as limiting to the scope of the invention as defined in the appended claims. Example 1 Primers were designed to amplify a 280 bp region of the G3 domain of aggrecan mRNA, a chondrocyte-specific marker. These primers were chosen not to amplify sequences from the chromosomal DNA template, as the region between the primers would be expected to contain intervening sequences that were too large to amplify under the reaction conditions here. Primers were also selected as a positive control for amplification. The primers were designed to co-amplify a 146 bp region of GAPDH, a housekeeping gene transcribed in all cell types. Subcultured human chondrocytes grown in monolayer on tissue-cultured plastic are released from the plastic with trypsin and inactivated by the addition of 5 volumes of cell culture medium containing 10% fetal bovine serum (FBS). I let it. Remove a small amount of the cell suspension for counting on a hemocytometer and pellet the remaining cells in a clinical centrifuge. The cell pellet is resuspended in 10 ml of phosphate buffered saline (PBS) and 10,000 cells are dispensed into 0.2 ml thin PCR tubes. The cells are pelleted by centrifugation in a PCR tube and the PBS supernatant is removed. The RT-PCR reaction is performed using a technique optimized for suppressing the degradation of mRNA as described later. Add the RT-PCR reaction mixture, which has been briefly preheated to 90 ° C. in a thermocycling device, directly to the cell pellet. The composition of this mixture is as follows: Tween-20 0.125 μl 0.5% (V / V) Placental RNA degrading enzyme inhibitor 1.25 μl 500 U / ml each primer 4 μl 11.25 pmol dNTPO 3 μl 300 μM 5 × buffer 5 μl 1 / 5 volumes Mn (OAc) ₂ 2.5 μl 24 μl with 2.5 mM water Place the reaction mixture in a thermocycling apparatus set at 90 ° C. The enzyme rTth (1 μl = 2.5 U) is added to the reaction, followed immediately by stirring and immediately returning the reaction to its original place in the thermocycler. The following program is as follows: 63.5 ° C (optimal annealing temperature of reverse primer) 15 minutes 92 ° C 1 minute or less 35 cycles 92 ° C 10 seconds 63.5 ° C (optimal reverse primer annealing temperature) 20 seconds 72 Hold at 4 ° C for 3 minutes at 4 ° C until ready for analysis. The specificity of the assay was established using human fibroblasts as a template for RT-PCR. The 280 bp band corresponding to aggrecan was only amplified from chondrocyte or purified chondrocyte RNA and not from fibroblast or purified fibroblast RNA. Similar analytical tests using purified RNA showed that there was no difference in the size of the fragments amplified from the isolated RNA template and from the whole cell template. The amplification products were analyzed by agarose gel electrophoresis, and the results are shown in FIG. Examples 2-9 The following general procedures were applied to Examples 2-9. Passaged human articular chondrocytes and dermal fibroblasts were grown in monolayer on tissue culture plastic and supplied with DMEM containing 10% fetal calf serum. Cells were released from tissue culture treated plastic with trypsin / EDTA and then inactivated by the addition of 5 volumes of medium containing 10% FBS. A portion of the cell suspension was taken for counting on a hemocytometer and the cells were pelleted in a clinical centrifuge. The cell pellet was resuspended in phosphate buffered saline and 10,000 cells were dispensed into 0.2 ml thin PCR tubes. The cells were pelleted by centrifugation in a PCR tube at 3000 rpm in a tabletop microcentrifuge and then the phosphate buffered saline supernatant was removed. Primers were designed using the Oligo 5.0 program (National Biosciences, Plymouth MN). The primer has a predicted melting temperature of 85 ° C or higher and a predicted optimum annealing temperature of 63-64 ° C. Each primer was chosen to anneal completely within a single exon of the mRNA sequence, and the region between the forward and reverse primers was expected to fill the intervening sequence. With this primer design, the sequence amplified from the reverse transcribed RNA template results in a smaller product than the sequence amplified from the chromosomal DNA template. Amplification products containing primers were 100-500 base pairs, allowing for short cycle times. The primer sequences (5 'to 3') are as follows: Primers were prepared and purified on reverse phase cartridges from Genosys Biotechnologies (Woodlands, TX). The