Methods for identifying and quantifying mRNA expression of androgen receptor splicing variants in prostate cancer

Constitutively active androgen receptor (AR) variants (AR-Vs) lacking the AR ligand-binding domain have been identified as drivers of prostate cancer resistance to AR-targeted therapies. A definitive understanding of the role and origin of AR-Vs in the natural history of prostate cancer progression requires cataloging the entire spectrum of AR-Vs expressed in prostate cancer, as well as accurate determination of their expression levels relative to full-length AR in clinical tissues and models of progression. Exon constituency differences at the 3′ terminus of mRNAs encoding AR-Vs compared with mRNAs encoding full-length AR can be exploited for discovery and quantification-based experiments. Here, we provide methodological details for 3′ rapid amplification of cDNA ends (3′ RACE) and absolute quantitative RT-PCR, which are cost-effective approaches for identifying new AR-Vs and quantifying their absolute expression levels in conjunction with full-length AR in RNA samples derived from various sources.