WO1997040189A1

WO1997040189A1 - HIGH VOLUME IN-SITU mRNA HYBRIDIZATION METHOD FOR THE QUANTIFICATION AND DISCOVERY OF DISEASE SPECIFIC GENES

Info

Publication number: WO1997040189A1
Application number: PCT/US1997/004710
Authority: WO
Inventors: David A. Jones; Douglas W. Harris; Michael K. Kenrick; Nicholas Thomas
Original assignee: Pharmacia & Upjohn Company; Nycomed Amersham Plc
Priority date: 1996-04-19
Filing date: 1997-04-03
Publication date: 1997-10-30
Also published as: JP2001509662A; EP0900286A1; CA2250105A1; AU2542497A

Abstract

A high volume substrate-based in-situ hybridization assay has been developed, utilizing scintillant containing microplates. The technique has the sensitivity to reliably detect specific mRNA transcripts at the level of 10-20 copies per cell. High specific activity antisense riboprobes specific for c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as a non-homologous vector derived control probe were used to compare mRNA levels in quiesced rat A-10 smooth muscle cells after stimulation with fetal calf serum (FCS) or platelet-derived growth factor (PDGF). Maximal c-fos induction was shown to occur following stimulation of with 30 ng/ml of PDGF or 10 % FCS, corresponding to a signal from the c-fos probe of 700 cpm. The non-homologous control background of 50 cpm and the GAPDH signals of 1700 cpm were independent of stimulation with PDGF or serum. Using PDGF, at 30 ng/ml, quiesced cells were stimulated at various times to provide an induction time-course for c-fos mRNA which peaked at 30 minutes and decreased to less than 50 % of this by 3 hours; a return to background level expression was apparent after 6 hours. Comparison with parallel northern blotting experiment showed this in-situ assay to be at least a 20-fold more sensitive and much more rapid to perform.

Description

HIGH VOLUME IN-SITU mRNA HYBRIDIZATION METHOD FOR THE QUANTIFICATION AND DISCOVERY OF DISEASE SPECD7IC GENES

BACKGROUND OF THE INVENTION 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.

Convenient methodologies, such as, differential display, subtractive cloning libraries, and microarray analysis are rapidly expanding the list of potential disease causing genes. Combined with the growing databases of expressed sequence tags, these technologies are defining numerous, molecular handles for understanding the basic cellular biology and the genesis of human diseases. The development of high throughput techniques for analyzing numerous genes, however, lags behind our abilities to identify those genes. In many cases, the first step in determining the role of novel transcripts in specific disease processes will be limited to correlating transcript levels with the disease phenotype of relevant cellular models.

Conventional analysis of gene expression generally centers around the use of northern blots, slot and dot blots, RNase protection assays and RT-PCR. Each of these techniques share cumbersome protocols for examining changes in mRNA expression levels and suffer from lack of quantification, poor sensitivity, and inadequate reproducibility. Further, each of these approaches requires isolating and manipulating the target mRNA pool prior to analysis. These factors severely limit sample throughput, increase analysis time and decrease reliability. In-situ mRNA hybridization on histological and cytological samples is a valuable tool for studying mRNA expression and discerning the role of specific genes in normal and diseased states. Current applications of in-situ hybridization protocols, however, suffer from technical drawbacks that limit its efficiency in examining numerous genes. Many of these technical drawbacks have been addressed by U.S. Patent 4,888,278 issued to Singer et al., the teachings of which are herein incorporated by reference.

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.

One drawback of the '278 patent is that it is limited to characterizing the expression profiles only of known genes. The demand for analysis of numerous unknown sequences, such as those being identified in various gene identification projects (e.g., the Human Genome Project, EST Databases, various food crop sequencing efforts, and human or animal pathogens) noted above, however, exceeds the capacity of the teachings of the '278 patent. Therefore, there remains a continuing need for the development of a methodology for the rapid and high volume characterization of genes of both known and unknown sequences.

SUMMARY OF THE INVENTION 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. In addition, 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. Specifically, 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. Specifically, 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. In a preferred embodiment, the method of the present invention utilizes a multiwell plate substrate for high volume sample throughput. Alternatively, 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.

