RNA Detection Method

About

RU 910 & 1030 Description The detection of coding and non-coding RNAs in tissue sections is important for expression analysis in molecular pathology. Alterations in gene expression are seen in many diseases, including cancer, metabolic, infectious, and several neurological diseases. In-situ hybridization (ISH) is one of the most common and widely used method for visualizing gene expression in specific cell types implicated in disease pathology. However, conventional ISH is not efficient for RNA detection in tissues. The principle drawbacks of conventional ISH are the release and diffusion of small- and even large-sized RNAs from thinly sliced paraffin-embedded and formalin-fixed tissues during the fixation process, mishybridization of the probes to other abundant RNAs and poor signal amplification resulting in inconsistent or false results. Our scientists have developed a novel fixation strategy to irreversibly crosslink or immobilize different RNAs for ISH, substantially improving their retention in tissues, thereby facilitating either their direct detection using fluorescently labeled oligonucleotide probes, or for less abundant and short RNAs, such as miRNAs, they have drastically enhancing the detection using signal amplification. They employ specific oligonucleotide probe design to avoid mishybridization to unrelated highly abundant RNAs. In addition, our scientists have designed several probes for miRNA, non-coding RNAs and mRNAs quantification. As proof of concept, we have visualized miRNAs expressed at high, medium and low levels in different tissues including the heart, liver, muscle, skin, pancreas, breast and brain and cancer tissues and cell lines. This improved ISH method is a broadly-enabling advance to visualize miRNAs, non-coding RNAs and mRNAs in paraffin-embedded tissues for diagnostic and research purposes. Applications An efficient research tool to study and detect RNA expression patterns in biological and clinical samples. A diagnostic kit for visualization of RNA in pathological samples with limiting availability. Novel probes for enhanced detection of miRNA using signal amplification Novel probes for direct detection of non-coding reference RNAs of distinct length and sub-cellular localization for normalization of RNA content or integrity in archived tissues. Advantage Highly sensitive method to visualize and quantify gene expression. Stage of Development Discovery  Patent Information US Patents 8,394,588 and 9,005,893 WO2013/016712, US-2013-0130260-A1, and US-2014-0220574 pending References Pena et al., Nature Methods (2009), 6, 139 - 141 Thum et al., Nature (2008), 456, 980-984 http://newswire.rockefeller.edu/?page=engine&id=872 Renwick, et al. Clin. Inv. (2013), doi:10.1172/JCI68760