Detection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem-loop array RT-PCR analysis

摘要

The small interfering RNA (siRNA)-mediated target mRNA cleavage activity generates cleaved mRNA fragments with varied termini, which creates major technical challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem-loop array reverse transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the siRNA-mediated target mRNA cleavage sites by determining precise sequences at the 3′- termini of cleaved mRNA fragments in human cells under physiological conditions. Our results demonstrated the great potential and broad applications of using the SLA-RT-PCR as a sensitive, cost-efficient, and high-throughput tool to systematically detect siRNA-targeted mRNA cleavage sites and fragments in human cells. Highlights ? SLA-RT-PCR identifies authentic siRNA-cleavage sites under physiological conditions. ? SLA-RT-PCR detects whether the siRNA therapeutic hits its predicted gene target. ? SLA-RT-PCR is the basis for the successful application of siRNA as a therapeutic for human disease.