FIGURE 1.

Schematics of primer and assay design. (A) Schematic representation of 25S rRNA showing relative positioning of forward and reverse primers for target and reference amplicons used in qRT-PCR. Sequences shown above and below the line are forward and reverse primers, respectively. (B) Overview of the assay depicting the SRL sequence with the depurinated adenine shown in bold. Depurination by an RIP (step 1) leaves behind an abasic site (*). Reverse transcriptase incorporates an adenosine at the position opposite the abasic site (step 2), resulting in a T to A transversion in the cDNA, which is propagated during PCR (step 3). The base change at this position is detected using reverse primer with and adenine at the 3′ terminus (in bold) and specificity is enhanced by a secondary mismatch (underlined).