Reverse Transcription Cycling Achieves Greater Than 100% cDNA Conversion With Implications For Ultra-High Sensitivity of Clinically-Actionable, Oncogenic RNA Mutations
Summary:
Oncogenic RNAs are biomarkers with diagnostic, prognostic and predictive clinical implications in many cancers and specimen types. Methods for detecting oncogenic RNAs typically require reverse transcription to cDNA, which is vulnerable to RNA drop-outs at low copy numbers. To improve the sensitivity and robustness of RNA detection, we developed “RT cycling,” a linear amplification method using a thermostable reverse transcriptase, an RNA-protective buffer and high-temperature cycling. Here, we demonstrate that cancer-associated RNAs can be amplified by at least 10-fold through cDNA synthesis, leading to accurate quantification of variants that are missed using conventional reverse transcription.
Authors: Melissa Church, Rebecca Rinehart, Shobha Gokul, Liangjing Chen, Stela Filipovic-Sadic, and Gary J Latham