Dear all,
I'm new in RNA extraction, cDNA synthesis and RT-PCR.
I have isolated RNA from 20 samples of Sorghum by Triazol and continued with SV total kit (Promega), then measured on Nanodrop.
the results of purity (A260:A280) were from 1.90 to 2.15, and the results of concentration, the highest one was 467 ng\µl and the lowest one was 62.8 ng\µl.
The bands on gel were good, sharp, clear, not degraded and not contaminated with genomic DNA.
My questions are:
1. Are the results of purity and concentration good?
2. Can I convert the lowest concentration of RNA (62.8 ng\µl) to cDNA, Will it give a good result in RT-PCR?
3. what is the best concentration to synthesis cDNA 1000 ng or 800 or 500 or 250?
Thank you
Nourhan Aiman
Hope you are fine.
Purity of RNA is good. You can convert the lowest concentration of RNA (62.8 ng\µl) to cDNA by RT-PCR, but need to adjust the RT-PCR reaction mixture concentration according to cDNA synthesis kit. But incase of RNA concentration (62.8 ng\µl) to cDNA is not good for research experiment. For this reason need to isolate RNA again. 600ng/800ng is the best concentration to synthesis cDNA.
Sharmin Suraiya, PhD
Hi,
the bigger concern for cDNA generation is linearity not the lowe rinput. As you have at least 63ng/µl and most kits allow 10µl input you could normalize your inputs to 200-500ng/rxn.
For qRT PCR this would meant, that you could normally use up to 10% of the cDNA reactions per qPCR so you would have ~50ng input which should be fine for all transcripts (as long as your RNA integrity is high enough). Of course if you need to run a lot of different qPCR assays you might consider to use more but i my hands cDNA synthesis with more than 10ng RNA input works robust.
Sven
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