WO1997040189A1 - Methode d'hybridation in situ d'arnm en grande quantite en vue de la quantification et de la decouverte de genes specifiques de certaines maladies - Google Patents

Methode d'hybridation in situ d'arnm en grande quantite en vue de la quantification et de la decouverte de genes specifiques de certaines maladies Download PDF

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WO1997040189A1
WO1997040189A1 PCT/US1997/004710 US9704710W WO9740189A1 WO 1997040189 A1 WO1997040189 A1 WO 1997040189A1 US 9704710 W US9704710 W US 9704710W WO 9740189 A1 WO9740189 A1 WO 9740189A1
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cells
probe
nucleic acid
fos
pdgf
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PCT/US1997/004710
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English (en)
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David A. Jones
Douglas W. Harris
Michael K. Kenrick
Nicholas Thomas
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Pharmacia & Upjohn Company
Nycomed Amersham Plc
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Priority to JP53806597A priority Critical patent/JP2001509662A/ja
Priority to AU25424/97A priority patent/AU2542497A/en
Priority to EP97916941A priority patent/EP0900286A1/fr
Publication of WO1997040189A1 publication Critical patent/WO1997040189A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • the present invention is concerned generally with improved methodologies for hybridization of nucleic acids within cells and tissues and methods for detection of such hybridization, and is specifically directed to improved in-situ hybridization methods and detection techniques for quantitative identification and analysis of specific nucleic acid sequences within whole cells and/or tissues.
  • the '278 patent reports an in-situ hybridization methodology and detection methodology having a simplified, efficient protocol that is rapid, sensitive, and reproducible such that a relatively unskilled person can perform it and, yet, remains non-destructive to cellular nucleic acids and cell morphology. Specifically, the '278 patent reports a rapid method for in-situ hybridization and several rapid methods for detecting a specific nucleic acid of interest in one or more test samples.
  • the in- situ hybridization method comprises the steps of obtaining at least one sample containing tissues or cells; fixing the sample using a fixative which preferably preserves and retains the nucleic acids of the cellular matrix such that the sample remains substantially in a condition for probe penetration; preferably avoiding proteolytic pretreatment of the sample; preparing a hybridization fluid comprising a labeled probe having at least one predetermined nucleotide sequence and an identifying label, this labeled probe ranging from about 20-4000 nucleotides in size; and combining the hybridization fluid with the fixed sample for not substantially less than 10 minutes and not substantially more than 24 hours.
  • the reported method for detecting a specific nucleic acid of interest within a sample comprises the steps of: obtaining at least one sample containing cells or tissues whose cellular nucleic acids have been hybridized in-situ using a radiolabeled probe comprising a predetermined nucleotide sequence and a radionuclide; and detecting the amount of radiolabel retained within the sample by radiation counting, the amount of radiolabeled probe retained by the sample being a measure of the nucleic acids of interest present within the sample which are substantially similar in composition to the predetermined nucleotide sequences of the probe.
  • the present invention provides a high throughput in-situ nucleotide hybridization method for cultured cells which allows quick and reliable quantification of targeted nucleotides in a variety cell-based systems.
  • the technique is suitable for the in-situ measurements of multiple transcripts and the effect of different treatments on transcript levels.
  • the information obtained through use of this method provides a critical understanding the molecular processes of disease.
  • the technique identifies potential disease causing genes that are candidates for therapeutic intervention.
  • the technique also can be employed to identify chemical entities which alter the expression of disease causing genes.
  • the present invention provides a method for quantifying the amount of a target nucleic acid sequence, such as mRNA, in morphologically intact cells.
  • the method involves the steps of: culturing not less than two physically distinct samples of cells on at least one substrate; contacting the cells with a fixative which preserves and retains the nucleic acids within the cellular morphology; exposing the fixed cells to a labeled nucleic acid probe under conditions whereby the labeled probe penetrates the morphologically intact cells and hybridizes to the target nucleic acid sequence; and measuring the amount of the probe hybridized to the target nucleic acid sequence.
