Contaminants present in samples are known to inhibit enzymatic reactions and, in the context of a reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) assay, are fully capable of distorting reported measurements. Such enzymatic reactions are a necessary part of the sample-preparation prior to the qPCR, such as nuclease/proteinase treatment and subsequent reverse transcription of mRNA to cDNA; inhibition of which often causes erroneous biological readouts, even though the qPCR amplification curves can look perfectly normal. The reason is these upstream reactions are usually exposed to a higher concentration of inhibitors than the PCR itself. Results may also be compromised by the degradation of RNA during sampling, transport, storage, and other handling processes.

The Universal RNA Spike from TATAA Biocenter is an easy to use and very effective tool for quality control throughout entire RT-qPCR experimental workflow. The Universal RNA Spike has a synthetic sequence that is not present in any known living organism. This RNA sequence is 1000 bases in length, which includes a 5’ Cap and a poly-A tail of approximately 200 bases, hence the TATAA Universal Spike mimics eukaryotic mRNA. Additionally, the Universal DNA Spike is available for DNA analyses.

The Spike assay, which is very robust and is optimized for high sensitivity for inhibition, amplifies a 300-base region towards the 3’ end of the synthetic transcript; see schematic below. The measured Cq and the shape of the amplification curve reflect inhibition. The Cq of the Spike assay also reflects losses during extraction, handling, transport, and storage of samples, including freeze-thaw events during RT-qPCR, which is another potential use of spike.