Real-time detection of mRNA splicing variants with specifically designed reverse-transcription loop-mediated isothermal amplification

Abstract

Alternative splicing is a ubiquitous and crucial process in cellular processes and has a specific linkage with diseases. To date, developing cost-effective methods with high sensitivity and specificity for detection of splicing variants has been needed. Herein, we report a novel splicing variant assay based on specifically designed reverse-transcription loop-mediated isothermal amplification. After reverse transcribing the splicing variant into cDNA, four DNA primers are specifically designed to recognize six distinct regions. The four DNA primers can hybridize with corresponding sequences for extension and strand displacement DNA synthesis to form stem-loop DNA and then LAMP amplification is started. The proposed method can detect as low as 100 aM splicing variants in real-time fashion with high specificity, showing great potential in biological function and clinical studies.