  • the method of the present invention utilizes a multiwell plate substrate for high volume sample throughput.
  • the method of the present invention utilizes particulates such as beads as the substrate. In a more preferred embodiment, these substrates contain a scintillant.
  • the probes used in the method of the present invention can be derived from characterized or uncharacterized gene sequences. These probes must be labeled to allow detection and quantitation. The amount of probe hybridized to target nucleic acid sequences is measured by spectroscopic techniques such as scintillation, fluorescence, ultraviolet, visible and luminescent.
  • a membrane pore-forming agent can be added to the cells following the contacting step.
  • the cells are treated during the culturing step with at least one chemical entity.
  • Fig. 1 illustrates specific hybridization of a GAPDH 316 probe by protection from ribonuclease degradation in rat A-10 vascular smooth muscle cells;
  • Fig. 2 is a 96-well saturation curve illustrating saturation of GAPDH 316 hybridization sites in rat A-10 vascular smooth muscle cells
  • Fig. 3 illustrates in-situ quantification of c-fos mRNA induction in quiescent rat A-10 smooth muscle cells after treatment with 10% fetal calf serum
  • Fig. 4 illustrates time-dependent profiles of c-fos induction in A10 cells following exposure to platelet-derived growth factor
  • Fig. 5 illustrates concentration-dependent profiles of c-fos induction in A 10 cells following 90 minute stimulation by exposure to platelet-derived growth factor
  • Fig. 6 illustrates the time dependent profiles of multiple PDGF-inducible genes as quantified by in-situ hybridization
  • Fig. 7 illustrates the utility of in-situ quantification in measuring transcriptional profiles of unknown gene sequences
  • Fig. 8 illustrates the utility of m-situ quantification in identifying agents that modulate gene expression.
  • the technique is suitable for parallel examination of multiple transcripts and the effect of different treatments on the expression of those transcripts.
  • This parallel hybridization of multiple genes will allow identification and confirmation of novel transcripts critical to disease processes.
  • This technique may also be useful in screening chemical libraries for agents which induce or repress the expression of desirable or undesirable genes.
  • nucleic acid e.g., recombinant DNA, recombinant RNA, synthetic DNA or synthetic RNA
  • probes with cells, microorganisms, or tissues whose structures are compatible with microscopic or biochemical examination such as is routinely performed in medical research laboratories.
  • the present invention applies a nucleic acid probe, either of a predetermined nucleotide sequence or of an undetermined nucleotide sequence, to the sample cells (or tissues) and then evaluates or measures the quantity of probe hybridization to the sample cells (or tissues).
  • the products of that gene and the RNA responsible for the making of the protein or polypeptide which the gene encodes can be quantified.
  • the product of a viral infection, the viral RNA, or even the viral DNA itself can be quantitated within the infected cells or tissues.
  • Such protocols provide enormous amounts of useful diagnostic and/or scientific information because the presence or absence of the specific nucleic acid of interest, or the relative amount of the specific nucleic acid of interest, can be correlated, directly and indirectly, with one or more cells of observable structure and morphology and in this way provide a basis for determining the potential importance of particular candidate genes in the genesis or etiology of disease.
  • the methodology of the present invention provides a useful method for clinical diagnosis and/or prognosis. It is apparent even to the casual reader that the present invention as a whole is heavily dependent upon a thorough knowledge and understanding of recombinant DNA technology and its many applications within molecular biology and clinical/diagnostic situations.
  • the recombinant DNA techniques employed when making and using the present invention are well established and constitute recognized methods for the isolation of specific plasmids; for the use of restriction endonucleases; for ligation of DNA fragments in-vitro; for the preparation of predetermined nucleotides in sequence as hybridization probes; and for the various methods of labeling such DNA (or RNA) probes using a variety of labels such as radionuclides.
  • this invention represents a significant improvement and extension of in-situ hybridization by providing greater sample throughput and allows the quantification of genes of unknown sequence.
