a qPCR amplification plot with no inhibition as the Spike control in water and in sample matrix shows the same Cq value and efficiency
The RT-reaction is inhibited but not the qPCR reaction, seen on the shifted Cq-value.
TATAA Universal RNA Spike II
- Reactions/SYBR or Probe:
TATAA universal RNA Spike is used to detect any inhibition of the cDNA synthesis/RT-PCR reaction.
TATAA Universal RNA Spike for quality Control
TATAA Universal RNA Spike II has an RNA sequence length of 2000 base pairs. The Spike II assay, which is used for detecting the Universal RNA Spike II template, is very robust and optimized for high sensitivity for inhibition. The assay amplifies a 286-base region towards the 3’-end of the synthetic template. The measured Cq-values and the shape of the amplification curves reflect the inhibition. The Cq-values of the Spike II assay also reflect losses during extraction, handling, transport, and storage of samples, including freeze-thaw events during RT-qPCR
The risk of compromised RT-qPCR results
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 the RT-qPCR often causes erroneous biological readouts, even though the qPCR amplification curves can look perfectly normal. The reason is that these upstream reactions usually are exposed to a higher concentration of inhibitors than the PCR itself.
The measured Cq and the shape of the amplification curve reflect inhibition. If the Cq value is greater in an experimental sample than in the control then the analytical process of that experimental sample is inhibited (Figure 3-6). The magnitude of the difference between these Cq values reflects the degree of inhibition.
Test extraction yield
The TATAA Universal RNA spike may be added to any stabilised and homogenized sample prior to RNA purification to test for material loss during isolation, transportation and storage of samples, including freeze-thaw events during RT-qPCR.
Equal quantities of the RNA spike are added to each experimental sample and to a control sample. The control sample should be based on RNase free water or elution buffer of the same volume as used for elution/dissolving in RNA purification protocol. An equal Cq of the TATAA Universal RNA Spike in the experimental and control samples reflects 100% yield. If the Cq values differ, the yield of the extraction can be estimated.