WO2005075669A1

WO2005075669A1 - Reverse transcription based method for detecting gene expression in a cell

Info

Publication number: WO2005075669A1
Application number: PCT/EP2005/001102
Authority: WO
Inventors: Albert Becker
Original assignee: Universität Bonn
Priority date: 2004-02-09
Filing date: 2005-02-04
Publication date: 2005-08-18

Abstract

A method for detecting gene expression in a cell is provided. A test sample comprising at least one single cell and an oligonucleotide binding to RNA, corresponding to a gene of interest with an unspecific tail sequence are provided, cDNA is prepared from the messenger RNA of the cell, which is part of a test sample. Further, at least one single cell of the sample is labeled to identify the cell. Particular sequences for the tail sequence are given. The inventive subject further comprises a kit for screening a test sample in order to locate a pathological condition and methods for identifying pharmaceutically active substances, for diagnosing a pathological condition in a patient in vitro and for screening a compound library for the identification of a pharmaceutically active substance for the treatment of a neurological disorder.

Description

REVERSE TRANSCRIPTION BASED METHOD FOR DETECTING GENE EXPRESSION IN A CELL

Background of the invention

Field of the invention

A method for detecting gene expression in a cell is provided. At test sample comprising at least one single cell and an oligonucleotide binding to RNA, corresponding to a gene of interest, and comprising an unspecific tail sequence are provided, cDNA is prepared from the messenger RNA of the cell, which is part of a test sample. Further, at least one single cell of the sample is labeled to identify the cell. Particular sequences for the tail sequence are given, and the inventive subject further comprises a kit for screening a test sample in order to locate a pathological condition and methods for identifying pharmaceutically active substances, for diagnosing a pathological condition in a patient in vitro and for screening a compound library for the identification of a pharmaceutically active substance for the treatment of a neurological disorder.

Description of the related art

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude.

Phenotypic characterization of cells in conjunction with single cell mRNA analysis, which yields information regarding expression of multiple genes in individual neurons, facilitates a detailed and comprehensive view of neuronal cell biology. More specifically, the aRNA amplification method has provided an approach to analyze mRNA levels in single cells that have been phenotypically characterized on the basis of electrophysiology, morphology, and/or protein expression. In this way, relative mRNA abundances can be directly assayed from a well-defined population of neurons. The concept of expression profiling led to the development of robotics methods for arraying thousands of cDNAs on microarrays. These cDNA arrays can be screened with labeled aRNA or cDNA to generate a molecular fingerprint of a specific cell type, disease state, or therapeutic efficacy. A broad view of how gene expression is altered in single neurons affected by a particular disease process may provide clues to pathogenetic disease mechanisms or avenues for therapeutic interventions. The use of mRNA profiles to produce diagnostics and therapeutics is called transcript-aided drug design (TADD). When coupled with single cell resolution, TADD promises to be an important tool in diagnosis of disease states, as well as provides a blueprint on which to develop therapeutic strategies. For example, mRNA abundances in an individual diseased cell may increase, decrease, or remain constant, and thus it is possible that a pharmaceutical alone or in combination with other drugs may be specifically designed to restore mRNA abundances to a normal state. Alternatively, if functional protein levels parallel the mRNA level changes, then drugs targeting the function of the proteins translated from these altered mRNAs may prove to be therapeutic. One promise of such an approach is that information about mRNA abundances that are altered in a diseased cell may provide new therapeutic indications for existing drugs. For example, if the abundance of mRNA for the beta-adrenergic receptor is altered as shown by the microarrays for a particular disease, already available adrenergic receptor agonists or antagonists that had not previously been used in this particular disease paradigm may prove to be therapeutically efficacious. The expression profile of a given cell is a measure of the potential for protein expression. Proteins are generally the functional entities within cells and differences in protein function often result in disease. The ability to monitor the coordinate changes in gene expression, in single phenotypically identified cells, that correlate with disease will provide unique insight into the expressed genetic variability of cells and will likely furnish unforeseen insight into the underlying cellular mechanisms that produce disease etiology. To examine the molecular components of certain regions of the human body, e.g. a given brain region, the antisense RNA amplification procedure was combined with microarray technology. This experimental approach permits the simultaneous detection and quantification of numerous mRNAs in fixed tissue sections.

The isolation of single cells or cell clusters from complex tissue sections has become a widely applied strategy strongly, supported by the introduction of laser assisted microdissection technology. After laser microdissection of single cells, cell-specific mRNA analyses of few isolated cells and even expression profiles of single cells have become possible. Microscopic discrimination of different cell types in routinely stained tissue sections is limited, whereas immunostaining enables a significantly more specific access to cells of interest. As a general problem, the majority of antibodies require immunostaining protocols that interfere with mRNA recovery. Therefore, the use of immunostaining prior to laser microdissection and gene expression analysis by PCR/real time PCR strategies has been limited.

Crino and co-workers reported on the characterization of multiple mRNAs within fixed, immunohistochemically labeled cells, which according to the authors' understanding provides a powerful tool for studying gene expression and the molecular pathophysiology of many neurological diseases. They investigated tuberous sclerosis (TSC), which is characterized by the presence of highly epileptogenic dysplastic cerebral cortex (tubers) composed of abnormally shaped neurons and giant cells. Mutation of the TSC gene may disrupt differentiation and maturation of neuronal precursors, since the TSC2 gene product tuberin is believed to regulate cellular proliferation. To test the hypothesis that cells in tubers may retain the molecular phenotype of embryonic or immature neurons, tubers from five TSC patients were probed with antibodies to proteins expressed in neuronal precursors (nestin, Ki-67, and proliferating cell nuclear antigen). Many dysmorphic neurons and giant cells in tubers were stained by these antibodies, while neurons in adjacent normal and control cortex were not labeled. To further characterize the molecular phenotype of cells in tubers, the authors developed a methodology in which poly(A)⁺ mRNA was amplified from immunohistochemically labeled single cells in paraffin- embedded brain specimen (Crino et al, Neurobiology 93 (1996), 14152-14157). While immunohistochemical labeling with specific markers provides some information regarding the developmental phenotype of individual cells, tubers are histologically heterogeneous lesions containing a variety of abnormal cell types. Indeed, one issue critical to studying tubers and providing insight into their epileptogenicity is that not all cells within tubers may be abnormal. Today, identifying differences at the molecular level between immature and normal cells is difficult because homogenization of tuber samples for mRNA analysis will include all cell types (neurons, giant cells, and glial cells), thereby precluding any possible conclusions regarding the molecular composition of only immunohistochemically labeled giant cells or neurons. Furthermore, in situ hybridization (ISH) or the in situ polymerase chain reaction on tissue sections is time consuming, since only one or two probes can be used at a time.

