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. Author manuscript; available in PMC: 2019 May 28.
Published in final edited form as: Eur Urol. 2017 Sep 1;73(5):727–735. doi: 10.1016/j.eururo.2017.08.009

Fig. 1 –

Fig. 1 –

Development of the BaseScope RNA in situ hybridization assay for detection of splice junctions specific to AR-FL and AR-V7. (A) Schematic illustration of the BaseScope assay and the splice junctions targeted for probe design. Top: Overview of the BaseScope assay workflow. Sections containing fixed tissues or cells were permeabilized, and exposed mRNA were hybridized with a single pair of BaseScope probes (ZZ pair) that straddle the exon/exon junction of interest. Following amplification by an advanced, next-generation signal amplification system, junction-specific signals can be visualized as punctate dots under a standard bright-field microscope. Bottom: AR splice junctions targeted for BaseScope probe design. The splice junction between AR exons 7 and 8 (E7/E8) was targeted for specific detection of the full-length AR (AR-FL), while the splice junction between exon 3 and cryptic exon 3 (CE3) (E3/CE3) was targeted for specific detection of AR-V7. (B) Specificity of the BaseScope AR probes as demonstrated by signals detected in prostate cancer cell lines with known AR-FL/AR-V7 profiles. The cell lines PC3 (AR-FL–negative, AR-V7–negative), LNCaP (AR-FL–positive, AR-V7–negative), and LNCaP95 (AR-FL–positive, AR-V7–positive) were stained using the following 1 ZZ BaseScope probes: AR-E7/E8 for AR-FL and ARE3/CE3 for AR-V7. (C) The BaseScope assay detects mature mRNA exclusively in cytoplasm. LNCaP95 cells (AR-FL–positive, AR-V7–positive) were stained with standard RNAscope (top) and BaseScope (bottom) assays. Both cytoplasmic and intranuclear (arrows) signals were detected with 18 ZZ AR-E1 and 20 ZZ AR-V7 probes used in the standard RNAscope (top) assay, while the 1 ZZ probes used in the BaseScope (bottom) assays detected punctate signals representing mature mRNA exclusively in the cytoplasm. (D) Comparison of AR-V7 signals detected by the standard RNAscope and BaseScope assays in a metastatic castration-resistant prostate cancer (mCRPC) biopsy specimen. The same mCRPC biopsy core was processed and stained for AR-V7 using 20 ZZ AR-V7 probes in the standard RNAscope assay (top) and the BaseScope assay (bottom) using the 1 ZZ AR-E3/CE3 probe. Note the intense intranuclear AR-V7 signal (arrow) with the RNAscope assay.