  • the improved in-situ hybridization methodology couples the ability of other methodologies, such as differential display, subtractive cloning libraries, and microarray analysis, to identify unknown genes with the ability of multiwell plates to allow high volume screening. Following the identification of a unknown gene, cultures of cells containing that gene are grown up in, e.g., 96-well microtiter plates and are then exposed to probes to these unknown genes made using standard techniques.
  • 96-well cell culture plates such as the Amersham Cytostar-TTM scintillating microtiter plate are preferred.
  • Cytostar-TTM microtiter plates have a clear polystyrene base plate into which a solid scintillant has been incorporated. Binding of a suitable radioligand to the base of the plate results in the generation of scintillation proximity counts.
  • the hybridized probe can be quantitated using various spectroscopic techniques. This technique allows the identification of changes in gene expression which provides one information as to the status of a gene in, e.g., a normal tissue state as compared to a disease tissue state.
  • the technique has the sensitivity to reliably detect specific mRNA transcripts at the level of 10-20 copies per cell.
  • One of the major advantages of this method is that identical cultures of cells can be grown in multiwell microtiter plates. Thus, each well can be tested in a different manner and each well potentially probed with different probes, or the identical probe, depending upon one's course of study. In this manner large numbers of probes as well as large numbers of treatments can be addressed at one time. Also the sequence of the probes need not be known in order to prepare and study changes in the hybridization of that probe which is a reflection of the mRNA changes that occur in a cell in response to different treatments.
  • the assay offers several attractive features that distinguish it from existing methodologies that include: (i) analysis of multiple experimental conditions or multiple transcripts in parallel, (ii) generation of an objective readout that allows statistical analysis on experimental data, (iii) target transcripts are fixed rather than isolated, thereby reducing manipulation artifacts, (iv) the high-volume format allows adaptation to automation and (v) sensitivity suitable for low abundance transcripts.
  • GAPDH transcript levels served as the "house keeping", control target because its expression is generally independent of cellular stimulation.
  • the c-fos gene is rapidly and dramatically induced in many cell types following stimulation with PDGF. Quantification of specifically hybridized riboprobes was performed in Cytostar-TTM plates using a microtiter plate scintillation counter using a correction of 25% efficiency for measuring 33 P. Results presented here are consistent with previous characterization of GAPDH and c-fos in growth factor stimulated cells.
  • the Cytostar-TTM assay does not require the long exposure times needed for autoradiography assays. Enhancement of sensitivity can be further achieved by using longer probe lengths with or without base hydrolysis into smaller fragments. Such probes provide a more complete coverage of the mRNA target transcript and thus provide increased label hybridized to each transcript.
  • c-fos a 2.5 fold increase in sensitivity was observed when hydrolyzed full length riboprobe (-1000 base pair) was used compared to the 236 base pair riboprobe. Unhydrolyzed full length c-fos probe gave even better sensitivity in some cases (data not shown).
  • the methodology as a whole is best described and most easily illustrated when used in the context of a single model test system.
  • the illustrative purpose of the hybridization methodology described hereinafter is to analyze the presence of a particular messenger RNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during growth factor stimulation of rat smooth muscle A10 cells.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • Rat smooth muscle A-10 cells were obtained from American Type Culture
  • Cells were passaged using 0.25% trypsin/EDTA (GibcoBRL 25200-056). Studies were conducted using cells (without regard for passage number) which were seeded into 96-well Amersham Cytostar-TTM scintillating microplates at 20,000 cells/well and grown until confluent.
  • Fixation and Permeabilization In the model test system, multiwell microtiter plates containing cultured cells are combined with a fixative, generally for about 10 minutes.
  • the purpose of fixing cells (and tissues sections) is to preserve the cells (or tissue section) in a morphologically stable state such that the mRNA is retained within the cellular matrix under the rigorous conditions present during in-situ hybridization.
  • overextensive insolubilization of the proteins by the fixative within the cellular matrix via cross-linking and/or precipitation renders the cytoplasm of the cell substantially impermeable to all but the smallest sized probes.
  • the preferred fixative is thus one which maintains and preserves the morphological integrity of the cellular matrix and of the nucleic acids within the cell as well as provides the most efficient degree of probe penetration.