Thus, Crino and co-workers devised a method to amplify and detect poly(A)⁺ mRNA from immunohistochemically labeled cells in tubers to directly assess the molecular pathology of single abnormal cells. These analyses were performed in fixed paraffin-embedded cortical tuber specimens. Specifically, poly(A)⁺ mRNA was amplified from individual nestin-labeled giant cells and dysmorphic neurons to identify mRNAs suggestive of cellular immaturity and to detect the TSC2 transcript. This approach permitted assessment of gene expression in a set of phenotypically defined individual cell types and the molecular pathophysiology of tuber formation (Crino et al., Neurobiology 93 (1996), 14152-14157).

However, this strategy is very elaborated and time consuming. In addition, the mRNA preservation after such fixation procedures is rather variable. No detectable results were obtained with transcripts longer than approximately 200 bp. This is probably due to unspecific strand breaks in the genomic DNA prior to cDNA production from mRNA.

Rapid protocols for immunohistochemistry and immunofluorescence with total incubation times of approximately 10 to 20 minutes or 25 to 40 minutes, and subsequent mRNA amplification without a preceding extraction step were suggested. Fink and co-workers aimed to complement microdissection techniques, which allow a cell-type or even cell- specific mRNA analysis within complex tissues and valid mRNA quantification by realtime reverse transcriptase-polymerase chain reaction from a few isolated cells by a cell- type specific immunostaining (Fink et al, Am. J. Pathol. 157 (2000), 1459-1466).

To evaluate its effect on mRNA quantitation, Fink and co-workers analyzed alveolar macrophages (AMs) from control rat lungs and those undergoing stimulation with lipopolysaccharide and interferon-γ nebulization. Whereas AMs from the left lung were directly harvested for mRNA extraction by bronchoalveolar lavage, tissue sections of the right lung were stained with an optimized immunofluorescence protocol detecting AMs. 15 AM profiles per sample were picked by laser-assisted sampling technique. Normalizing to a standard gene, nitric oxide synthase II (NOSH) and tumor necrosis factor (TNF)-α mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. In stimulated lungs, the percentage of picked samples positive for NOSII or TNF-α mRNA increased significantly. Moreover, a marked increase in the ratio of target gene mRNA to standard gene mRNA was noted for both NOSII and TNF-α in picked AMs from stimulated lungs, which matched very well the increase detected in the lavaged AMs undergoing direct RNA extraction.

However, the authors experienced serious problems. Because long-term immersion of tissue in aqueous media is deleterious for RNA preservation, short-term indirect immunofluorescence proved to provide the best mRNA efficiency rates. Using an antibody to stain AMs, a specific detection was obtained within 15 minutes. However, the additionally used anti-NOSII and anti-TNF-α antibodies did not detect their specific epitopes. Pretreatment with citrate buffer and microwave did indeed unmask these epitopes and allowed specific staining. This, however, was shown to be disadvantageous for nucleic acids and amplification. In consequence, the suitability and shortest possible incubation time for staining had to be tested for each primary antibody. Finally, the influence of the fluorochrome conjugated to the secondary antibody on the real-time PCR technology, based on fluorescence detection, must be taken into consideration with respect to unspecific background signals. In an additional publication, Fink and co-workers demonstrate that an mRNA amplification is only successful when a combination of short-term formalin fixation, reduction of antibody incubation time, application of immunofluorescence, and digestion of proteinase K is used (Fink et al, Lab. Invest. 80 (2000), 327-333).

Fink and co-workers further suggest to minimize the loss of RNA recovery by using formalin fixation of the tissue, a most possible reduction of incubation times, which is less than 20 minutes, and a mild digestion of proteinase K. However, the vast majority of antibodies require a significant longer incubation time than 10 to 20 minutes. Most primary antibodies indeed require at least over-night incubation and therefore, the protocols suggested by Fink and co-workers are not be appropriate for use in routine molecular pathological diagnostics.

In contrast to tissue samples which are fixed in formalin, fresh frozen tissue samples are in general the first bioptic tissue sample available after surgery. They are commonly regarded to offer better mRNA quality than fixed tissue samples. Tissue microdissection has become one of the key tools in molecular biomedicine and modern pathology. In combination with various downstream applications, this technique provides the possibility of cell-type or even cell-specific investigation of DNA, RNA, and proteins. For reliable mRNA analysis, in microdissected samples of complex tissues, several preconditions have to be fulfilled: 1) cells of interest have to be detected unambiguously. Thus, in many cases, an immunostaining procedure is inevitable required. 2) Microdissection and isolation of single cell profiles must be performed in a precise manner, without destruction of the relevant mRNA and without contamination by adjacent tissues. 3) Next to qualitative mRNA detection by reverse transcriptase-polymerase chain reaction (RT- PCR), a sensitive and reliable mRNA quantitation has to be established when transcriptional regulatory events are targeted with the number of mRNA copies increasing or decreasing. None of the protocols of the prior art fulfill these requirements sufficiently.

