I'm working with a new primer for RT-PCR. Even after running gradient PCR with +10 to -10 deg C from annealing temp, we cannot get any outcome. We blasted the primer and found it to be specific. What else can be done to get the desired product after PCR amplification?
Thank you Nikonov. It will be more good if u can suggest some paper describing this method (or) if u have complete protocol, and if you can send it to me, it will be more more good to understand it completely. Many thanks in advance-
Are you carrying out the reverse transcription step with random primers, oligo-dT, a mix of the two or with a target specific primer?
If target-specific, it is just possible that the RT step is very inefficient, because the primer happens to bind to a strong stem structure in the RNA. You may ant to analyse your primer binding sites via mFold. Use of random primers will also help.
It is very unusual not to get anything at all for the PCR step. I assume that you have a positive control that you know expresses your target RNA. You should recheck the primer sequences, make sure you resuspended them in the appropriate amount of water/TE; you may be using too little/much of primer and, depending on the master mix you may want to modify the PCR conditions.
Dear Stephen Bustin.
We are reverse transcribing using random primers, oligo DT mix. Next we do have the positive control that contains target RNA, and blasted primer sequence, found it to be matching the transcript variants of the RNA. However we should work with primer conc and also PCR conditions.
Thank you for your valuable suggestions -
maybe consider trying RNA concentrations too. The reaction should not be overloaded. Maybe also try using your reverse primer only in the RT step to use it as specific reverse primer. You could check if you can add additiva (more DTT in RT reaction). Then i would advise to use the single stranded cDNA fresh as possible and check your PCR conditions. Here you could try to use a higher buffer concentration too (1.5-2X Buffer, if its a standard KCl based Buffer). good luck
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