RNAscope® ISH as a validation tool for high-throughput transcriptomic analyses
Validation of Single-Cell RNA-seq – Silberstein et al. applied this technology in single cells to identify the secreted factors produced by transplanted niche cells that regulate stem cell function. RNAscope ISH was used to validate the expression of IL18 proximal to the transplantation site.
Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators Silberstein et al. Cell Stem Cell. 2016 Aug 9. pii: S1934-5909(16)30199-0. doi: 10.1016/j.stem.2016.07.004. PMID:27524439
Researchers in the Spotlight--Download interviews to read how researchers use RNAscope ISH to validate RNA-seq results.
Validation of NanoString nCounter – Chen et al. performed a NanoString assay to identify genes differentially expressed between control and LKB1 mutant lung cancer samples. The RNAscope ISH assay was used to validate these results, showing that the lncRNA LINC00473 is associated with LKB1 inactivation in NSCLC. Given the poor specificity of LKB1 antibodies, this paper demonstrates that LINC00473 could be used as a surrogate biomarker for LKB1 in lung cancer samples.
cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth Chen et al. J Clin Invest. 2016 Jun 1;126(6):2267-79. doi: 10.1172/JCI85250 PMID: 27140397
Validation of LncRNA Microarray – In a recent study lncRNA microarrays identified over 2800 lncRNAs that were differentially expressed between triple-negative breast cancer (TNBC) tissue and normal adjacent tissue. Lin et al. used RNAscope ISH to confirm that expression of the lncRNA LINK-A is indeed significantly increased in TNBC tissues compared to adjacent normal tissues, and also were able to further localize the expression to the cytoplasm and close to the cellular membrane.
The LINK-A lncRNA activates normoxic HIF1α signalling in triple-negative breast cancer Lin et al. Nat Cell Biol. 2016 Feb;18(2):213-24. doi: 10.1038/ncb3295 PMID: 26751287
Validation of Digital Transcriptome Subtraction (DTS) — This recently developed method has been used to identify pathogenic viruses in cancer, but can also be applied to non-cancer related diseases. Whole transcriptome sequencing is applied to a tumor sample followed by in silico removal of host sequence fragments. The remaining sequences are aligned and blasted against known pathogen sequences to identify candidate sequences present in the sample. Highly sensitive methods such as RNAscope ISH are then performed to validate the presence of the pathogenic sequence. Cimino et al. described this application in their 2014 publication.
Detection of viral pathogens in high grade gliomas from unmapped next-generation sequencing data Cimino et al. Exp Mol Pathol. 2014 Jun;96(3):310-5. doi: 10.1016/j.yexmp.2014.03.010 PMID: 24704430