  • the preferred method thus utilizes a fixative which is able to preserve and retain the nucleic acids of the cell and concomitantly restricts the cross-linking and/or precipitation (insolubilization) of the proteins in the cellular matrix such that the cell (or tissues) remain substantially in an open configuration for probe penetration and subsequent hybridization.
  • a fixative is paraformaldehyde, a solid formaldehyde polymer which can be solubilized by dissolving the solid as a 4% solution in phosphate buffered saline containing 5mM MgC12. While it has been noted that bottled formalin solutions can be unpredictable in their RNA retention properties, for the instant application, a particular formaldehyde available from Sigma Chemicals (St. Louis, Missouri) a 4% formaldehyde in phosphate buffered saline (Sigma HT-50-1-2), was found to satisfy our requirements.
  • the preferred method for permeabilization involves exposing the fixed cellular protein matrix to a solution containing 0.25% Triton X-100 in phosphate buffered saline (PBS) for 20 minutes at room temperature.
  • PBS phosphate buffered saline
  • the probes useful for the Methodology are labeled and can be of known or unknown nucleotide sequence.
  • the preferred identifying label is a radionuclide.
  • Other preferred labels for probes are fluorescent labels, enzyme labels, or haptens (e.g., biotin, digoxin, etc.).
  • the most preferred radionuclide label is ⁇ 33> P.
  • Other preferred radionuclide labels are ⁇ 32> P, ⁇ 125> I, ⁇ 35> S, ⁇ 14> C and ⁇ 3> H.
  • any of the other known and conventionally used radionuclides which can be chemically bonded or enzymatically incorporated into a nucleic acid fragment or nucleotide sequence using either the preferred method, RNA transcription, the nick-translation methodology or other suitable methodology may also be used without limitation.
  • the nucleotide sequence may be substantially similar to at least a portion of the nucleic acids normally present within the fixed cell or tissue or may be substantially similar to a specific nucleic acid of interest which is not normally present within the cell and is associated with an abnormal or pathological state.
  • the probe is a RNA (or DNA) fragment ranging in size from about 20-4000 nucleotides in size. Probe fragments about 4000 nucleotides in length are presently the largest sized probes believed capable of penetrating the cell (or tissue) for in-situ hybridization. Nevertheless, if larger sized probes can be prepared and utilized, these are deemed to be within the scope of the invention. These larger sized probes are advantageous because they are able to provide large increases in signal and allow detection of much smaller numbers of molecules within the cell.
  • a [ 33 P] labeled probe was prepared using a commercially available transcription kit (Ambion Maxiscript # 1326).
  • a pBlueScript II-SKX+) plasmid vector (Stratogene # 212205) was constructed containing a 236 base pair insert (into the Pstl restriction site) of rat c-fos (355 to 587 of Accession #X06769).
  • the vector containing c-fos insert was linearized using BamHl (GibcoBRL #15201-023), phenol/CHClg extraction purified, ethanol precipitated, resuspended in water at a concentration of 0.5 ⁇ g/ ⁇ l and transcribed with T7 RNA polymerase.
  • Another pBlueScript II-SK(+) plasmid vector was constructed containing a 316 base pair insert (between Sad and BamHl) of rat GAPDH (369 - 685 of Accession #X02231).
  • the vector containing GAPDH was linearized using BamHl, phenol/CHCl 3 extraction purified, ethanol precipitated, resuspended in water at a concentration of 0.5 ⁇ g/ ⁇ l and transcribed with T3 RNA polymerase (GibcoBRL18036-012).
  • a transcribable, non-homologous sequence was also prepared by restriction digestion of pBlueScript II-SK(+) with PvuII. This resulted in the excising of a fragment which contains the sequences between the T7 and T3 promoter regions of the vector and thus provided a linearized transcribable region with no sequence homology to eucaryotic DNA.
  • the cut BlueScript vector was phenol/CHCl 3 extraction purified, ethanol precipitated, resuspended in water at a concentration of 0.5 ⁇ g/ ⁇ l and transcribed with T7 RNA polymerase.
