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

Abstract

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201-2208, 2016. © 2016 Wiley Periodicals, Inc.

Keywords: AUTOMATION; BIOMARKER; GENE EXPRESSION; IN SITU HYBRIDIZATION; RNA; RNAscope.

Figure 1

Automated RNAscope technology overview. (A) RNAscope probe design. (i) A standard target probe consists of a pool of 20 double Z probes targeting a region of 1000 bases. Each Z target probe contains three elements: the lower region is complementary to the target RNA and is selected for target specific hybridization and uniform hybridization properties; a spacer sequence links the lower region to an upper region; the two adjacent upper regions from a double Z target probe form a 28 base binding site for the pre‐amplifier. (ii) Once the Z probe pairs hybridize to the RNA target the pre‐amplifier binds to the upper regions of the Z probe pairs. (iii) Hybridization of multiple amplifiers per pre‐amplifier. (iv) Hybridization of multiple labeled probes per amplifier. Labeled probes contain a chromogenic enzyme to generate one punctate dot per RNA target. The size of the dot is directly proportional to the number of Z probe pairs hybridized onto the RNA target. Hybridization of only three Z probe pairs is sufficient to obtain a detectable signal by brightfield microscopy. (B) Current (v2.5) and previous (v1.0) versions of the manual RNAscope single‐plex assay were tested on FFPE sections of HeLa cells using either the negative control probe dapB or the positive control probe Hs‐TBP. Images are shown at 40× magnification. (C) General overview of the workflow for the automated single‐plex chromogenic RNA ISH assay for FFPE samples. *Ventana DISCOVERY ULTRA and XT platforms have an additional amplification step (Amplification Step 7).

Figure 2

Automated RNAscope ISH assay detects high target signal with low background in human head and neck tumor on multiple platforms. Serial FFPE sections of a head and neck cancer case were stained with the automated RNAscope assay using the following probes: dapB (negative control), Hs‐PPIB (positive control), HPV‐HR18, and Hs‐PD‐L1. The assay was performed using either the Leica BOND RX (LS) or Roche Ventana DISCOVERY ULTRA (VS) automated platform. Signal was detected using DAB (brown) chromogen. Images are shown at either 10× (A–D, F–I) or 40× (E, J) magnification. Black boxes indicate regions shown at 40× magnification.

Figure 3

Simultaneous detection of two target genes within the same cell using the automated RNA ISH duplex assay. Serial FFPE sections from a head and neck (A, B, E, F, I, J) and cervical (C, D, G, H, K, L) cancer case were stained with the automated RNAscope duplex assay using the Leica BOND RX platform. The automated duplex RNAscope assay was performed to simultaneously detect p16 and the pooled probe HPV‐HR18 (which detects 18 high‐risk genotypes of HPV E6 and E7) (I–L). HPV‐HR18 was detected using DAB (brown) chromogen and p16 was detected using fast red (red) chromogen. (A–H) Sections were probed with either p16 only or HPV‐HR18 only, but detection of both chromogens was performed. Images are shown at either 10× (A, C, E, G, I, K) or 40x (B, D, F, H, J, L) magnification. Black box indicates region shown at 40× magnification.

Figure 4

Simultaneous detection of two distinct cell populations using the automated RNAscope ISH duplex assay. The RNAscope automated duplex assay was performed using the Leica BOND RX platform on FFPE sections of human tumors from multiple tissues. KRT19 was detected using DAB (brown) chromogen and PECAM1 was detected using fast red (red) chromogen in breast, cervix, colon, esophagus, kidney, lung, ovary, stomach, and tonsil. Images are shown at 20× magnification.

Figure 5

The automated RNAscope ISH assay performs consistently and reproducibly. (A) Three different lots of automated RNAscope assay reagents were generated for the Leica BOND RX automated platform and tested on FFPE sections of HeLa cells with either dapB or Hs‐TBP probe. (B) The average TBP signal per cell, indicating the average number of dots per cell, was quantified for every lot tested on the Leica BOND RX automated platform. Data are presented as mean + SEM; n = 3–8 replicates. (C) Three independent runs of the automated RNAscope assay were performed on FFPE sections of HeLa cells using either dapB or Hs‐TBP probes on the Leica BOND RX automated platform. (D) The average TBP signal per cell, indicating the average number of dots per cell, was quantified for every run performed on the Leica BOND RX automated platform. Data are presented as mean + SEM; n = 3–5 replicates. (A, C) Images are shown at 40× magnification.