Therefore, there is a need for a fast, convenient and easy to handle protocol for gene expression analysis, which can be easily automized and provides the opportunity to be employed with virtually any primary antibody. Such protocol can find a wide use in molecular pathological diagnostics and molecular genetic research.

Brief summary of the invention

The unexpectedly developed novel method for detecting gene expression in a cell comprises the steps of

(i) providing a test sample comprising at least one single cell;

(ii) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest;

(iii) preparing cDNA from mRNA of said sample with said oligonucleotide; (iv) labeling at least one single cell of said sample to identify at least one specific cell.

In a further aspect, the invention provides a kit for screening a test sample for the presence of at least one cell indicating a pathological condition or a susceptibility to a pathological condition, comprising

(i) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest;

(ii) a labeling compound specific for a cellular marker indicating a pathological condition; and

(iii) cell permeabilizing and cell stabilizing agent.

Yet another aspect of the invention provides a method for identifying a pharmaceutically active substance for the prevention or treatment of a disease, comprising the steps of (i) providing a test sample comprising at least one single cell;

(ii) contacting a test substance with said sample;

(iii) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest;

(iv) preparing cDNA from mRNA of said sample with said oligonucleotide; (v) labeling at least one single cell of said sample to identify at least one specific cell;

(vi) isolating said identified specific cell from said sample; and (vii) detecting an increase or a decrease of at least one disease specific gene in said specific cell, wherein said increase or decrease qualifies said test substance as a pharmaceutically active substance.

Still another aspect of the invention is directed to a method for diagnosing in vitro a pathological condition in a patient comprising the steps of

(i) providing a test sample of said patient comprising at least one single cell;

(ii) providing an oligonucleotide binding to RNA, corresponding at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest;

(iii) preparing cDNA from mRNA of said sample with said oligonucleotide;

(iv) labeling at least one single cell of said sample to identify at least one specific cell; and

(v) detecting a pathological condition, wherein said detection of said pathological condition is based on the presence or absence or the amount of said mRNA in said test sample.

Yet another aspect of the invention provides for a method for screening a compound library for the identification of a pharmaceutically active substance for the treatment or the prevention of a neurological disorder, comprising the steps of

(i) providing a test sample comprising at least one single cell expressing at least one gene associated with a neurological disorder; (ii) contacting said sample with candidates of a compound library; (iii) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest;

(iv) preparing cDNA from mRNA of said sample with said oligonucleotide; (v) labeling at least one single cell of said sample to identify at least one specific cell expressing said gene associated with said neurological disorder; (vi) isolating said identified specific cell from said sample; and (vii) detecting an increase or a decrease of the expression of said gene associated with said neurological disorder in said specific cell, wherein said increase or decrease qualifies said candidates of said compound library as a pharmaceutically active substance.

Finally, the invention provides for a method for detecting gene expression in a cell, comprising the steps of

(i) providing a test sample comprising at least one single cell;

(ii) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2; (iii) preparing cDNA from mRNA of said sample with said oligonucleotide; (iv) incubating said sample with an antibody under conditions sufficient for the production of a detectable signal to identify at least one specific cell; (v) isolating said identified specific cell from said sample; and (vi) analyzing the expression profile of said specific cell.

It has been surprisingly found that by employing the inventive method for detecting gene expression in a cell virtually any primary antibody can be used. The incubation time for the antibody may be as long as required by the specific antibody to produce a detectable signal to identify at least one specific cell.

Brief description of the drawings

Fig. 1 shows amplification plots demonstrating real time PCR detection of GAPDH (A), GFAP (B) and NFM (C) in a tissue sample after in situ-RT and immunostaining using a short time incubation period (<1 h, open squares) versus a long time incubation period (> 24 h, dark triangles) for the primary antibody against CD34. The integrity of both, high (GAPDH) as well as low abundant transcripts (NFM) is conserved independent of the incubation time of the primary antibody.

Fig. 2 shows (A) laser microdissection of a single CD34-expressing cellular element in a ganglioglioma (black arrow, upper left corner, magnification: x40). By immunostaining, CD34-expressing cellular elements can be clearly distinguished from adjacent CD-34 negative cells (grey arrow, lower right corner). (B) The same slice after laser microdissection of the CD-34 positive cell. (C) PCR analysis for a variety of neuroepithelial lineage marker genes of laser microdissected CD34 positive cellular elements after initial in situ-RT. The co-expression of CD34 (lane 2) and neurofilament (lane 6) suggests a neuronal origin. No expression of glial fibrillary acid protein (lane 4) and myelin basic protein (lane 5) is observed (lane 1 - length standard, lane 3 GAPDH).

Detailed description of the invention

The present invention relates to a novel method for detecting gene expression in a cell. A test sample comprising at least one single cell and a oligonucleotide, which binds to RNA and corresponds to a specific gene of interest is combined with an unspecific tail sequence. With the help of this oligonucleotide, cDNA is prepared from mRNA of the cell, which is part of the test sample. Further, at least one single cell of the sample is specifically labeled to identify the cell.

The experiments performed by the inventor led to the surprising result that virtually any primary antibody for the labeling procedure can be employed and, most surprisingly, the incubation time for the antibody could be chosen according to the needs of the primary antibody. Even overnight incubation of the primary antibody according to immunostaining protocols known in the art was possible without interfering with the quality of recovered mRNA. This finding leads to the unexpected result that, contrary to the observations in the prior art, even a long term incubation with a primary antibody for immunostaining is possible. Such long incubation times are supposed to be deleterious for high quality mRNA recovery. However, according to the present invention, long incubation times can be performed without any negative effects especially in RNA recovery.

Without intending to be bound by theory, it is believed that the combination of an oligonucleotide which binds to RNA and corresponds to a gene of interest and an unspecific tail sequence leads to a strong fixation of the test sample on the support layer (e.g. an microscope or object slide), which in turn leads to the formation of physical bridges, either between individual the tail sequences and/or between tail sequences and the tissue sample on the support layer (e.g. object slide). Additionally, the formation of physical bridges between the tail sequences and the support layer directly is possible. It is believed that such bridges lead to the strong fixation of the tissue on the support layer, thus avoiding loss of tissue and RNA during hybridization of the oligonucleotide to its target transcript and further washing and recovery steps.

