Friday, Mar 27, 2015

How RNA ISH assays can solve the difficulties in other technologies?

RNAscope® in situ hybridization assays

Non-Coding RNA
Unlike protein coding genes, for which immunohistochemistry (IHC) and RNA in situ hybridization are complementary for mapping gene expression to specific cells in situ, long non-coding RNA gene expression can only be interrogated by RNA in situ hybridization. The generally lower expression levels of lncRNAs than their protein coding counterparts demand the highest sensitivity from RNA in situ hybridization methods. The single-molecule sensitivity and rapid assay development time (<2 weeks) of ACD's RNAscope technology make RNAscope® ideally suited for localizing lncRNAs expression to specific cell types and sub-cellular structures. RNAscope in situ assays will undoubtedly accelerate lncRNA research and become an indispensable tool for lncRNA-based molecular diagnostics.

Human prostate cancer: PCA3 expression (brown dots) in FFPE tissue with RNAscope® 2.0 HD Detection Kit

Single-Cell Analysis
Unlike quantitative reverse-transcription polymerase chain reaction (qRT-PCR), RNA next generation sequencing (NGS) and microarrays, RNA FISH technology counts on direct hybridization of probes to target that does not involve any cDNA synthesis and enzymatic amplification of target template and so quantification should theoretically be free from enzyme bias. Also, the destruction of cells and the cell compartments during RNA extraction from pooled sample for qRT-PCR, RNA NGS and microarrays consequently provides the average number of transcripts while omits the transcription information in individual cells. Conversely, detection of individual RNA molecules without the cell lysis in RNA FISH allows the studies of variation in gene expressions between individual cells.

Gene fusion
Current diagnostic techniques for gene fusion detection is mainly based on fluorescent in situ hybridization (FISH) detecting the translocation events at the DNA level. However, development of DNA FISH assays is time consuming and may be difficult or impossible for events involving subtle genomic alterations. The RNAscope in situ hybridization technology provides a universal assay format for directly detecting gene fusions at the RNA level. In a typical assay design, two probes are designed to hybridize to the two partner gene sequences of the fused transcripts in a duplex fluorescence format. In wild type cells, the signals from the two probes will be distinct and rarely co-localize. However, in cells harboring the gene fusion, the two probes will hybridize to the same fusion transcripts, and the two signals will co-localize and can be detected as merged fluorescent dots.


No Antibody, No Problem
Because over 70% of protein-coding genes have no reliable antibody for immunohistochemistry (IHC), your research often comes to a screeching halt while you wait for new antibodies to be developed. We say, forget about your antibody problems with RNAscope® in situ assays. With rapid probe design and universal assay workflows for any gene, RNAscope® Technology frees you from the hassles of antibody screening, saving you precious time and effort, while delivering publication quality data.

ApoE and Lgr5 : mouse cerebellum FFPE

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