Reverse Transcription Based Method for Detecting Gene Expression in a Cell

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This patent describes a novel reverse transcription-based method for detecting gene expression at the single-cell level. The method involves using an oligonucleotide with a gene-specific region and an artificial, unspecific tail sequence to enable conversion of single-cell mRNA to cDNA, followed by cell identification via antibody labeling. The approach overcomes previous limitations by allowing prolonged antibody incubation times without compromising RNA integrity, making it compatible with virtually any primary antibody and allowing precise gene expression analysis in individually labeled cells. The invention also includes kits and protocols for screening pathological conditions, drug responses, and identifying pharmaceutically active substances.

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• Routine molecular diagnostic testing of tissue samples for cancer or neurological disorders by analyzing gene expression in specific, immunolabeled single cells.
• Research into cell-specific gene expression profiles in complex tissues, such as distinguishing different cell types in tumor microenvironments.
• Screening of drug candidates for efficacy and mechanism of action by monitoring changes in gene expression in targeted single cells after drug exposure.
• Detection of viral infections at the single-cell level by using virus-specific gene probes combined with antibody-based cell labeling.
• Profiling of multidrug resistance genes to guide personalized cancer therapy by identifying resistant cells in a heterogeneous tumor population.
• Automated, high-throughput screening for biomarkers or therapeutic targets in pathology and pharmaceutical research.

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• Enables precise detection and quantification of gene expression in individual cells rather than bulk tissues, improving diagnostic accuracy.
• Compatible with a full range of primary antibodies regardless of required incubation times, ensuring protocol flexibility and broad applicability.
• Preserves the integrity of mRNA even after long antibody staining steps, overcoming a key limitation of previous methods.
• Supports use with fresh frozen or fixed tissue sections, facilitating integration into standard pathology workflows.
• Applicable to a wide variety of diseases, including tumors, neurological disorders, viral infections, and drug resistance phenotypes.
• Facilitates advanced research and diagnostics, including high-resolution expression profiling, drug screening, and biomarker discovery at the single-cell level.

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Patent 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.