To facilitate the entry of the nucleic acid probes into the cells, a membrane pore-forming agent can be added to the cells following the contacting step. When the goal is to identify potential therapeutics, the cells are treated during the culturing step with at least one chemical entity.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates specific hybridization of a GAPDH₃₁₆ 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 GAPDH₃₁₆ 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; and

Fig. 8 illustrates the utility of m-situ quantification in identifying agents that modulate gene expression.

DETAILED DESCRIPTION OF THE INVENTION In-situ mRNA hybridization on histological and cytological samples is a valuable tool for studying mRNA and discerning the role of specific genes in various growth conditions or disease states. It suffers, however, from drawbacks that limit its usefulness, in addition to being technically challenging for casual users. In view of the limitation on current mRNA analysis techniques, we have developed a high throughput m-situ nucleotide hybridization method for cultured cells. We have utilized this system to examine c-fos induction in A-10 smooth muscle cells after stimulation with PDGF. The general methodology is widely applicable and will allow quick and reliable quantitation of specific mRNAs, of either known or unknown sequence, in numerous cell-based systems. In addition, 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.

The novel and unique high volume in-situ hybridization method described herein allows the use of 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). Thus, for cells (or tissues) expressing a particular gene, the products of that gene and the RNA responsible for the making of the protein or polypeptide which the gene encodes can be quantified. Similarly, for cells and tissues infected by a virus, 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. In addition, 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. Accordingly, it is presumed that the reader is familiar with the applications and limits of the various techniques and will recognize that minor changes in reagents, concentrations, temperature, reaction times, and similar alterations of known methods are merely obvious variations of choice. Any major differences from the published and accepted techniques will be identified and described in detail as necessary. However, no description or repetition of the many compositions, protocols, and manipulative techniques will be given here for these well known procedures. For a complete description and recitation of the protocols and for more detailed information regarding recombmant DNA techniques and expression vectors, the reader is directed to the following publications: Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Davis et al., Advanced Bacterial Genetics, Cold Spring Harbor Laboratory (1982); Primrose and Dimmock, Introduction To Modern Virology, Second Ed. (1980); and Old and Primrose, Principles Of Gene Manipulation (1980), the texts of which are expressly incorporated herein by reference.

In addition it is presumed and understood that the terminology, technical and otherwise, used herein follows their usual, well understood meanings as they are used and applied in common parlance or the technical literature. For these reasons, only if a term has not been used previously within the field generally or if an unusual definition is employed, then and then only will a formal definition be provided as part of the text. For this reason, technical terms such as nucleotide, nucleic acid, plasmid, genome, expression vector, transcription, translation, expression and similar technical terms are presumed to be commonly understood by the practitioner ordinarily skilled within this art.

To fully and properly appreciate the subject matter as a whole which comprises applicants' present invention, one must comprehend that 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-T™ scintillating microtiter plate are preferred. Cytostar-T™ 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. Following the techniques described herein, 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.

The invention described herein was made feasible by adoption of a novel experimental approach for rapidly and objectively quantifying results. Samples with equivalent numbers of cells are hybridized with labeled probes and results obtained immediately by counting radioactive decay in a scintillation counter or, alternatively, by detecting chromogenic enzyme reaction products.

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.

In developing the assay we utilized a rat A-10 cell model system and examined the transcriptional profile of GAPDH and c-fos in response to PDGF treatment. GAPDH transcript levels served as the "house keeping", control target because its expression is generally independent of cellular stimulation. In contrast, 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-T™ plates using a microtiter plate scintillation counter using a correction of 25% efficiency for measuring ³³P. Results presented here are consistent with previous characterization of GAPDH and c-fos in growth factor stimulated cells.

