Using In Situ Padlock Probe Technology to Detect mRNA Splice Variants in Tumor Cells

Advanced prostate cancer (PC) patients commonly receive anti-hormonal drugs targeting the androgen receptor (AR) signaling pathways. However, almost all patients acquire therapy resistance that can be caused by AR amplification or expression of AR splice variant 7 (AR-V7). Therefore, AR-V7 and AR expression are potential biomarkers for early detection of therapy resistance. Here, we present our padlock probe (PLP)-based approach for the in situ detection of AR full length, AR-V7, and prostate-specific transcripts in PC cell lines, which is applicable for circulating tumor cells (CTCs) isolated from cancer patients. First, PC cell lines are seeded on glass slides. Then, cDNA is created using target-specific reverse transcription primers. PLPs are hybridized to the cDNA and ligated to form circular single-stranded DNA molecules. The PLP sequence is ligated and amplified by rolling circle amplification and the resulting rolling circle products can be detected using fluorescently labeled probes. Quantification can be automated using the image analysis software CellProfiler.