It is a particular advantage of the present invention that virtually any primary antibody can be employed in the invention for specifically labeling a particular cell since incubation times for the antibody may be chosen according to the requirements of the particular antibody.

It is of particular advantage that the present invention has numerous practical applications. First of all, the present invention can be used for a broad range of routine molecular pathology diagnostic applications. PCR based analysis of allelic losses on chromosomes lp and 19q in oligodendroglioma as a predictive parameter for chemotherapy response serves as an intriguing example. This analysis is frequently compromised by contaminating cellular elements of infiltrated normal brain tissue, since oligodendroglial cells do not exhibit the characteristic "honeycomb" appearance in fresh frozen tissue sections. Using the protocol of the present invention with an antibody against MAP2c, oligodendroglial cells can be immunostained in fresh frozen tissue sections after in situ- RT. A PCR based analysis of loss of chromosomes lp/19q can subsequently be performed in laser-microdissected and - due to immunolabeling - highly enriched oligodendroglioma cell elements.

Further, the present invention can be employed for research applications, i.e. expression analyses of individual cell types in complex tissue samples. For a variety of scientific questions, a cellular resolution of gene expression patterns in complex tissue samples requires prior immunostaining. The origin of CD34 expressing cellular elements in gangliogliomas constitutes an example for this application. Gangliogliomas represent highly differentiated glioneuronal tumors (Fink et al. Lab Invest. 80 (2000), 327-333). . The expression of CD34 in gangliogliomas suggested an origin from a developmentally compromised precursor lesion. However, it was not clear which cellular elements express CD34. Therefore, the present inventor has performed PCR analysis for a variety of neuroepithelial lineage marker genes of laser microdissected CD34 positive cellular elements after initial in situ-RT. It was surprising to observe a substantial expression of neurofilament but not other cell type specific genes in CD34 positive cells. This finding is depicted in Figure 2. These experimental data suggest a neuronal origin of the CD34 positive cellular components in gangliogliomas.

Additionally, a combination of the present protocol in concert with subsequent aRNA amplification is possible. The present protocol therefore also allows the combination with aRNA amplification strategies and subsequent genomic profiling using of polyT/T7 primers.

Thus, the present invention provides, in its broadest sense, a diagnostic method which allows that cells, which are specifically labeled e.g. by immunostaining, can be used for a qualitative and quantitative expression analysis. According to the inventive method, the degradation sensitive mRNA is transferred into stable cDNA. For this, either individual sequences may be transcribed or the whole cellular mRNA may be transcribed with the help of oligo-dT-primers. Particularly chosen tail sequences, which are unspecific for the gene of interest so that they do not bind to the particular mRNA, are used as elongation sequences for the gene specific oligonucleotide. With the help of these tail sequences, a transcribed cDNA is fixed to the target structures, i.e. the specific mRNA sequences. Scattering, diffusion or drifting away of particular target structures is efficiently prevented, since the tail sequences lead to bridges between each other and thereby hold the target structures in their places. It is ensured that the cDNA, transcribed from mRNA, may be analyzed quantitatively on its actual and specific location. After specifically labeling the desired cells e.g. by immunostaining with specific antibodies, a particular target cell may be isolated by any appropriate means, e.g. laser assisted microdissection, and the isolated cell can be analyzed according to its expression profile. It is a further advantage that shock frozen cryo-conserved tissues sections can be used for expression analyses. This is especially true if an oligo-dT-primer is combined with the specific tail sequence, so that whole cellular mRNA is transferred into its stable cDNA form.

Labeling by antibody staining (immunohistochemistry) is one of the major diagnostic routine methods. A huge number of different antibodies is commercially available, which may be incubated with tissue or cell material in order to determine particular cellular or to molecular characteristics.

Expression profiling and expression analysis is also already wildly used but it is estimated that especially expression profiling will gain further relevance in differential diagnostics. Expression analysis may be performed on the level of mRNA/cDNA or on the protein level. Expression analysis is particular relevant for diagnostics in which no suitable antibodies are available, which is often the case with membrane associated proteins or some intracellular proteins. Expression analysis is further most suitable for the determination of mutations, as the detection of mutations by antibodies is rather difficult. Expression analysis can also be used for sub-typing of pathogens and the detection of target molecules which are present in low concentration. Example for such target molecules are viruses and bacteria. Expression analysis can also be employed if routine antibody staining of two different molecules probably lead to cross reactivity of antibodies or an overlay, interaction or overlap of staining signals.

However, in routine expression analysis protocols problems in reproducibility and sensitivity are frequently reported. This is at least in part due to the inhomogenicity of the cell probes which are mainly composed of a mixture of normal and pathological cells.

Another major problem is the sensitivity and susceptibility of mRNA towards degradation.

In order to circumvent the above-stated problems, the present invention is directed towards a combination of specific labeling, which may be immunohistological staining and expression analysis. With the help of the present invention it is possible to analyze the expression profiles of cells, which are immunohistochemically exactly characterized. As many antibodies require incubation periods at least for several hours or overnight, such antibodies cannot be use to date in combination with expression profiling, as the incubation conditions for the antibody leads to the complete destruction and degradation of RNA. Therefore, methods described in literature aimed to shorten antibody incubation times, however, only a very limited set of antibodies is able to produce suitable signals after short incubation times like 10 or 20 minutes.

To the knowledge of the inventor the inventive concept of the present invention to combine expression analysis and data observed by immunohistochemistry by virtually employing any primary antibody has not been reported before.