We obtained a signal of 600-700 cpm for the hybridization of c-fos probe under maximal condition for stimulation and 1300-1800 cpm for the hybridization of GAPDH probe. Based on the specific activities of these probes, this corresponds to an approximate gene expression level for c-fos of 80-100 copies per cell and GAPDH of 150-200 copies per cell. These values are in close agreement with values quoted for PDGF-stimulated expression levels in BALB/c 3T3 cells (refs). Statistically significant increases in c-fos expression levels were detectable above control cells at levels that corresponded to 20-30 transcripts/cell. This level of expression was readily detectable in the Cytostar-T™ assay format and indicates its utility in monitoring transcripts of low abundance. The sensitivity of the assay by comparison with parallel northern blotting experiments indicated that the Cytostar-T™ assay was at least 20-fold more sensitive (data not shown). In addition to being more sensitive than northern blotting techniques, the Cytostar-T™ 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. In the case of 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). MATERIALS AND METHODS Cell Culture

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. Because mRNA presents particular difficulties to the user because of its lability, its solubility and its dispersal throughout the cytoplasm of a single cell, the use of a simple and highly efficient method for the detection of cellular mRNA (and by implication cytoplasmic DNA sequences as well) via assessment of critical parameters, the use of an in-situ hybridization method as disclosed in the Singer et al. patent (US 4,888,278) is advised. It will be expressly understood and appreciated, however, that the novel methodology disclosed in the instant application is equally applicable to a wide variety of other systems, cells, and tissues for the quantification of specific nucleic acids of interest. On this basis, the described empirical experiments involving the rat smooth muscle A10 cells are merely illustrative and representative of the many applications in which the novel quantification method may be used advantageously.

The bulk of the work to determine the applicability of the methodology was done using rat smooth muscle A 10 cells. To determine the general applicability of the methodology to other cell types, however, work was also done using MCF7 cells and CHO cells. The novel in-situ quantification methodology was found to be equally useful and accurate for each of these cell types. Rat smooth muscle A-10 cells were obtained from American Type Culture

Collection and grown in high glucose Dulbecco's modified Eagles medium (GibcoBRL #11965-068) supplemented with 15% fetal calf serum (GibcoBRL 16000-028), IX antibiotic-antimycotic (GibcoBRL 15240-013) and ImM sodium pyruvate (GibcoBRL 11360-013). Cells which are described as having been serum starved were grown in identical media except 1% fetal calf serum was used. Cells were cultured in T-75 flasks at 37°C in a humidified atmosphere of 95% air/5% CO₂. The medium was changed at 3 day intervals. 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-T™ 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. However, 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. Such 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.

There are several generally used methods one can use to achieve permeabilization of the fixed cellular protein matrix. These methodologies include the use of protease, acid, detergents, and/or heat denaturization. Use of proteases may be problematic in view of the possibility their digestive action may affect the integrity of the fixed cellular protein matrix. In the instant application, 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.

Preparation of Probes 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. In addition to these preferred radionuclides, 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.

Within the model test system utilizing primary cultures of rat smooth muscle A10 cells, a [³³P] 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 0.5μg/μl 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/CHCl₃ extraction purified, ethanol precipitated, resuspended in water at a concentration of 0.5μg/μl 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/CHCl₃ extraction purified, ethanol precipitated, resuspended in water at a concentration of 0.5μg/μl 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 0.5μl water, lμl 10X transcription buffer, 0.5μl 20 mM DTT, 0.5μl ATP, 0.5μl CTP, 0.5μl GTP, 0.5μl RNase inhibitor, 4μl [³³P]UTP, lμl of 0.5μg/μl linearized vector and lμl 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 lμl of DNase 1 and continued incubation for 15 minutes. Riboprobes were purified by diluting the incubation mix to 50μl 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 ReadySafe™ (Beckman #270-453718-D). Probe specific activities were approximately >4 x IO⁹ cpm/μg. 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/m² 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.

Following elution, fragments were reamplified and subcloned into a PCR 2.1 plasmid (Invitrogen, San Diego, California). Candidate genes were analyzed by dideoxynucleotide sequencing to verify novelty by comparison with publicly available cDNA databases such as Genbank. Probes were labeled as described above for c-fos and used for in-situ hybridization.

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^β 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). In-Situ Hybridization

Permeabilization solution was aspirated and cells were covered with 50 μl of hybridization buffer containing about lxlO⁶ 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). Plates were sealed (Costar #3095) and incubated overnight at 50°C. Hybridization buffer and unhybridized probe were removed by aspiration and each well was washed twice with 250μl 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 lOOμl 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.

