When I extracted total RNA from cell (tumor and normal) and I made one amount of RNA to convert to cDNA, I got no reverse transcription reaction. I diluted cDNA to 100 microliters, in addition to my cDNA work with a GAPDH primer, but when I studied gene expression by syprgreen in RT-PCR , the result was no amplification with my interest primer and GAPDH primer although I did RT-PCR two times and I got the same result.
Dear Asma
First of all check your cDNA conversion that was successfull or not (with spect. or runing on gel).
If it was ok change your dilution final volume and dilute it with lower volume.
If it was not ok, check your conversion process again.
Regards.
Dear Asma,
I am happy to know that GAPDH primer works with your cDNA, Hopefully you should first check the primer amplification with simple PCR. Do the electrophoresis on agarose gel then check for the amplification then use the same for the real time, If Amplified.
thanks
I did simple PCR with my interest primer , did gel electrophoresis and got beautiful band . also I did the same condition in rt-pcr but no amplification . I will try to change the dilution of cDNA
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