Development of a BC120-specific RT-qPCR assay. (A) Schematic outlining the primer, probe, and blocking LNA-binding sites on the BC120 and BC200 RNA linker-ligated templates. (B) Twenty-five nanograms of ligated MCF-7 RNA was used as a template for RT-qPCR using the primer/probe sets shown in (A) in the presence and absence of the blocking DIG-labeled LNA. In parallel, purified in vitro transcribed (IVT) BC120 and BC200 were also used as RT-qPCR templates. Data are relative to the untreated (no LNA) reactions and represent the mean of four replicates +/− SD. (C) Amplification products from (B) were separated by agarose gel electrophoresis and stained with SYBR Safe nucleic acid dye. (D) Absolute quantification of BC120 expression in ligated RNA from the indicated human tissues using T4 RNA ligase at a temperature of 37°C. Quantification was performed using a standard curve generated with serial dilutions of linker-ligated in vitro transcribed BC120 RNA and represents the mean of three replicates +/− SD.