Residual RNase A was removed by a 10 minute incubation (with shaking) with an additional 250μl RNase buffer solution (see above) followed by aspiration. Non- homologous riboprobe was washed away using two washes with 250μl 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.

The following examples are offered by way of illustration and are not intended to limit the invention in any manner. In all examples, all percentages are by weight if for solids and by volume if for liquids, and all temperatures are in degrees Celsius unless otherwise noted.

EXAMPLE 1 Ribonuclease protection of a GAPDH316 probe hybridized to formaldehyde fixed rat A-10 vascular smooth muscle cells.

We first optimized the hybridization of a high specific-activity, antisense probe specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to A10 cells cultured in the Cytostar-T™ 96-well microtiter plates. The target GAPDH mRNA is present at high copy number in most cell types and frequently serves as a control for gene expression studies. Rat A10 vascular smooth muscle cells were cultured in 96- well Cytostar-T™ 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. 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 GAPDH₃₁₆ 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). These conditions correspond well with fixation and permeabilization protocols for other cell types fixed on glass coverslips. RNase A treatment substantially decreased the total signal obtained with GAPDH₃₁₆ as the probe and revealed specific binding at RNase A concentrations above 1.0 μg/ml (Fig. 1). Higher concentrations of RNase A failed to decrease the GAPDH₃₁₆ 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 GAPDH₃₁₆ 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-T™ 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 lxlO⁷ cpm of probe to each well in hybridization buffer. Fig. 2 illustrates a typical saturation curve for the binding of GAPDH₃₁₆ 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. 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. Probe amounts at 1x10^s cpm/well saturated the target GAPDH mRNA. This corresponds to a probe concentration of approximately 40 pM. In comparison, the non-homologous probe increased minimally, showing less that 300 cpm, even at lxlO⁷ cpm of probe added per well. This minimal background provided a greater than 25 fold separation between the GAPDH₃₁₆ probe and the non-homologous probe and confirmed specific hybridization of to GAPDH transcripts.

EXAMPLE 3 In-situ measurement of c-foβ induction in serum-deprived rat A-10 smooth muscle following addition of 10% fetal calf serum.

We next examined the ability of the assay to monitor changes in gene expression levels. To accomplish this, quiescent, rat A-10 smooth muscle cells were stimulated with 10% FCS. Following stimulation, induction of c-fos was measured using the in-situ hybridization protocol. 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.

We further characterized PDGF induced c-fos expression by examining the temporal profile of c-fos expression and its dependence on the concentration of 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. At maximal stimulation, c-fos levels increase 10X above that observed in control cells. GAPDH₃₁₆ and non-homologous probe hybridization remained unchanged during this time course and confirmed a specific effect on c-fos expression. In similar experiments, 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 5

Temporal transcript profile analysis of multiple genes.

The ability to analyze multiple transcripts under a variety of conditions will be critical to efficient characterization of disease causing genes. In view of this, we measured the temporal induction of five distinct transcripts in A10 cells responding to PDGF. Quiescent A 10 cells were stimulated with 30 ng PDGF/ml for 0 - 12 hrs. Following stimulation, cells were fixed and an in-situ hybridization assay performed using ribonucleotide probes specific for jun(A), c-fos, egr-1, c-myc and erb-B2. Fig. 6 illustrates the parallel analysis of each of these target mRNA. In each case, excess probe was removed by RNase digestion and stringency washes as described in materials and methods. Specific hybridization was determined by comparison of the non-homologous probe binding with that of c-fos. Results were obtained in triplicate and are presented as mean ± S.E. The non-homologous control showed little hybridization in control or PDGF stimulated cells. A rapid induction of c-fos was accompanied by upregulation of egrl. Both transcripts were present at basal levels in control cells and increased 20-30 fold following addition of PDGF. The temporal profile of egr-1 induction was delayed with respect to c-fos with maximal induction seen at 45 min. Probes specific for jun(A) and erbB2 hybridized at levels significantly above background but remained comparable to unstimulated cells throughout the experiment. A single gene, c-myc, was undetectable in control and PDGF stimulated cells during the time-course analyzed.

