Fully Automated RNAscope In Situ Hybridization Assays for Formalin‐Fixed Paraffin‐Embedded Cells and Tissues

Anderson, Courtney; Zhang, Bingqing; Miller, Melanie; Butko, Emerald; Wu, Xinfeng; Laver, Thomas W; Kernag, Casey; Kim, Jeffrey; Luo, Yuling; Lamparski, Henry G.; Park, Emily; Su, Nan; Ma, Xiao‐Jun

doi:10.1002/jcb.25606

Cited by 90 publications

(78 citation statements)

References 19 publications

(21 reference statements)

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“…The assay was performed by using automated Ventana Discovery Ultra slide-staining system (Roche) as previously described. 27 PRECSIT-specific probe (catalog number 559389) was designed and purchased at Advanced Cell Diagnostics (ACD, Newark, CA). Specific probes for human PPIB (Cyclophilin B) and bacterial DapB (4hydroxy-tetrahydrodipicolinate reductase) mRNAs were used as positive and negative controls, respectively (both from ACD).…”

Section: Rna In Situ Hybridizationmentioning

confidence: 99%

p53-Regulated Long Noncoding RNA PRECSIT Promotes Progression of Cutaneous Squamous Cell Carcinoma via STAT3 Signaling

Piipponen

Nissinen

Riihilä

et al. 2020

The American Journal of Pathology

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Long noncoding RNAs (lncRNAs) have emerged as putative biomarkers and therapeutic targets in cancer. The role of lncRNA LINC00346 in cutaneous squamous carcinoma (cSCC) was examined. The expression of LINC00346 was up-regulated in cSCC cells compared with normal human epidermal keratinocytes. Elevated expression of LINC00346 was noted in tumor cells in cSCC tissue sections in vivo, as compared with cSCC in situ, and actinic keratosis by RNA in situ hybridization; and the expression in seborrheic keratosis and normal skin was very low. Immunohistochemical analysis of cSCC tissue sections and functional assays of cSCC cells in culture showed that LINC00346 expression is down-regulated by p53. Knockdown of LINC00346 inhibited invasion of cSCC cells in culture and suppressed growth of human cSCC xenografts in vivo. Knockdown of LINC00346 inhibited expression of activated STAT3 and resulted in down-regulation of the expression of matrix metalloproteinase (MMP)-1, MMP-3, MMP-10, and MMP-13. Based on these observations LINC00346 was named p53 regulated carcinoma-associated STAT3-activating long intergenic non-protein coding transcript (PRECSIT). These results identify PRE-CSIT as a new p53-regulated lncRNA, which promotes progression of cSCC via STAT3 signaling.

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Section: Rna In Situ Hybridizationmentioning

confidence: 99%

p53-Regulated Long Noncoding RNA PRECSIT Promotes Progression of Cutaneous Squamous Cell Carcinoma via STAT3 Signaling

Piipponen

Nissinen

Riihilä

et al. 2020

The American Journal of Pathology

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“…RNAscope technology provides single-molecule sensitivity and resolution, enabling quantification of specific RNA molecules at the single cell level (20). Recent development of proprietary RNAscope probes complementary to MCPyV LTAg/sTAg mRNA permits direct visualization of viral transcripts with spatial and morphological context.…”

Section: Introductionmentioning

confidence: 99%

Age and Gender Associations of Virus Positivity in Merkel Cell Carcinoma Characterized Using a Novel RNA In Situ Hybridization Assay

Wang

Harms

Palanisamy

et al. 2017

Clinical Cancer Research

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Purpose-Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Merkel cell polyomavirus (MCPyV) plays an oncogenic role in the majority of MCCs. Detection of MCPyV in MCCs has diagnostic utility and prognostic potential. We investigated whether RNAscope, an RNA in situ hybridization (ISH) assay for detection of RNA transcripts in tissues, is useful for MCPyV detection.Experimental Design-We applied an RNAscope probe targeting MCPyV T antigen transcripts on tissue microarrays (TMAs) and whole tissue sections encompassing 87 MCCs from 75 patients, 14 carcinomas of other types, and benign tissues. For comparison, quantitative PCR (qPCR) was performed on 57 cases of MCC from 52 patients.Results-RNA-ISH demonstrated the presence of MCPyV in 37/75 (49.3%) cases. Notably, tumors from younger patients (< 73 years) had a significantly higher virus positivity than those from elderly patients (≥ 73 years) (64.9% vs. 34.2%, P =0.011). Female patients had a higher positive rate of MCPyV than male patients (66.7% vs. 39.6%, P =0.032). Data from both RNA- HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptdetermining MCPyV status, RNAscope had 100% sensitivity and 100% specificity. There was a strong correlation between qPCR copy number and RNA-ISH product score (Spearman's correlation coefficient R 2 = 0.932, P < 0.0001).Conclusions-RNA-ISH is comparably sensitive to qPCR for detection of MCPyV and allows for correlation with tissue morphology. This study also reveals a significant association between age, gender, and MCPyV positivity.

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“…An RNAscope in situ hybridization assay was performed as described previously (Anderson, Zhang et al, 2016). The sample sections were processed according to the manufacturer's instructions using an RNAscope Red kit (ACDBio).…”

Section: Rnascope In Situ Hybridization Assaymentioning

confidence: 99%

Stromal ISLR promotes intestinal regeneration and cancer by suppressing epithelial Hippo signaling via FAT1

Tang

Sheng

et al. 2019

Preprint

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The YAP signaling activation in epithelial cells is essential for intestinal regeneration and tumorigenesis. However, the molecular mechanism linking stromal signals to YAP-mediated intestinal regeneration and tumorigenesis is poorly defined. Here we report a stroma-epithelia YAP signaling axis essential for stromal cells to modulate epithelial cell growth during intestinal regeneration and tumorigenesis. Specifically, upon inflammation and in cancer, an oncogenic transcription factor ETS1 in stromal cells induces expression of a secreted protein ISLR that can directly binds to a transmembrane protocadherin FAT1 on the surface of epithelial cells. This binding suppressed the Hippo signaling by disrupting MST1-FAT1 association, resulting in YAP signaling activation. Deletion of Islr in stromal cells in mice markedly impaired intestinal regeneration, and suppressed tumorigenesis in the colon. Moreover, the expression of stromal cellspecific ISLR and ETS1 significantly increased in inflamed mucosa of human IBD patients and in human colorectal adenocarcinoma, accounting for the epithelial YAP hyperactivation. Collectively, our findings uncovered a molecular mechanism governing signals for communication between stroma and epithelium during tissue regeneration and tumorigenesis.

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Fully Automated RNAscope In Situ Hybridization Assays for Formalin‐Fixed Paraffin‐Embedded Cells and Tissues

Cited by 90 publications

References 19 publications

p53-Regulated Long Noncoding RNA PRECSIT Promotes Progression of Cutaneous Squamous Cell Carcinoma via STAT3 Signaling

p53-Regulated Long Noncoding RNA PRECSIT Promotes Progression of Cutaneous Squamous Cell Carcinoma via STAT3 Signaling

Age and Gender Associations of Virus Positivity in Merkel Cell Carcinoma Characterized Using a Novel RNA In Situ Hybridization Assay

Stromal ISLR promotes intestinal regeneration and cancer by suppressing epithelial Hippo signaling via FAT1

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