In RT-PCR for mRNA expression study, we take the equal amount of RNA for this, but some researchers, on their personal experience, take equal cDNA instead RNA. So, which is better and more accurate?
both can be taken and will work because after all you need to validate the expression of the gene of the interest with the Housekeeping Gene of the same sample which will remove any errors or bias.
both can be taken and will work because after all you need to validate the expression of the gene of the interest with the Housekeeping Gene of the same sample which will remove any errors or bias.
I usually quantify my RNA by NanoDrop then make duplicate cDNAs for each RNA template. This helps average out any inconsistencies in cDNA synthesis efficiency. Make sure you use at least 2 house keeping genes to normalise your qPCR results.
Quantifying RNA and using equal amount of RNA for RT is much better.
RT alone is already a question mark. Even if you make a duplicate RT from the same sample, there will be differences in these two reactions. Therefor using different amount of RNA for RT and then equalizing cDNA for qPCR is a big NO for me.
If you have a Qubit, it is very good to quantify cDNA with this flurorimeter. I use this to quantify dna, rna e cdna..the Qubit is very very precise thanks to dyes.....and for the purity (280, 260, 230) I use a nanodrop or similar. Best....and merry Christmas and y New Year, Olga
In case you are planning to quantify cDNA after reverse transcription keep in mind that you will need to remove non-incoporated dNTPs from your sample otherwise you will not be able to accurately determine the amount of cDNA at 260 nm with a nano-drop. In my opinion an additional cleaning step would likely do you rather harm than good. Therefore, I use column purified RNA for my first 'standardization' via UV-VIS going into the RT reaction and an appropriate housekeeper gene in my actual qPCR experiment.
If you use a quubit, you have not this problem as reported in a previous answer, i.e. non-incorporated dNTP.
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