what is the best time to convert rna into cdna? I have extracted my rna using trizol, stored it in -80 for maybe a month- month and a half before I synthesized my cdna. Does it affect my cdna and hence, the qpcr?
I dont think so that stored rna will affect your qpcr. One question is are you using any kit for cDNA synthesis?
It is best to synthesize cDNA from fresh RNA (without freezing). cDNA is more stable than RNA and you can store cDNA at -20.
Freeze and thaw damages the RNA. It affects the RNA integrity which can be measured by gel electrophoresis or by bioanalzyer (but NOT by nanodrop). Low RNA integrity means the RNA has broken down into smaller fragments and degraded. So, cDNA synthesized from that is not good and you might have difficulty to detect low abundance target by qPCR.
What you could do when you have samples collected at different time points:
1. store cell pellets at -80 or
2. store RLT (qiagen kit) cell lysates
Extract RNA for all the samples at once in the morning, keep on ice and convert all to cDNA in the afternoon. This ensures all the cDNA have the same quality and not affected by the freeze and thaw of RNA.
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