  • Linearized vector was transcribed in a 1.5 ml conical centrifuge tube (DOT Scientific #509-FTG) in a transcription mix consisting of 0.5 ⁇ l water, l ⁇ l 10X transcription buffer, 0.5 ⁇ l 20 mM DTT, 0.5 ⁇ l ATP, 0.5 ⁇ l CTP, 0.5 ⁇ l GTP, 0.5 ⁇ l RNase inhibitor, 4 ⁇ l [ 33 P]UTP, l ⁇ l of 0.5 ⁇ g/ ⁇ l linearized vector and l ⁇ l T3 or T7 RNA polymerase (according to the Ambion protocol).
  • the DNA was transcribed for 1 hour at 37°C and then digested (according to the Ambion protocol) with the addition of l ⁇ l of DNase 1 and continued incubation for 15 minutes.
  • Riboprobes were purified by diluting the incubation mix to 50 ⁇ l using lOmM Tris- HCl/lmM EDTA buffer and separating the remaining free nucleotide from incorporated nucleotide using a commercially available Sephadex G-50 spin column (Pharmacia Biotech #27-5335-01) and the protocol provided. A small sample of the purified probe was counted using 15 ml of ReadySafeTM (Beckman #270-453718-D). Probe specific activities were approximately >4 x IO 9 cpm/ ⁇ g. In general, probes in the range of 200 to 400 base pairs were chosen in an effort to maximize the penetration of riboprobe into the cell.
  • Probes to genes of undetermined sequence are amplified by differential display PCR according to the teachings of Welsh et al. (1992) Nucleic Acids Research 20(19):4965-4970. MCF7 cells were stimulated with 50 joules/m 2 of UV light and allowed to recover for 4 hrs. Total RNA isolated from UV treated cells or parallel untreated cells were subject to reverse transcription for generation of single- stranded cDNA. Arbitrarily chosen primer sets were used in polymerase chain reaction to amplify cDNA fragments from both control and UV treated cells in the presence of radiolabeled nucleotides. Products were resolved on a urea containing polyacrylamide gel and observed by autoradiography. Eight candidate fragments with differential intensity revealed by autoradiography were excised and eluted.
  • Hybridization Fluid The composition of the hybridization fluid is a mixture of components which optimize the conditions of in-situ hybridization. It will be understood that individual hybridization fluid mixtures are prepared comprising probes of known or unknown nucleotide sequence and the control probe respectively. The quantity of total probe used is a predetermined amount based upon a saturation binding curve determined for GAPDH (Fig. 2).
  • a preferred hybridization fluid comprises 1 X 10 ⁇ cpm of riboprobe, either known or unknown, or control probe, respectively, (prepared in the antisense orientation relative to the target mRNA as described above) in a solution of 10% dextran sulfate (Sigma #D-8906), 50% formamide (GibcoBRL #15515-018), 0.3M NaCI (Sigma #S-5150), lOmM Tris, pH 8 (Sigma #T-3038), ImM EDTA (Sigma #E- 7889), IX Denhardts (Sigma #D-2532), lOmM DTT (Sigma #D-9779), 0.5mg/ml yeast tRNA (Sigma #R-8508), lOmM vanadyl ribonucleoside complex (GibcoBRL #15522- 014).
  • Permeabilization solution was aspirated and cells were covered with 50 ⁇ l of hybridization buffer containing about lxlO 6 cpm riboprobe (prepared in the antisense orientation relative to the target mRNA as described above) in a solution of 10% dextran sulfate (Sigma #D-8906), 50% formamide (GibcoBRL #15515-018), 0.3M NaCI (Sigma #S-5150), lOmM Tris, pH 8 (Sigma #T-3038), ImM EDTA (Sigma #E-7889), IX Denhardts (Sigma #D-2532), lOmM DTT (Sigma #D-9779), 0.5mg/ml yeast tRNA (Sigma #R-8508), lOmM vanadyl ribonucleoside complex (GibcoBRL #15522-014).
