Question 13 of 20 Fundamentals Concept
Why does an RT-qPCR experiment need a no-RT control?
Short answer
A no-RT control contains the same RNA sample but no reverse transcriptase, so no cDNA is made. Any signal in it comes from DNA, usually contaminating genomic DNA, and shows how much of the sample's signal may not come from RNA at all.
Why
RNA preparations almost always carry some genomic DNA. Primers that can bind that DNA will amplify it, and the result looks just like expression. Running the RNA through a mock reverse transcription without the enzyme separates the two sources.
If the no-RT well is negative, or comes up many cycles later than the matching sample, the DNA contribution is negligible. If it comes up close behind the sample, part of the measured signal is DNA, and the result is inflated. For example, a sample at Cq 24 with its no-RT at Cq 36 has roughly 4000 times less DNA template than total template at 100 percent efficiency, so DNA barely matters.
A no-RT at Cq 26 would mean about a quarter of the signal could be DNA. The no-template control answers a different question, contamination of reagents. MIQE 2.0 notes that once an extraction has been shown to be DNA-free, a no-RT control for each sample is desirable but no longer essential.
Junction-spanning primers lower the risk but do not remove it, because processed pseudogenes have no introns.
A strong answer covers
- Same RNA, no reverse transcriptase, so only DNA can amplify
- Detects genomic DNA contributing to apparent expression
- Judge the Cq gap between sample and no-RT, not just presence
- Different purpose from the no-template control
- DNase treatment and junction primers reduce but do not remove the need
Common mistakes
- Treating the no-template control as if it also checked for genomic DNA
- Assuming exon-junction primers make genomic DNA impossible to amplify
- Reading any no-RT signal as a failed sample without comparing its Cq to the sample's
The interviewer might ask next
How large a Cq gap between a sample and its no-RT control would you accept, and why?