Dear colleagues,
I have a problem in my RT-PCR reaction, recently. I have used my PCR primers for ERK1 and ERK2 genes for several time and the results was OK. in gradient PCR reactions sharp product bands were always seen. But recently I can't see any Bands in my PCR in both two genes while b-actin band is always achieved. and it means my PCR reaction is compeleted and all PCR components including PCR master mix, c-DNA and thermal cycler work correctly. So what is the problem? I also can not see any bands in recent gradient reactions and I also have bought new primer pairs for Erk1 and Erk2 genes.
Have you changed samples or cDNA synthesis conditions? Is there any way your most recent RNA samples were partially degraded and b-actin is just more stable than ERK1/2? Or are your samples exactly the same as when you got positive results?
It seems either your cDNA synthesis has not been efficient or you have had RNA degradation. I would make new cDNA (as test) and if this does not work extract RNA again under RNAs free conditions. Sometimes changing the reverse transcriptase might be helpful.
Thank you for your guidance. But the purity and OD of extracted RNAs were measured using agarose electrophoresis and nonodrop assay, respectively. all parameters seemed OK. I am fully confused....
You might also consider cleaning up your RNA after extraction, in case there is still some residues available which might inhibit the subsequent reactions.
So, your RNA seems OK, but have you checked the purity and integrity of cDNA? Also, is there any chance that your primers got contaminated? Do you made alicuots? The recommended storage for long-term is -80ºC, short-term -20ºC. Hope it helps.
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