  • Hybridization buffer and unhybridized probe were removed by aspiration and each well was washed twice with 250 ⁇ l IX SSC (GibcoBRL #15557-044) at room temperature with shaking at 150 rpm for thirty minutes.
  • Residual single stranded unhybridized riboprobe was digested by adding lOO ⁇ l 20 mg/ml RNase A (Sigma #R- 6513) (in a buffer consisting of lOmM Tris, pH 8.0 (Sigma #T-3038), 0.5 M NaCI (Sigma #S-5150) and ImM EDTA (Sigma #E-7889)) and incubating at room temperature for 30 minutes with shaking at 150rpm followed by aspiration.
  • RNase A Sigma #R- 6513
  • Residual RNase A was removed by a 10 minute incubation (with shaking) with an additional 250 ⁇ l RNase buffer solution (see above) followed by aspiration.
  • Non- homologous riboprobe was washed away using two washes with 250 ⁇ l 0.25X SSC and incubating the plates at 65°C for 45 minutes with shaking at 150 rpm followed by aspiration.
  • Probe which remains associated with the cells represents specifically hybridized probe and was counted directly using a Wallac MicroBeta plate counter.
  • GAPDH glyceraldehyde-3-phosphate dehydrogenase
  • Excess probe was removed by washing as described in materials and methods. Following the removal of bulk unbound probe, triplicate wells were incubated with 0 - 100 ug/ml RNase A for 30' at 50oC. Digested probe was removed with two stringency washes and remaining undigested probe determined by scintillation spectroscopy. Each condition was performed in triplicate and specific hybridization determined by subtracting the remaining non-homologous counts from counts obtained with the GAPDH probe. Results presented are mean ⁇ S.E. Maximal specific binding of GAPDH 316 was obtained following fixation with 4% formaldehyde for 10 min and permeabilization with 0.25% Triton X-100 in PBS for 20 minutes (data not shown).
  • RNase A treatment substantially decreased the total signal obtained with GAPDH 316 as the probe and revealed specific binding at RNase A concentrations above 1.0 ⁇ g/ml (Fig. 1). Higher concentrations of RNase A failed to decrease the GAPDH 316 or non-homologous probe binding further and assured complete digestion of excess ribonucleotide probe in each case.
  • the use of RNase Tl gave nearly identical results (data not shown). Stringency washes at 65°C with 0.25X SSC provided a maximal signal to noise ratio when comparing GAPDH 316 to the non-homologous probe.
  • EXAMPLE 2 Saturation of GAPDH316 binding in rat A-10 vascular smooth muscle cells.
  • Rat A10 vascular smooth muscle cells were cultured in 96-well Cytostar-TTM plates until confluent. Cells were fixed and incubated with hybridization solution containing a non-homologous 33P-labeled riboprobe or a 33P-labeled 316 bp antisense probe specific for rat GAPDH at concentration between 18000 to lxlO 7 cpm of probe to each well in hybridization buffer.
  • Fig. 2 illustrates a typical saturation curve for the binding of GAPDH 316 in A 10 cells using optimal hybridization and wash conditions. Excess probe was removed by RNase digestion and stringency washes described in materials and methods.
  • Fig. 3 demonstrates induction of c-fos in A 10 cultures responding to FCS. Levels of c-fos expression in unstimulated cells were similar to levels achieved with control, non-homologous probe. In contrast, treatment with FCS resulted in a 10-fold increase in c-fos expression over basal levels. Further inspection of Fig. 3 reveals that the signals obtained for the non- homologous control were independent of stimulation with FCS. Results presented are mean ⁇ S.E.
  • EXAMPLE 4 Time and concentration dependent induction of c-fos in A10 smooth muscle cells treated with PDGF.
  • Fig. 4 illustrates a typical time-dependent induction patterns of c-fos in response to 30 ng/ml PDGF stimulation.
  • Expression of c-fos mRNA was detectable within 15 minutes, maximal at 30 minutes and returned to background levels within 120 min following stimulation by PDGF.
