I have RNA samples that I will be using for cDNA synthesis, and analysis of expression levels. The RNA has been DNAse treated, both in column during extraction, and post column extraction (AMPD1).
I designed my primers to overlap exon junctions anyway, but I'd like to stick to the MIQE guidelines and submit validation supplementary data anyway showing no DNA contamination.
I designed one set of primers which amplifies the reference gene within an exon (i.e non-exon gap junction), for this purpose specifically.
Instead of running each of my RNA samples through a -RT cDNA sysnthesis, and then running a PCR, could I simply run the PCR on the RNA samples?
It would save me cDNA synthesis reagents such as the OligodTs and Random Hexamers, and RNAse inhibitor. I dont see the point in running a -RT cDNA synthesis reaction on samples that I would like to confirm contain no DNA.
The postive control for this PCR would be a non-DNASe treated RNA sample.
Yes you can. In fact, if we are using the Quanta bio qScript XLT cDNA SuperMix for RT, a mastermix with all of the RT components including RT enzyme, their instruction manual says do do just what you're proposing since you can't remove the RT from the mix. Pasted from the kit manual:
Guidelines for Reverse Transcription-qPCR
Minus RT-controls: Accurate quantification of gene expression by RT-qPCR requires testing and reporting the extent of contamination of genomic DNA in each RNA sample for each gene of interest. The presence of trace amounts of gDNA does not usually interfere with quantification of high copy reference genes. However, it can have a significant contribution on signal for low copy genes. Even when using primers that are separated by intronic sequence or bridge exon junctions, the presence of genomic DNA can produce positive signals from amplification of pseudogene or off-target PCR product. Therefore, it is important to always include the appropriate “no RT” or “minus RT” control reactions in your experimental design.
Since the reverse transcriptase is an integral component of qScript XLT cDNA SuperMix, it is not feasible to construct a formal cDNA synthesis control that includes all components except the RT. The most direct method to test for the presence of genomic DNA is to bypass the RT step and use an equivalent amount of the RNA preparation directly for PCR amplification. For example: if you start with 1 μg of total RNA for cDNA synthesis and use 1/10th of the first-strand reaction as template for qPCR; then use 100 ng of total RNA as template for the minus RT-control qPCR. Any signal from the RNA only reaction is attributable to the presence of genomic DNA.
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