qPCR Gene Expression Assays and an Exogenous RNA control for indication of reaction inhibition in the RT-qPCR workflow

Jaakko Kurkela Thermo Fisher, Finland

Abstract
Reverse transcriptase quantitative PCR (RT-qPCR) is a routine laboratory technique for the measurement of RNA that is both sensitive and specific with appropriate assay design. Thermo Scientific Solaris qPCR Gene Expression Assays are probe-based assays that are ideal for routine molecular applications; they combine minor groove binder technology (MGB) with a rigorous algorithm that permits detection of all known splice variants of a gene target, while distinguishing among closely related family members, on a genome-wide scale. Inhibitors of RT-qPCR may be present in experiments and not easily recognized. The primary challenge is that methods such as spectrophotometric techniques do not always detect RT-qPCR inhibitors. The Thermo Scientific Solaris RNA Spike Control Kit was designed as an exogenous control to identify the presence of reaction inhibition including those commonly carried through processing steps to isolate RNA such as EDTA, phenol, heparin and EtOH. Here we describe applications for this exogenous control to identify the presence of reaction inhibition and illustrate the impact inhibition can have on results and data interpretation. Experimental results demonstrate RNA Spike Control provides accurate identification of RT-qPCR inhibition and the ability to predict when inhibition has been removed, for example, by dilution of the RNA sample into the reverse transcription reaction. The RNA Spike Control identified RT-qPCR inhibition in a gene expression assay, explaining the variable results obtained. Furthermore, the use of RNA Spike Control Kit was compared to current inhibition identification protocols and was found to be an easy and succinct workflow, offering a clear advantage of this technology.