My lab has been using the Qubit ssDNA kit to quantify cDNA prior to qPCR input, but we have doubts about the accuracy of this kit. The MFR explicitly states that the Qubit ssDNA kit does not bind exclusively to ssDNA, and they don't provide any further information as to how the quantification may be skewed by gDNA and/or RNA contamination in cDNA samples post RT-rxn.
From my understanding, most people quantify RNA via Qubit or Nanodrop prior to cDNA synthesis (to evaluate mRNA expression via qPCR), thereafter standardizing the RNA inputs into cDNA synthesis. I agree with this practice, but our team likes to view qPCR data in copies/ng of cDNA, so we like to get an accurate [as possible] quantification of our cDNA samples.
Is anyone aware of another method/kit used to quantify ssDNA? I imagine we could treat our cDNA samples with RNase to minimize RNA contamination, which I believe to be the biggest culprit affecting the accuracy of our ssDNA quantification. We already treat the samples with on-column DNase I during RNA prep, and a second DNase digestion prior to the RT rxn, so I am doubtful dsDNA contamination would be skewing our cDNA quantification so significantly.
Thanks so much for the help all!!!
If your measurements are way off w Qubit, do RNAse treatment, will be cheaper/simpler than getting another machine.
As for which way is the best for quantification, I am afraid it will be 'it depends' - cost, time, sample type, DNA length, GC content etc.
We use Qubit with mostly satisfactory results. If you need higher precision, try BioAnalyzer, but even that does not show much improvement compared to Qubit:
Article Comparison of techniques for quantification of next-generati...
https://www.nature.com/articles/s41598-018-19574-w
I personally think the BioAnalyzer is LESS accurate than the Qubit for sample quantification because it's based on a comparison to the ladder, which can fade over time in storage. This is less of an issue for the DNA kits but it's a huge issue with the RNA kits.
I think you've already identified the control that will tell you how much of an issue the presence of RNA is for the quality of quantification: do a few RTs from practice RNA, and then for each cDNA sample, do a column purification only on one half (should capture both RNA and DNA) and an RNase digestion followed by column purification on the other half. Then qubit them both with the ssDNA kit and see if there's any meaningful difference. Even if there IS a meaningful difference, if it's very consistent between samples you can probably just compensate for it mathematically rather than having to run RNase digestions and cleanups all the time.
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