RT-PCR reaction is an rTth enzyme and buffer optimized by Perkin Elmer Cetus (EZ rTth RNA PCR Kit, Roche Molecular Systems, Branchburg NJ) to allow both reverse transcriptase and DNA polymerase activity. Performed using liquid conditions. The reaction volume is 25 μl and consists of 0.5% (v / v) Tween 20, 11.25 pmol of each primer, 300 μM of each dNTP, 1 × reaction buffer, 2.5 mM manganese acetate, and water. Samples shown were 500 U / ml placental RNase inhibitor (Gibco, Grand Island NY), RNase A / T ₁ (20 U / ml RNase A and 800 U / ml RNase T ₁ ) Or 80 U / ml of DNase I (Ambion, Austin TX). A master mixture was prepared and heated in a 90 ° C. heating block, then 24 μl was dispensed into a PCR tube containing 10,000 cells. The reaction was placed in a MJ-Research PTC-100 thermocycle set at 90 ° C. One microliter (2.5 U) of the enzyme rTth was added to the reaction, which was then immediately stirred and immediately returned to the thermocycling apparatus. A reverse transcription step was performed at 63.5 ° C. for 5 minutes, followed by a denaturation step at 92 ° C. for 60 seconds. The cDNA was then amplified by 35 cycles of a denaturation step at 92 ° C for 10 seconds and an annealing / extension step at 63.5 ° C for 20 seconds. Complete the thermocycler program with a final extension step at 72 ° C for 3 minutes and hold the reaction at 4 ° C until ready for agarose gel electrophoresis. Some samples were incubated prior to the above program for digestion with RNase or DNase. One-fifth to one-third of each reaction was run on a 3% NuSieve 3: 1 agarose gel (FMC, Rockland ME) in ethidium bromide-containing TBE buffer, and the bands were visualized by UV illumination. Southern blotting was performed using a PosiBlot apparatus (Stratagene, La Jolla CA) according to the manufacturer's instructions. Random primed probes were made using Stratagene's Random PrimeIt II procedure. Hybridization of the label probe to the membrane was performed using Amersham Rapid-Hyb solution, followed by high stringency washing in 0.1% SSC and 0.1% SDS at 65 ° C. The template used to generate the aggrecan probe was a plasmid having a sequence encoding the G3 domain of human aggrecan. The template used to generate the GAPDH probe is a plasmid having a sequence encoding human GAPDH. Example 2 After the general procedure shown above, analysis was performed on a given amount of purified total RNA from chondrocytes (top) and fibroblasts (bottom), as shown in FIGS. 2a and 2b. The aggrecan band is 280 bp and the GAPDH band is 146 bp. The lower band is about 70 bp and is the result of primer-primer interaction. FIG. 2a shows an agarose gel stained with ethidium bromide, and FIG. 2b shows a Southern blot of a gel hybridized with human aggrecan and a GAPDH probe. Example 3 20 ng of purified total RNA from chondrocytes and fibroblasts was rTth or primer in 1 × RT-PCR buffer in the presence or absence of RNase A / T ₁ (20 U / 800 U per ml) Digested at 45 ° C. for 135 min. After digestion with RNase, aggrecan, GAPDH primers and rTth enzyme were added and the RT-PCR thermocycling program started. In FIG. 4, bands corresponding to the amplified aggrecan (280 bp), GAPDH (146 bp), and primer-primer interaction (about 70 bp) are visible on an agarose gel stained with ethidium bromide. Example 4 100 ng of purified total RNA from chondrocytes and fibroblasts in 1 × RT-PCR buffer without rTth or primers without enzyme (lanes 1 and 2), RNase A / T ₁ (per ml) 20 U / 800 U) (lanes 3 and 4), DNAse I (80 U / ml) (lanes 5 and 6), or both (lanes 7 and 8) were digested at 37 ° C. for 50 minutes. After digestion, the reaction was heated at 90 ° C. for 2 minutes. Aggrecan, GAPDH primer and rTth enzyme were added and the thermocycling program started. In FIG. 5, bands corresponding to amplified aggrecan (280 bp), GAPDH (146 bp), and primer-primer interaction (about 70 bp) are visible on an agarose gel stained with ethidium bromide. Example 5 400 ng of purified total RNA from chondrocytes and fibroblasts was incubated at 37 ° C. with RNase A / T ₁ (20 U / 800 U per ml) without rTth or primers in 1 × RT-PCR buffer. Digested for minutes. After digestion, aggrecan and GAPDH primers were added, the reaction was heated to 90 ° C., the rTth enzyme was added, and the thermocycling program started. The reaction solution sample was run on an agarose gel, blotted on a charged nylon membrane, and hybridized with a human GAPDH probe. FIG. 