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. We therefore evaluated the effects of a specific PDGF receptor tyrosine kinase inhibitor (tyrphostin A9) on the temporal profile of c-fos gene expression in A10 cells responding to PDGF. Quiescent A10 cells were preincubated overnight with 0 - 1 μM tyrphostin A9. Cells were stimulated with 30 ng PDGF/ml for 30 minutes in the presence of 10 μg/ml of cyclohexamide. Following stimulation, cells were fixed and an in-situ hybridization assay performed using ribonucleotide probes specific for GAPDH and c-fos. 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.lμM tyrphostin and saturated by 1.0 μM. 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

Temporal transcript profile analysis of multiple, unknown genes.

The ability to analyze multiple transcripts resulting from multiple unknown genes will be critical to the efficient characterization of disease causing genes. In view of this, we measured the induction of ten previously undescribed gene transcripts in MCF7 cells responding to UV radiation. MCF7 cells were stimulated with 50 joules/m² 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.

UV treatment induced mRNA for p21 at both three and six hours while 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.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The components, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary, and are not intended as limitations on the scope of the present invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims.

Claims

CLAIMS WE CLAIM:

-1- A method for quantifying the amount of a target nucleic acid sequence in morphologically intact cells, said method comprising the steps of:

A. culturing not less than two physically distinct samples of cells on at least one substrate;

B. contacting said cells with a fixative which preserves and retains the nucleic acids within the cellular morphology of said cells; C. exposing said fixed cells to a labeled nucleic acid probe under conditions whereby said labeled probe penetrates the morphologically intact cells and hybridizes to the target nucleic acid sequence; and

D. measuring the amount of said probe hybridized to said target nucleic acid sequence. -2-

The method of claim 1, further comprising the step of, prior to Step C: adding to said cells a membrane pore-forming agent.

-3- The method of claim 2 wherein said pore-forming agent is a detergent selected from the group consisting of polyoxyethylene 23 lauryl ether (Brij 35); polyoxyethylene 20 cetyl ether (Brij 58); sodium dodecyl sulfate; 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (CHAPS TM ) and polyoxyethylene ether CAS# 9002-93-1 (Triton X-100).

-4- The method of claim 1, wherein Step A further comprises: treating said cells with at least one chemical entity.

-5- The method of claim 1 wherein said probe is derived from uncharacterized gene sequences. -6-

The method of claim 1 wherein said substrate is a multiwell plate.

-7- The method of claim 6 wherein said plate contains a scintillant.

-8- The method of claim 1 wherein said substrate is particulate.

-9- The method of claim 8 wherein said particulate contains a scintillant.

-10- The method of claim 1 wherein the amount of said probe hybridized to said target nucleic acid sequence is measured by a spectroscopic technique. -11-

The method of claim 10 wherein the spectroscopic technique is selected from the group consisting of scintillation, fluorescence, ultraviolet, visible and luminescent.

-12- A method for quantifying the amount of a target nucleic acid sequence in morphologically intact cells, said method comprising the steps of:

A. culturing not less than one physically distinct sample of cells on a multiwell plate;

B. contacting said cells with a fixative which preserves and retains the nucleic acids within the cellular morphology of said cells;

C. exposing said fixed cells to a labeled nucleic acid probe under conditions whereby said labeled probe penetrates the morphologically intact cells and hybridizes to the target nucleic acid sequence; and

D. measuring the amount of said probe hybridized to said target nucleic acid sequence.

-13- The method of claim 12, further comprising the step of, prior to Step C: adding to said cells a membrane pore-forming agent.

-14- The method of claim 3 wherein said pore-forming agent is a detergent selected from the group consisting of polyoxyethylene 23 lauryl ether (Brij 35); polyoxyethylene 20 cetyl ether (Brij 58); sodium dodecyl sulfate; 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (CHAPS TM ) and polyoxyethylene ether CAS# 9002-93-1 (Triton X-100). -15-

The method of claim 12, wherein Step A further comprises: treating said cells with at least one chemical entity. -16- The method of claim 12 wherein said probe is derived from uncharacterized gene sequences.

-17- The method of claim 12 wherein said plate contains a scintillant.