As used herein, the term "tail sequence" refers to any nucleotide sequence, which is unspecific for a particular gene of interest. The tail sequence being "unspecific" means that the sequence does essentially not bind to the mRNA of the gene of interest. According to a preferred embodiment of the present invention, the tail sequence is an artificial sequence, which forms a free tail sequence.

In particular preferred embodiments, the tail sequence is G- or C-rich. The inventor has successfully employed tail sequences composed of G- or C-rich sequences. Particular preferred are the sequences of SEQ ID NO: 1 and/or SEQ ID NO: 2. However, all combinations of stretches of 3, 4, 5 or more G nucleotides and/or C nucleotides can be employed in all combinations with A and/or T nucleotides. In particular, the present invention shall not be limited to the use of sequences SEQ ID NO: 1 and/or SEQ ID NO: 2.

For the preparation of cDNA from RNA of a test sample with the help of the oligonucleotide comprising parts which correspond to a gene of interest and the unspecific tail sequence, it is particularly useful to use in situ reverse transcription (in situ RT).

For labeling of at least one single cell of the test sample, the sample can be incubated with an antibody under conditions sufficient for the production of a detectable signal. In particular, an antibody which is specific for a cellular marker can be used. The marker, which is preferably capable of distinguishing different cells, e.g. normal cells and tumor cells, may also indicate a pathological condition.

The term "pathological condition" as used herein, refers to a disease and/or the susceptibility to a disease which can either be treated or prevented.

In a further preferred embodiment of the present invention labeling of at least one single cells can also comprise any specific antibody, a single chain antibody, a tag like a histidin tag (HIS -tag) or specifically binding proteins and/or aptameres.

Further, receptor/ligand interactions can be used for recognition and labeling purposes.

The inventive method may further comprise the step of isolating the specifically labeled and identified cell from said sample. The isolated cell may later be further analyzed, by for example determining the expression profile of the cell.

In case an antibody is used for labeling a particular cell of interest, the antibody may any monoclonal antibody either commercially available for made to order from hybridoma cells, which is a well-known procedure in the art. Any primary monoclonal antibody specific for any gene of interest can be employed. For visualization of the binding of the antibody to its target structure a second antibody, which produces a detectable signal, is preferable. The secondary antibody may be enzyme labeled or fluorescence labeled, and the labeling may comprise alkaline phosphatase, peroxidase, fluorescein or the like.

It is particularly preferable to use Map2c, GFAP or an antibody specific or CD34. CD34 is an expression pattern in gangliogliomas, which represent highly differentiated glioneuronal tumors. Blϋmcke an co-workers performed an immunohistochemical study and examined epilepsy-associated lesions for CD34, a stem cell marker transiently expressed during early neurolation (Blϋmcke et al., Acta Neuropathol 97 (1999), 481- 490). Most examined tissue sample from patients with chronic epilepsy revealed neural cells immunoreactive for CD34. Prominent immunoreactivity was detected in gangliogliomas, low-great astrocytomas and oligodendrogliomas. The majority of CD34- immunoreactive cells co-localized with S-100 protein and a small subpopulation was also immunoreactive for neuronal antigens.

Thus, CD34 may represent a valuable marker for the diagnostic evaluation of neoplastic and/or malformative pathological chances in epilepsy patients. It is expected that CD34 immunoreactivity of these lesions indicates an origin from dysplastic or atypically differentiated neuronal precursors.

In a particular preferred embodiment of the present invention the pathological condition is a disease or a susceptibility to a disease and preferably, the pathological condition is a neurological disorder. The pathological condition may be selected from the group consisting of glioneuronal tumors, gangliogliomas, oligodendroglioma and astrocytoma. The pathological condition can also be epilepsy and patients with pharmacologically resistant forms of epilepsy like temporal lobe epilepsy (TLE) can also be employed.

The pathological condition can also be a viral infection, in particular mediated by oncogenic viruses like herpesviruses, papillomaviruses, retroviruses, hepadnaviruses or adenoviruses. In this case, the specific oligonucleotide is a virus specific primer. Viral nucleotide sequences can be specifically detected even in a mixture of cells or in tumors, which harbor viral sequences only in very specific regions of the tumor.

Major representatives of the family of herpesviruses are herpes simplex virus type I and type II calling gingivostomatitis (primary disease) and the recurrent forms, which are usually herpes labialis (fever blisters) and herpes genitalis (gential and anal lesions). The major transforming herpesvirus is the Epstein Barr virus, which primarily causes infectious mononucleosis, but which is strongly related to the tumor of Burkitt's lymphoma, a human cancer with a great incident in Central Africa and New Guinea.

Papillomaviruses are another group of human oncogenic viruses, which are connected with skin papillomas, skin cancer and cancerous lesions of the mucosa. More than 100 types of human papillomaviruses have been characterized, which are all highly similar in their genomic organization. Among this huge number of viruses, in particular HPN16 and 18 are linked to cervical cancer and cervical dysplasias. This is why especially types 16 and 18 are called high risk types of human papillomaviruses.

Hepadnaviruses, among which the hepatitis B virus is the most prominent representative, are also associated with human cancer and tumors, in particular liver cell carcinoma (primary hepatocellular carcinoma).

Adenoviruses, which show a high oncogenic potential in rodents (new born hamsters) also comprise B and JC viruses. Their oncogenic potential is not yet fully elucidated but both types of viruses are supposed to be associated with human cancer and tumor formation.

The present invention may be used to specifically detect viral DNA or RNA in lesions or tumors and the method of the present invention is particular advantageously if tumors are not solidly grown but are distributed diffuse in the tissue so that the tumor has no clear boundaries and is therefore difficult to analyze or remove by surgery.

