In our lab we are performing an miRNA isolation from rat liver with Trizol (including the extra step for helping in the recovery of miRNAs) and then we perform the RT reaction with Taqman microRNA reverse transcription kit and specific RT primers for our miRNAs of interest. Then we perform the qPCR reaction with the PCR specific primers too, also from Taqman.
We never had this problem before but, in this experiment, working with the same tissue and techniques our Ct values are really variable between and inside groups and also our endogenous control (snU6) is really variable (from 20 to 26 cycles) so it is totally not reliable.
Has someone experienced the same problem? Could it be that we are loosing miRNAs during isolation (if it was the reason, it would be the first time because it has worked for us before). The ratios in nanodrop for tota RNA are >1,8 for 260/280ratio and >1,7 for 260/230 ratio and the concentrations are always above 1000 ng/ul. We always work with RNAse free environment and with the same kits and protocols as before.
Thank you all in advance.
Unsure about the exact problem you might be having but when we faced sporadic Cts from our miRNA expression work we went back and DNase treated everything to ensure no gDNA contamination.
In addition to this, ensure you have not added to much TE to the reaction mix. When doing your RNA to cDNA conversion, make sure that you are adding the same volume of RNA to each reaction. If one has 5uL of RNA in TE and another has 8uL, this will make a big difference in terms of the cDNA conversion efficiency, due to the differing levels of EDTA.
Hope that helps.
Dear Michael, I don't think we use any TE, as far as I know. We elute RNA with RNAse free water and we also dilute it for the RT reaction with water. The only thing we can do about your suggestions is try the DNase treatment.
Thank you very much for your help!
Hi Aïda, happy to help. If I think of anything else I will be sure to let you know. Hope the DNase treatment helps though.
Hi Aïda, you also may test your qPCR machine - time to time they have problems and need service.
Do you have RNA from pervious experiments that you indicated worked well previously? If yes, then test the performance of the kit. Are these livers from a specific treatment arm of a study? The 260/280 and 260/230 look good but have you run the RNA on a gel or a Bioanalyzer to get an idea of integrity?
For how many micrornas you performed the cDNA synthesis at the same time? I already had problems using more then three. Then i stablished just two for each cDNA.
Thank you very much to all of you! We finally repeated the RNA and microRNA isolation with a specific kit for microRNA isolation and PCR were much better. Gerardo Silva, we perform one cDNA synthesis of one microRNA in each tube, so this couldn't be the problems. Jeffrey the integrity was okay for RNA but for microRNA we didn't have the tool to test it, We think that our problem was in the RNA isolation protocol, we did it with clorophorm/ethanol/isopropanol method and maybe we were losing microRNAs during the process,
Sergei, our machine has been tested recently so this was not the problem.
Thank you all anyway for your suggestions, they are good to know to take in to account for future experiments!
Good to hear you got things worked out. I have seen by adjusting the salt (GuSCN) and Ethanol that I can certainly affect the species of RNA that are recovered. Best of luck in all your future miRNA research.
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