I'm trying to use sno202 as an endogenous control for miR relative quantification in cardiac tissue. I'm using Taqman primers (Reverse Transcriptase and Real-Time) and BioRad Thermocycler for both reactions.
Starting RNA: 10 ng
RT cycle annealing temp: 42 C (15 uL volume)
q PCR annealing temp: 60 C (20 uL volume with triplicates from RT reaction)
Thermofisher estimates the Ct values for sno202 to be in the early 20s, but I consistently get Ct values of 31-33 in my samples, rendering them unreliable.
Is there a reason for these high consistent Ct values, despite my lab mates getting reliable lower values in past experiments?
It's possible that your RNA wasn't treated nicely somewhere upstream. Have you seen similar results from RNA isolates from different days?
Also, have you check purity of your RNA? (A260/280, A260/230)?
I assume that your miR is the typical 19-21 bp species, so less utility in checking RNA integrity
--Matt
If you did not dilute your ~0.66667 ng/uL cDNA samples prior to qPCR you could be inhibiting the Taq in the qPCR phase... --- try a serial 1:2 dilution series of a representative sample and identify the dilution range over which the reaction comes to life. Once you know your optimal dilution range for each target of interest - work within that range...
Hi
In my laboratory, a 1:10 dilution of the cDNA extract is used routinely. We only try other ratios if this does not work.
Regards
It definitely seems like something in your reaction is not optimized. What do your melt curves look like? It's not uncommon for commercial or previously published primer sets to work slightly differently in your own lab due to RNA extraction, Reverse Transcription reactions, and simple pipetting technique. If your RNA is of good quality (260/230 and 260/280 ratios check out) and good integrity (2 strong bands on an RNA gel) and you don't see inhibition (prepare a serial dilution covering 5 logs of concentrations -- if you see the Cq values coming up later than expected at high concentrations, you might have inhibition), you could try running a temperature gradient or vary the concentration of forward and reverse primers. There is an example of how to optimize your primer concentrations here: http://www.sigmaaldrich.com/technical-documents/articles/biology/assay-optimization-and-validation.html
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