6 shows the amplified chromosomal DNA (250 bp) and a band (146 bp) corresponding to the cDNA. Example 6 20,000 healthy chondrocytes and fibroblasts pelleted in a PCR tube and resuspended in 1 × RT-PCR buffer without rTth or primers were treated with RNase A / T ₁ ( Digestion at 37 ° C. for 5 minutes at 20 U / 800 U per ml) or 40 U / ml RNase inhibitor was added. Separate reactions containing only the aggrecan primer or only the GAPDH primer were performed. The reaction was heated to 90 ° C., the rTth enzyme was added, and the thermocycler program was started. In FIG. 7, bands corresponding to aggrecan (280 bp), GAPDH (146 bp), and primer-primer interaction (about 70 bp) are visible on an agarose gel stained with ethidium bromide. Example 7 Serial dilutions of healthy chondrocytes and fibroblasts in phosphate buffered saline were pelleted at the bottom of a PCR tube and the phosphate buffered saline supernatant was removed. A reaction mixture containing aggrecan, GAPDH primers, and rTth enzyme, preheated to 90 ° C., was added to the cell pellet and the thermocycling program started. In FIG. 8, bands corresponding to aggrecan (280 bp), GAPDH (146 bp), and primer-primer interaction (about 70 bp) are visible on an agarose gel stained with ethidium bromide. Example 8 Analysis was performed as in Example 7, with serial dilutions of healthy chondrocytes and fibroblasts. Reaction samples were run on agarose gels, blotted onto a charged nylon membrane, and hybridized with human aggrecan and GAPDH probes. FIG. 9 shows bands corresponding to aggrecan (280 bp), GAPDH (146 bp), and primer-primer interaction (about 70 bp). Example 9 10,000 healthy chondrocytes were pelleted in a PCR tube, RNase A / T ₁ (20 U / 800 U per ml), or 1 × RT-PCR buffer containing 40 U / ml RNase inhibitor Resuspended in liquid (containing aggrecan and GAPDH primers). Samples were immediately placed at 90 ° C or digested at 37 ° C for 5 minutes before heating to 90 ° C. The enzyme rTth was added and the thermal cycler program started. In FIG. 10, bands corresponding to aggrecan (280 bp) and GAPDH (146 bp) are visible on an agarose gel stained with ethidium bromide.

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Claims (1)

[Claims]   1. Whether the target cell is present in the cell population, consisting of the following steps a) to d) How to determine if:     a) Identify a marker for the target cell and identify the mRNA encoding the marker.   Make primers specific to     b) RT-PCR reaction solution, cell lysing agent, primer made in step (a), and target   Prepare a reaction mixture of cells and mix at a temperature sufficient to prevent mRNA degradation.   Maintain the compound,     c) Use the primers in RT-PCR to create specifically amplified DNA.   Amplifying the mRNA, and     d) The presence of the specific amplified DNA as an indicator of the presence or absence of the target cells in the cell population   Detect presence / absence.   2. 2. The method of claim 1, wherein the cell population contains one or more types of cells.   3. (B) maintaining the temperature in the range of about 60 to 90 ° C. The method of claim 1.   4. Target cells are chondrocytes, keratinocytes, cells containing viral RNA, Cells containing bacterial RNA, mRNA isoforms containing deletion or insertion mutations, or transcribed in cancer 2. The method according to claim 1, wherein the method is selected from cells expressing an RNA.   5. The target cell is a chondrocyte or a keratinocyte, 4 ways.   6. 6. The method according to claim 5, wherein the target cell is a chondrocyte.   7. 2. The method of claim 1, wherein the marker is aggrecan or keratin. .   8. 8. The method according to claim 7, wherein the marker is aggrecan.   9. Wherein the primer has the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Item 8.   Ten. The method of claim 1, wherein the target cells are obtained from a biopsy performed in a human patient. Method.   11． 11. The method of claim 10, wherein the biopsy contains human cartilage tissue.   12． 12. The method according to claim 11, wherein the target cells are human chondrocytes.   13. Claims wherein the number of target cells in the cell population is less than about 1.000 Item 1.   14. The method of claim 1, wherein the number of target cells in the cell population is less than about 100. 3 ways.   15． The method according to claim 1, wherein the reaction mixture also contains an RNase inhibitor. Law.