In another preferred embodiment of the present invention the tumor expresses a multidrug resistance gene. In cancer treatment, a major problem to be overcome is the resistance of tumor cells to anti cancer drugs. An intensely studied type of cellular drug resistance is the multidrug resistance phenotype, which is characterized by a reduced intracellular drug level and an over expression of certain genes, like individual members of the ABC (ATP binding cassette) superfamily of membrane transporters, including members of P- glycoprotein encoded by the MDR1 gene and MRP (multidrug resistance associated protein). However, several atypical multidrug resistant tumor cell lines and tumors have been described that lack over expression of P-glycoprotein and MRP, but nevertheless have reduced intracellular drug levels, suggesting the presence of other drug transport mechanisms. As such other mechanisms are not elucidated yet, the present invention can be used to further elucidate molecular situations behind the well-known phenotype of multidrug resistance.

In a preferred embodiment of the present invention the labeling of at least one single cell and the identification of the associated detectable signal employs a process selected from the group consisting of immunohistochemistry (IHC), multi parameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, and automated cell analysis.

For the isolation step of the particularly labeled cell laser assisted microdissection can be used. For this, UN-laser microbeam technology based on a nitrogen laser with a wavelength of 337 nm (PALM, Bernried, Germany) can be used.

The analysis of the expression profile of the identified specific cell is preferably done by amplification of nucleic acids of said specific cell by polymerase chain reaction (PCR). The PCR may also be a quantitative PCR.

The test sample can either preferably be a cryosection of a snap frozen tissue sample or a section of a paraffin embedded tissue sample. In both cases, the unspecific tail sequence which forms part of the oligonucleotide specific for a gene of interest binds to parts of the tissue and leads to a fixation of the primer sequence to the tissue, thereby avoiding any replacements during analysis.

The test sample, in particular the tissue sample may be derived from a mammal, which in a particular preferred embodiment of the present invention is a human.

The subject matter of the present invention further comprises a method for detecting gene expression in a cell, comprising the steps of (i) providing a test sample comprising at least one single cell;

(ii) providing an oligonucleotide comprising a tail sequence, an oligo-dT sequence and a T7 polymerase recognition sequence;

(iii) preparing cDΝA form mRΝA of said sample with said oligonucleotide;

(iv) labeling at least one single cell of said sample to identify at least one specific cell. Again, the tail sequence is preferably SEQ ID NO: 1 or SEQ ID NO: 2. It is another object of the present invention to provide a kit for screening a test sample for the presence of at least one cell indicating a pathological condition or a susceptibility to a pathological condition comprising

(ii) a labeling compound specific for a cellular marker indicating a pathological condition; and (iv) cell permeabilizing and cell stabilizing agent.

The kit can also comprise tail sequences according to SEQ ID NO: 1 and/or SEQ ID NO: 2 and may further comprise at least one transcriptase, buffers and agents suitable for reverse transcription.

It is a further object of the present invention to be provide a method for identifying a pharmaceutically active substance for the prevention or treatment of a disease, comprising the steps of

(i) providing a test sample comprising at least one single cell;

(ii) contacting a test substance with said sample;

(iii) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising tail sequence unspecific for said gene of interest;

(iv) preparing cDNA from mRNA of said sample with said oligonucleotide;

(v) labeling at least one single cell of said sample to identify at least one specific cell;

(vi) isolating said identified specific cell from said sample; and

(vii) detecting an increase or a decrease of at least one disease specific gene in said specific cell, wherein said increase or decrease qualifies said test substance as a pharmaceutically active substance.

The pharmaceutically active substance is preferably a drug candidate.

Further, the present invention provides a method of diagnosing in vitro a pathological condition in a patient comprising the steps of (i) providing a test sample of said patient comprising at least one single cell;

(ii) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest; (iii) preparing cDNA from mRNA of said sample with said oligonucleotide; (iv) labeling at least one single cell of said sample to identify at least one specific cell; and (v) detecting a pathological condition, wherein said detection of said pathological condition is based on the presence or absence or the amount of said mRNA in said test sample.

The invention is further directed to a method for screening a compound library for the identification of a pharmaceutically active substance for the treatment or the prevention of a neurological disorder, comprising the steps of

(i) providing a test sample comprising at least one single cell expressing at least one gene associated with a neurological disorder;

(ii) contacting said sample with candidates of a compound library;

(iii) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest; (iv) preparing cDNA from mRNA of said sample with said oligonucleotide; (v) labeling at least one single cell of said sample to identify at least one specific cell expressing said gene associated with said neurological disorder; (vi) isolating said identified specific cell from said sample; and (vii) detecting an increase or a decrease of the expression of said gene associated with said neurological disorder in said specific cell, wherein said increase or decrease qualifies said candidates of said compound library as a pharmaceutically active substance.

In a preferred embodiment, the method for screening a compound library involves high throughput screening (HTS).

Finally, the present invention is directed to a method for detecting gene expression in a cell, comprising the steps of (i) providing a test sample comprising at least one single cell;

The above disclosure generally describes the present invention. Several documents are cited throughout the text of this specification. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The content of all cited references are hereby expressly incorporated by reference; however, there is no admission that any documents cited is in deed prior art to the present invention.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purpose of illustration only and are not intended to limit the scope of the invention, which is described in the attached claims.

Examples

The following examples will further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed description of conventional methods, such as those employed herein can be found in the cited literature; see also "The Merck Manual Of Diagnosis And Therapy" 17^th ed. by Beers and Birkow (Merck & Co., Inc. 2003).

The practice of the present invention will employ unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA technology and immunology, which are all within the skill of the art. Methods of more molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual (Sambrook et al., (1987) Molecular Cloning: A Laboratory Manual 2^nd ed., Cold Spring Harbor Laboratory Press) and Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3^rd ed. (Ausubel et al).

Detailed Description of the Experiments

The protocol is composed of four subsequent steps, i.e. in situ reverse transcription (RT), immunostaining, laser assisted microdissection, and PCR or real time RT-PCR. The procedures up to the in situ reverse transcription should be carried out under RNase-free precautions. Since mRNA is converted to cDNA prior to immunostaining, the protocol allows the use of virtually any antibody independent of its incubation time.

