If you have not used the same amount (ng) of RNA for cDNA synthesis, but you have diluted the samples equally for qPCR analysis, could you get reliable results, as you normalize the results with HK genes and control samples?
If you have less than 500 ng of RNA it is worth it using it or it will be already too little concentrated for qPCRs and Ct values will be very high? If not what would be the best dilution?
The answer to your first question is yes, if youre using a housekeeping gene to normalize you don't need to quantify the RNA before hand. I usually do it anyway because its easier to have more similar TMs for the housekeeping gene.
For the second question, I usually use between 250 and 500 ng of rna to make into cDNA. The 250ng gives me ct values of around 24 for GAPDH and 500 is closer to 15.
Hope that helps!
Ermela
I agree with Ermela. The reference genes should normalize your signals and account for any variation in the template.
In regards to your second question, less than 500 ng of RNA should be more than enough. You should get ~20ng of cDNA with that amount of RNA and you should get ct values for GAPDH within 18-25.
Cheers!
The intra- and inter-normalization of the Cts will account account for the variations in between conditions(control vs test) but it is always recommended to start with the same concentration of RNA used for the reverse transcription reaction. We dilute the cDNA for better efficacy of the PCR reagents.
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