RNAscope: A Novel in Situ RNA Analysis Platform for Formalin-Fixed, Paraffin-Embedded Tissues

Number of probe pairs required for generating visible signal in chromogenic RNAscope assay. HeLa cell FFPE pellet was used as a model system for testing the number of probe pairs needed to detect POLR2A mRNAs in FFPE tissue specimens. A: Chromogenic staining was performed with the full PolR2A probe set consisting of 20 target probe pairs or with dapB probe set as negative control. Nuclei were counterstained with hematoxylin. B: The PolR2A probe set was divided into 20 individual target probe pairs, numbered 1 to 20 based on their location along the target (5′ to 3′). Chromogenic staining was then performed using one target pair (TP 10; left) or a combination of two target pairs (TP 10 and TP 11; middle) or a combination of three consecutive target pairs located at the middle of the full probe set (TP 9, TP 10, and TP 11; right). Insets: Higher magnification views. Similar experiments were also performed using one, two, or three target pairs located at the 5′ or 3′ end of the probe set; similar results were observed.

Detection of HPV E6/E7 mRNA. Cells from three cervical cancer cell lines known to contain different HPV subtypes [ie, SiHa (HPV-16), HeLa (HPV-18), and MS751 (HPV-45)] were stained by RNAscope with probe sets targeting HPV-16, HPV-18, and HPV-45. Positive staining was detected with an alkaline phosphatase label probe followed by reaction with Fast Red substrate (red), which was visualized under a fluorescence microscope. Cells were counterstained with DAPI (blue).

Single-copy HER2 mRNA detection in HeLa cells. A: The same fluorescently labeled HER2 probes and signal amplification system were used to detect HER2 genomic DNA (see Figure 3A) and HER2 mRNA (green) in HeLa and SK-BR-3 cells. B: Relative signal intensities of individual fluorescent dots in HeLa cells were quantified using CellProfiler image analysis software for identifying and quantifying cell phenotypes (Broad Institute, Cambridge, MA; http://www.cellprofiler.org) and were plotted as histograms for RNA and DNA.