Hello,
I hope you are fine.
I met a problem in qRT-PCR. I prepared my cDNA from 1500 ng RNA. After I get cDNA I did not dilute it and used 0.5 ul from it but still my ct values for GAPDH is 27. I want my ct values to be around 15-22. It is strange that I did not dilute my cDNA so the cDNA is fairly concentrated but still ct values are so high.
What should be the optimal amount of cDNA/microliter in one well of the 96 well plate?
Please suggest.
Thank you
Best regards
Your concentrated sample might contain PCR inhibitors and that is the reason why you are getting lower CT values than expected. You should run multiple different dilutions to determine what is optimal ex. 1:10, 1:20 etc. this will also allow to assess how good your primers are since they may not be optimal for your target gene.
What cDNA kit are you using? The protocol should tell you the optimal amount of RNA to use. Ours requires 2000ng of RNA.
Did you measure your cDNA before use? What concentration was it?
What qPCR Master mix are you using? The protocol should tell you the optimal amount of cDNA to use. Ours requires 20ng.
In my opinion, could be because of the cDNA quality. You may check for the quality of cDNA by using standard primer for example for Bombyx mori we use A3 (cytoplasmic actin gene) primer and do PCR to see whether cDNA is fine to be used. Wish you all the best!
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