  • c-fos levels increase 10X above that observed in control cells.
  • GAPDH 316 and non-homologous probe hybridization remained unchanged during this time course and confirmed a specific effect on c-fos expression.
  • we observed c-fos induction 90 minutes after stimulation with as little as 3 ng/ml PDGF with maximal induction occurring at 100 ng/ml PDGF Fig. 5).
  • EXAMPLE S Inhibition of immediate early gene expression.
  • Ligand binding by PDGF receptors like other growth factor receptor tyrosine kinases, induces receptor autophosphorylation on tyrosine residues within the receptor cytoplasmic domain. This receptor activation initiates a cascade of signaling events that ultimately converges on the transcription of immediate early genes.
  • tyrphostin A9 a specific PDGF receptor tyrosine kinase inhibitor
  • Quiescent A10 cells were preincubated overnight with 0 - 1 ⁇ M tyrphostin A9.
  • Fig. 8 illustrates a tyrphostin A9 inhibition curve which demonstrates a concentration-dependent blockade of c-fos gene expression in cells pretreated with tyrphostin A9. Inhibition of c-fos expression was evident with O.l ⁇ M tyrphostin and saturated by 1.0 ⁇ M. In contrast to c-fos, GAPDH mRNA levels remain unchanged at each concentration examined and eliminated cytotoxic effects as the mechanism for inhibition of c-fos. EXAMPLE 7
  • MCF7 cells were stimulated with 50 joules/m 2 and allowed to recover for three or six hours. Following stimulation, cells were fixed and an in-situ hybridization assay performed using ribonucleotide probes specific for GAPDH, p21 and the ten genes previously identified from a differential display analysis. These ten probes were isolated using the teachings of Welsh et al. (1992) Nucleic Acids Research 20(19):4965-4970. Fig. 7 illustrates the parallel analysis of each of these target transcripts. The non- homologous control showed little hybridization in either control or UV treated cells.
  • GADPH and the non-homologous probe remained constant.
  • Several of the unique target transcripts showed altered expression levels following UV treatment and verified the utility of the assay for quantifying and identifying uncharacterized potential disease causing genes.

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Abstract

Analyse d'hybridation in situ à partir d'un substrat volumineux, à l'aide de microplaques contenant du scintillant. La technique est suffisamment sensible pour qu'on puisse détecter des transcrits d'ARNm spécifiques au taux de 10-20 exemplaires par cellule. On utilise des ribosondes antisens à activité spécifique élevée spécialisées pour c-fos et la glycéraldéhyde-3-phosphate déshydrogénase (GAPDH) ainsi qu'une sonde de contrôle dérivée d'un vecteur non homologue pour comparer les taux d'ARNm dans des cellules de muscle lisse A-10 d'un rat mises en état de quiescence après stimulation au moyen du sérum embryonnaire de veau (FCS) ou de facteur de croissance d'origine plaquettaire (PDGF). Il a été montré qu'une induction c-fos maximale se produit après une stimulation de 30 ng/ml de PDGF ou de 10 % de FCS, ce qui correspond à un signal émis par la sonde c-fos de 700 cpm. Le milieu de contrôle non homologue de 50 cpm et les signaux GAPDH de 1700 cpm étaient indépendants de la stimulation par PDGF ou par sérum. Avec l'apport de PDGF, à 30 ng/ml, les cellules mises en état de quiescence étaient stimulées à différents moments pour donner un schéma temporel d'induction pour l'ARNm c-fos qui culminait à 30 minutes et s'abaissait à moins de 50 % de la valeur de crête au bout de 3 heures; un retour au même taux d'expression que le niveau du milieu a été observé au bout de 6 heures. La comparaison avec une analyse réalisée par transfert de Northern menée en parallèle a montré que la méthode d'analyse in situ décrite était au moins 20 fois plus sensible et également beaucoup plus rapide.