1. In situ reverse transcription The in situ reverse transcription has to be carried out under RNase-free precautions. Membrane-covered slides are pretreated with poly-L-lysine to prevent the tissue from floating away. Fresh frozen tissue are cut into 12 μm sections, mounted on membrane-covered slides and are air-dried for 30 min. Slides are fixed in 4 % PF A/PBS (pH 7.4) at 4°C for 10 min and washed. All washes are static for 2x 5 min in PBS at room temperature. A dissolution of the secondary structure of mRNA was carried out with a magnetic stir bar for 10 min in 0.1 M TEA (÷NaCl) (pH 8) and 0.5 % acetic anhydride. After 5 min 0.5 % acetic anhydride was pipetted slowly to TEA again. Slides are washed with PBS. In the next step slides are subjected to 0.1 % Triton/PBS to enlarge the membrane passage for 20 min. Slides are washed with PBS.

A reverse transcription preparation (MBI Fermentas), containing 8 μl of 5x reaction buffer, 2 μl of ribonuclease inhibitor (20 U/μl), 4 μl of dNTPs (10 mM each), 4 μl of reverse transcriptase (200 U/μl), 2 μl of each reverse primer (10 pmol) and 18 μl DEPC-water was placed on each slide. Primers contained a nonspecific tail for concatamerization, e.g. 5'-CCCCAAACCCCAAACCCC-3' and/or 5'-GGGGTTTGGGGTTTGGGG-3'. Also poly-T primers can be used. This modification yields a scaffold of resulting cDNAs fixed to the site of the reverse transcriptase reaction (Behrens et al, J. Pathol. 194 (2001), 43-50). This approach resulted in a high cellular specificity of signals. A coverslip was used on the sections, which were placed in a humid box to prevent drying out. Incubation at 4°C was performed for 1 hour, followed by an 2 hours incubation at 37°C. After a wash with PBS and gentle removal of the cover slips, slides were fixed in 4 % PF A/PBS (pH 7.4) at 4°C for 10 min again.

2. Immunohistochemistry After the reverse transcription step, slides were stained with a monoclonal mouse antibody (Immunotech) directed against human CD34. The short time staining started with a block (PBS with 10 %FCS, 2 % NHS, non-fat dry milk) for 30 min at 37°C. Primary antibody (CD34, ready to use) was added and the slides were incubated at 37°C for 15 min. After a wash in PBS (2x 2 min) the slides were covered with the biotinylated second antibody anti-mouse IgG (vector laboratories) (1:100, PBS with 10 % FCS) and incubated at 37°C for 15 min. This was followed by a wash (2x 2 min) and a 15 min incubation at 37°C with the AB complex (vector laboratories) (PBS with 10 % FCS, 1 % A, 1 % B). The development of the staining with DAB (0.05 M Tris/HCl pH 7.4 with DAB 1 :50, H₂O₂ 1 :2000) was performed after a wash (2x 2 min).

The long time staining was performed with the same materials, but with different dilutions and incubation times. The slides were incubated for 1,5 hours with the block (PBS with 10 % FCS, 0.5 % NHS, non-fat dry milk) at 37°C, overnight with the primary antibody at room temperature, 2 hours with the second antibody (1:200, PBS with 10 % FCS) at 37°C and 2 hours with the AB complex (PBS with 10 % FCS, 0.7 % A, 0.7% B) at 37°C. Different incubation times did not result in significant mRNA degradation, as is shown in Figure 1.

3. Laser assisted microdissection UV-Laser microbeam technology based on a nitrogen laser (PALM, Bernried, Germany) was applied for microdissection and separate harvesting of individual cellular components and subsequent PCR amplification.

4. PCR/Real time RT-PCR Conventional PCR was applied for the qualitative detection of mRNA in microdissected tissue samples. Real time PCR was used for quantification of target mRNAs. cDNA extraction from laser microdissected cells was carried out according to the manufacturer's guidelines (Qiagen, Hilden) with slight modifications. After cDNA elution into 60 μl of AE buffer, the same 60 μl were pipetted onto the column a second time and centrifuged again after incubation for 5 min.

For PCR amplification from this starting material, a final volume of 10 μl was used containing 1.0/1.5 mM(0.2/0.3 μl) MgCl₂ (50 mM), 5.75/5.65 μl H₂O, 1 μl lOx PCR buffer, 1 μl dNTPs (2 mM each), 0.5 μl of each primer (10 pmol/μl), 0.05 μl of Taq polymerase and 1 μl cDNA template. PCR experiments were performed under the following conditions: denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 30 sec, 55/60°C for 40 sec and 72°C for 1 min and a final cycle at 72°C for 10 min. PCR products are visualized by gel electrophoresis using 2 % agarose gels containing ethidium bromide.

Real time PCR (ABI PRISM™ 7700 Sequence Detection) was performed in MicroAmp^®OpticalTubes (Applied Biosystems, Foster City, California, USA) in a 13μl reaction volume containing 6.25 μl SYBR Green PCR Master Mix (Applied Biosystems), 0.375 μl forward primer (10 pmol/μl), 0.375 μl reverse primer (10 pmol/μl), 3.0 μl DEPC-H₂O and 2.5 μl of cDNA template. For each cDNA sample, real time PCR reaction was performed in duplicate for each gene (Table 1) and no- template controls were included. Pre-incubation was performed for 10 min at 95 °C to denature the target cDNA and activate AmpliTaq Gold DNA polymerase. cDNA was amplified for 50 cycles of 20 sec at 94°C, 30 sec at 59°C, and 40 sec at 72°C. The fluorescence signal is determined within each PCR cycle and quantification was carried out according to the ΔΔCt method, described in Fink et al., Am. J. Pathol. 157 (2000), 1459-1466.

In the following tables, the sequences (table 1 - forward primer, table 2 - reverse primer) and the annealing temperatures and MgCl₂ concentrations of primers (table 3) that were used for PCR/real time PCR, are depicted.

