We are optimizing protocols for plasma miRNA isolation and amplification. However for some miRNA, Ct Values fall in the range of 37-40. Negatives are clean. Should we ignore such low Cts as indicators of no or low expression (as we often do for qRT-PCR of mRNA)? We make cDNAs using 10 ng total RNA and are sure that there is no issue with the RNA quality since other miRNAs do show amplification. Neither higher input (more than 10ng) for cDNA synthesis nor dilution of cDNA has been of help.
Important question: are these Cq values consistent?
If all your replicate wells report 37 (or thereabouts), and the melt curves look solid, I would be inclined to trust the values. It likely represents a very low level of miRNA, but also a trustworthy low level. It may simply be an inefficient PCR. It's also likely to low to be of major biological consequence.
If the replicate wells report "37, 34, nothing, nothing, 40" then I would dismiss it as noise.
Presumably the important output for this analysis is comparative, i.e. do these miRs increase in some samples? If they do (say "28, 28.1, 27.6" vs "37, 37.1, 36.5") then this miR is clearly important, and massively upregulated under some circumstances: keep all the data.
If they don't, and are ~37 for all samples, then probably this miR simply isn't really present to any meaningful amount, and you shouldn't worry about measuring it.
Thanks a lot. Your answer makes a lot of sense.
In our case, Ct values are low (~ 37) for all samples.
Hi,
I would add to John answer that you perform your RT on only 10 ng of RNA. I suppose there is a linear relation between starting RNA amount and cDNA yield. I usually do a RT on 500ng RNA for mRNA relative expression.
We can assume that if you started with this amount, your Ct would drop to 32-33 instead of 37, not so bad.
Thanks Philippe. We did try using higher amount of input RNA for cDNA synthesis but to no avail. It is likely that the miRNA of our interest is present in extremely low abundance in the plasma.
Hi Mam, it would be better if you run one negative control in the same plate, where you add water instead of cDNA. Because sometime we get false positive result when we consider higher Ct values.
15 to 35 ct values are in good range.
So long as ur std curve fits.
When working with plasma or serum is better to use a relationship for volume recovered in the RNA isolation.
When we start with 100 microL of plasma, we use 1-2 microL of RNA elution.
If you do nano drop quantification below 15 ng/ml is not very precise...
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