RNAscope™ ISH as a validation tool for high-throughput transcriptomic analyses

High-throughput transcriptomic analyses, such as microarray, RNA sequencing (RNA-Seq) and NanoString nCounter, enable researchers to study the complete set of RNA transcripts produced by the genome. These analyses can generate a wealth of data, but most often need to be validated within the tissue microenvironment. RNAscope™ ISH and the recently introduced (Aug 2016) BaseScope™ ISH assays can both be applied as methods to validate high-throughput findings at the single cell level with spatial information in tissue.

  • Confirmation of results - RNAscope assays have been used in numerous publications to confirm the results of high-throughput analyses and provide further information on the cellular localization of RNA species
  • Alternative splicing - BaseScope ISH is capable of detecting splice variants using probes that span the exon junctions present in a variant.  Information derived from RNA-seq on alternative splicing events can be validated in cells and tissues by BaseScope ISH.
  • Co-expression - RNAscope Multiplex Fluorescent Assays and Duplex Chromogenic Assays are routinely applied to simultaneously visualize expression of up to three RNA targets. Co-localization of two or three specific markers in the same or different cells can be determined by RNAscope ISH.

Validation of Single-Cell RNA-seq - Silberstein et al. applied this technology to 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.

Interview with Dr Rohit Mehra:
Realising the potential of Iong noncoding RNA as a cancer biomarker - From NGS discovery to validation with RNA in situ Hybridization
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Interview with Dr Nallasivam Palanisamy:
Development of Prostate Cancer Personalized Medicine - Advancing analysis of prostate tumor molecular heterogeneity by combined immunohistochemistry and novel RNA in situ hybridization
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Interview with Dr Alexy Pronin:
Exploring GPCR expression in the eye. From discovery with NGS to spatial and quantitatvie expression confirmation with RNAscope technology
Download Interview

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

RNAscope ISH has been cited in many publications as a validation tool applied downstream of discoveries made with NGS, Microarrays, NanoString nCounter.

Validate and localize expression of your transcriptomic results. We can provide consultation.

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