Table 1

Table 2

Table 3

Results

The results are summarizes in the attached figures.

Figure 1 shows amplification plots demonstrating real time PCR detection of GAPDH (A), GFAP (B) and NFM (C) in a tissue sample after in situ-RT and immunostaining using a short time incubation period (<1 h, open squares) versus a long time incubation period (> 24 h, dark triangles) for the primary antibody against CD34. The integrity of both, high (GAPDH) as well as low abundant transcripts (NFM) is conserved independent of the incubation time of the primary antibody.

Figure 2 shows (A) laser microdissection of a single CD34-expressing cellular element in a ganglioglioma (black arrow, upper left corner, magnification: x40). By immunostaining, CD34-expressing cellular elements can be clearly distinguished from adjacent CD-34 negative cells (grey arrow, lower right corner). (B) The same slice after laser microdissection of the CD-34 positive cell. (C) PCR analysis for a variety of neuroepithelial lineage marker genes of laser microdissected CD34 positive cellular elements after initial in situ-RT. The co- expression of CD34 (lane 2) and neurofilament (lane 6) suggests a neuronal origin. No expression of glial fibrillary acid protein (lane 4) and myelin basic protein (lane 5) is observed (lane 1 - length standard, lane 3 GAPDH). References

1. Behrens et al, J. Pathol., 194 (2001), 43-50 2. Blumcke et al, Acta Neuropathol. 97 (1999), 481 -490

3. Crino et al., Proc. Natl. Acad. Sci. USA., 93 (1996), 14152-14157

4. Fink et al., Am. J. Pathol. 157 (2000), 1459-1466.

5. Fink et al. Lab Invest. 80 (2000), 327-333.

Claims

1. A method for detecting gene expression in a cell, comprising the steps of (i) providing a test sample comprising at least one single cell; (ii) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest; (iii) preparing cDNA from mRNA of said sample with said oligonucleotide; (iv) labeling at least one single cell of said sample to identify at least one specific cell.

2. The method of claim 1, wherein said tail sequence is an artificial sequence forming a free tail sequence.

3. The method of claim 1, wherein said tail sequence is G- or C-rich.

4. The method of claim 1, wherein said tail sequence comprises the sequence of 5'- GGGGTTTGGGGTTTGGGG-3' (SEQ ID NO:l) or the sequence of 5'- CCCC AAACCCC AAACCCC-3' (SEQ ID NO:2).

5. The method of claim 1, wherein step (iii) comprises in situ reverse transcription (in situ RT).

6. The method of claim 1, wherein said labeling in step (iv) comprises incubating said sample with an antibody under conditions sufficient for the production of a detectable signal.

7. The method of claim 6, wherein said antibody is an antibody specific for a cellular marker.

8. The method of claim 7, wherein said marker is capable to distinguish different cells.

9. The method of claim 7, wherein said marker indicates a pathological condition.

10. The method of claim 1, wherein said labeling in step (iv) comprises an antibody, a single chain antibody, a tag, specifically binding proteins and/or aptameres.

11. The method of claim 1, further comprising the step of (v) isolating said identified specific cell from said sample.

12. The method of claim 1 , further comprising the step of (vi) analyzing the expression profile of said specific cell.

13. The method of claim 6, wherein the antibody is a monoclonal antibody.

14. The method of claim 6, wherein said method further comprises a second antibody for the visualization of said detectable signal.

15. The method of claim 14, wherein said secondary antibody is enzyme labeled or fluorescence labeled.

16. The method of claim 15, wherein said labeling is by alkaline phosphatase, peroxidase or fluorescein.

17. The method of claim 6, wherein said antibody is Map2c, GFAP or an antibody specific for CD34.

18. The method of claim 9, wherein said pathological condition is a disease or a susceptibility to a disease.

19. The method of claim 9, wherein said pathological condition is a neurological disorder.

20. The method of claim 9, wherein said pathological condition is selected from the group consisting of glioneural tumors, gangliogliomas, oligodendroglioma, and astrocytoma.

21. The method of claim 9, wherein said pathological ^' condition is epilepsy, a viral infection or a tumor.

22. The method of claim 21, wherein said viral infection is by oncogenic viruses.

23. The method of claim 21, wherein said viral infection is by herpesviruses, papillomaviruses, retroviruses, hepadnavirus, or adenoviruses.

24. The method of claim 21, wherein said tumor expresses a multidrug resistance gene.

25. The method of claim 1, wherein said labeling of at least one single cell in step (iv) is identified by a process selected from the group consisting of immunohistochemistry (IHC), multi parameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, and automated cell analysis.

26. The method of claim 11, wherein said isolating in step (v) is by laser assisted microdissection.

27. The method of claim 12, wherein said analyzing in step (vi) is by amplification of nucleic acids of said specific cell by polymerase chain reaction (PCR).

28. The method of claim 27, wherein said PCR is quantitative PCR.

29. The method of claim 1, wherein said test sample is a cryosection of a snap frozen tissue sample.

30. The method of claim 1, wherein said test sample is a section of a paraffin embedded tissue sample.

31. The method of claim 29 or 30, wherein said tail sequence binds to said tissue.

32. The method of claim 1, wherein said sample is derived from a mammal.

33. The method of claim 32, wherein said mammal is a human.

34. A kit for screening a test sample for the presence of at least one cell indicating a pathological condition or a susceptibility to a pathological condition, comprising (i) providing an oligonucleotide binding to RNA, corresponding to at least one gene of interest, and comprising a tail sequence unspecific for said gene of interest; (ii) a labeling compound specific for a cellular marker indicating a pathological condition; and (iii) cell permeabilizing and cell stabilizing agent.

35. The kit of claim 34, wherein said tail sequence is SEQ ID NO: 1 or SEQ ID NO:2.

36. The kit of claim 34, wherein said kit further comprises at least one transcriptase, buffers and agents suitable for reverse transcription.