PCT/US1997/004710 1996-04-19 1997-04-03 Methode d'hybridation in situ d'arnm en grande quantite en vue de la quantification et de la decouverte de genes specifiques de certaines maladies WO1997040189A1 (fr)

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JP53806597A JP2001509662A (ja) 1996-04-19 1997-04-03 疾患特異的遺伝子の定量および発見のための高容量ｉｎ―ｓｉｔｕ ｍＲＮＡ ハイブリダイゼーション法
AU25424/97A AU2542497A (en) 1996-04-19 1997-04-03 High volume (in-situ) mrna hybridization method for the quantification and discovery of disease specific genes
EP97916941A EP0900286A1 (fr) 1996-04-19 1997-04-03 METHODE D'HYBRIDATION IN SITU D'ARNm EN GRANDE QUANTITE EN VUE DE LA QUANTIFICATION ET DE LA DECOUVERTE DE GENES SPECIFIQUES DE CERTAINES MALADIES

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* Cited by examiner, † Cited by third party
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WO2001088184A2 (fr) * 2000-05-18 2001-11-22 Centre National De La Recherche Scientifique Compositions et procedes pour analyses genetiques
WO2002097390A2 (fr) * 2001-06-01 2002-12-05 Biovitrum Ab Dosage par hybridation quantitative en vue de l'analyse d'acide nucleique
WO2008121927A1 (fr) 2007-03-30 2008-10-09 High Throughput Genomics, Inc. Mesure d'un analyte insoluble dans un échantillon
US8741564B2 (en) 2011-05-04 2014-06-03 Htg Molecular Diagnostics, Inc. Quantitative nuclease protection assay (QNPA) and sequencing (QNPS) improvements

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EP0357436A2 (fr) * 1988-08-31 1990-03-07 Research Development Foundation Essai d'hybridation "in situ" en une seule étape
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WO1992009704A1 (fr) * 1990-11-29 1992-06-11 Diagnostic Hybrids, Inc. Procede de detection et d'identification in situ de sequences d'acide nucleique
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US4888278A (en) * 1985-10-22 1989-12-19 University Of Massachusetts Medical Center In-situ hybridization to detect nucleic acid sequences in morphologically intact cells
EP0357436A2 (fr) * 1988-08-31 1990-03-07 Research Development Foundation Essai d'hybridation "in situ" en une seule étape
WO1990002204A1 (fr) * 1988-08-31 1990-03-08 Research Development Foundation METHODE MANUELLE D'HYBRIDATION $i(IN SITU)
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001088184A2 (fr) * 2000-05-18 2001-11-22 Centre National De La Recherche Scientifique Compositions et procedes pour analyses genetiques
EP1158057A1 (fr) * 2000-05-18 2001-11-28 Centre National De La Recherche Scientifique Compositions et procédés applicables à l'étude du dosage génétique
WO2001088184A3 (fr) * 2000-05-18 2002-03-07 Centre Nat Rech Scient Compositions et procedes pour analyses genetiques
WO2002097390A2 (fr) * 2001-06-01 2002-12-05 Biovitrum Ab Dosage par hybridation quantitative en vue de l'analyse d'acide nucleique
WO2002097390A3 (fr) * 2001-06-01 2003-10-30 Biovitrum Ab Dosage par hybridation quantitative en vue de l'analyse d'acide nucleique
WO2008121927A1 (fr) 2007-03-30 2008-10-09 High Throughput Genomics, Inc. Mesure d'un analyte insoluble dans un échantillon
JP2010523943A (ja) * 2007-03-30 2010-07-15 ハイ・スループット・ジェノミックス・インコーポレイテッド サンプル中の不溶性検出対象の測定法
AU2008232523B2 (en) * 2007-03-30 2013-05-16 Htg Molecular Diagnostics, Inc. Measurement of an insoluble analyte in a sample
US8741564B2 (en) 2011-05-04 2014-06-03 Htg Molecular Diagnostics, Inc. Quantitative nuclease protection assay (QNPA) and sequencing (QNPS) improvements

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CA2250105A1 (fr) 1997-10-30
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AU2542497A (en) 1997-11-12

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