I am working on expression levels of beta defensins in epididymis. The RNA is fine as far as A260/280 or A260/230 is concerened. I fixed the RNA conc. at 2000ng while going for cDNA conversion. For qPCR it was diluted 3x. However, the reference genes and the genes of interest show Ct in the range of 29-32. I have repeated the experiment fixing at 1000ng and using it undiluted and got the same results. Any suggestions would be appreciated.
Dear Vipul
- Have you checked to make sure your RNA is un degraded by running on an agarose gel or Agilent biochip ?
- Have you pre screened your primers by agarose gel electrophoresis and made sure your annealing temp in qPCR is not too high ?:
- This tends to be Tm-2C but I verify my primer annealing by screening on an agarose gel and setting up 6 x reactions from Tm-3C to Tm+2C varying the annealing temp from reaction to reaction by 1C intervals; That is Tm-3C for one set of duplicates; Tm-2C for another etc...up to the 6th set of duplicates @ Tm+2C
- I then run an a 1-2% agarose gel and look for the Ta with a specific single band but also the strongest band (i.e. highest efficiency of PCR)
- I then carry this temp over to my actual sybr green 'customised' qPCR reaction
- I concur with Chinmaya: This is likely to a PCR efficiency issue so select the optimal Ta as I have suggested and then perform an initial standard curve reaction and make sure your verified primer efficiencies are > 85%
- Incidentally, I say up to 2% agarose gel for pre screening because your products should ideally be ~ 100-200bp and DEFINITELY no more than 300bp
Hope this helps
Dear Laurence,
Besides nanodrop, I have checked the RNA quality on agarose. However, I do not get the conventional 18S, 28S bands but a smear-like continuous column of transcripts, which i am told is fine (I am not sure if it actually is ). I decided the Ta of my primers by a gradient PCR starting from Tm-5c to Tm, so I can say that I am sure about the Ta of my primers.
Dear Vipul
- That's good. I would say that a smear is OK providing you can see some semblance of the 18S and 28S bands
- If your smear approximates to where the 18s/28S ribosomal subunits should be then yes for RT/qPCR you are probably fine
- However, if your RNA is a giant blob at the bottom of the gel then you could have problems
- To offset degradation - clearly you have some - I would:
- make cDNA using random primers
- Bias your primers towards the 3' end of your transcript and in particular the last 1-2kb of your transcript
- To elaborate, RNA tends to degrade from the 5' end so if your amplicons are derived from the 3' end then even a degraded population might be fully intact in this neighbourhood
- Thus, your primers will have a full copy number of transcripts to amplify from and this will not only maximise PCR efficiency but also ensure that the relative copy number of your gene in different populations (cDNA preps) is maintained thus rendering your qPCR valid
Perhaps too obvious, but besides the above you might want to check the performance of your RT reaction itself. Perhaps the problem lies there? I suggest to perform a control experiment with a different sample (e.g. derived from a different tissue). If there is no or little cDNA produced... then your Ct will remain low. And as suggested really check for fragmentation, e.g. by bioanalyzer. As a side note, I found that random decamers sometimes can help to increase the yield, thus giving lower Ct numbers when dealing with fragmented RNA. Also check if the expression of your genes of interest is not low (gene expression databases, BIOGPS, REFEXA, NCBI GEO profiles)I've seen people trying to look for RNA that was simply not there....
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