RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues

Abstract

In situ analysis of biomarkers is highly desirable in molecular pathology because it allows the examination of biomarker status within the histopathological context of clinical specimens. Immunohistochemistry and DNA in situ hybridization (ISH) are widely used in clinical settings to assess protein and DNA biomarkers, respectively, but clinical use of in situ RNA analysis is rare. This disparity is especially notable when considering the abundance of RNA biomarkers discovered through whole-genome expression profiling. This is largely due to the high degree of technical complexity and insufficient sensitivity and specificity of current RNA ISH techniques. Here, we describe RNAscope, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. RNAscope is compatible with routine formalin-fixed, paraffin-embedded tissue specimens and can use either conventional chromogenic dyes for bright-field microscopy or fluorescent dyes for multiplex analysis. Unlike grind-and-bind RNA analysis methods such as real-time RT-PCR, RNAscope brings the benefits of in situ analysis to RNA biomarkers and may enable rapid development of RNA ISH-based molecular diagnostic assays.

Figure 1

Schematic of the RNAscope assay procedure. In step 1, cells or tissues are fixed and permeabilized to allow for target probe access. In step 2, target RNA-specific oligonucleotide probes (Z) are hybridized in pairs (ZZ) to multiple RNA targets. In step 3, multiple signal amplification molecules are hybridized, each recognizing a specific target probe, and each unique label probe is conjugated to a different fluorophore or enzyme. In step 4, signals are detected using an epifluorescent microscope (for fluorescent label) or standard bright-field microscope (for enzyme label).

Figure 2

Validation of RNAscope. A: HeLa cells were hybridized with either the full set of probes to 18S rRNA, the left half of the set, or the right half of the set (as shown in the schematic along the top). A no-probe control was performed in parallel as an indicator of background staining. Cells were counterstained with DAPI (blue), which masks nucleolar 18S RNA. Scale bar = 10 μm. B: HCV-uninfected (left) or HCV-infected (right) HuH-7 cells were hybridized with probe sets to HCV mRNA (green). Cells were costained with 18S rRNA target probes (red) as an internal control for assay success. Nuclei were counterstained with DAPI (blue). Original magnification, ×40. C: HeLa cells were hybridized with probes to β-actin, RPLP0 (60S acidic ribosomal protein P0), PPIB (peptidylprolyl isomerase B), and HPRT-1 (hypoxanthine phosphoribosyltransferase 1) in multiplex fluorescence format. Nuclei were counterstained with DAPI. Original magnification, ×40.

Figure 3

Single RNA molecule detection. A: HER2 genomic DNA in HeLa and SK-BR-3 cells was detected using the RNAscope probes and signal amplification system. Nuclei were costained for IL-8 for diploid genome Original magnification, ×40. B: HER2 mRNA detection in HeLa cells using the same probes and signal amplification system. A probe set to 18S rRNA was used as internal control for RNA detection. HER2 mRNA dots were counted; mean dots per cell (±95% confidence limit) are indicated. Nuclei were counterstained with DAPI (blue). Original magnification, ×40. C: HER2 mRNA copies per cell in HeLa cells determined by QuantiGene 2.0 (QG2) using a standard curve from in vitro transcribed RNA. Mean copies per cell (±95% confidence limit) are indicated (green dot), as calculated from triplicate measurements using linear regression.

Figure 4

RNAscope detection of RNA in FFPE tumor tissues. A: Chromogenic staining (DAB) of primary tumor tissues (breast, lung, and prostate) hybridized with either probes to ubiquitin C (UBC) or probes against the bacterial gene dapB as negative control. Nuclei were counterstained with hematoxylin. Original magnification, ×40. B: Fluorescent detection of low-copy transcripts in FFPE samples. Breast tumor tissue section was hybridized with either no probes or with Alexa Fluor 488-labeled probe sets (green) to HPRT1 or POLR2A. Nuclei were counterstained with DAPI (blue). Scale bar = 10 μm.

Figure 5

Detection of Ig κ chain expression in B lymphocytes in FFPE human tonsil tissue. κ light chain mRNA transcripts were stained using RNAscope or a commercial non-radioisotopic RNA ISH kit. For RNAscope, a negative control (bacterial gene dapB) was also included. The dotted line outlines the mantle zone. Original magnification, ×40.

Figure 6

Multiplex fluorescence detection of uPA and PAI mRNAs in breast cancer. Merged pseudo-colored image of a metastatic breast cancer tissue section stained with probes specific to cytokeratins [PanCK (CK-8, CK-18, and CK-19), labeled with Alexa Fluor 647], uPA (labeled with Alexa Fluor 546), and PAI-1 (labeled with Alexa Fluor 488). Both uPA expression (arrowhead and right inset) and coexpression with PAI-1 (arrow and left inset) were detected. Nuclei were counterstained with DAPI (blue). Original magnification, ×40.