cDNA not enough-variability - (Oct/31/2013 )
I'm starting a new qPCR experiment and I would like to minimize the intra-plate and inter-plate variabilities, so I'm having several doubts.....
I've synthesised my cDNA (20 ul/diluted 1:5), but I've just realized that it won't be enough for all the genes that I want to analyse (30 genes in triplicate- 2ul each well). I know that it may sound silly, but I have two options but I'm not sure that I will avoid the RT variability:
1. do a second RT from the same patient and finish the last batch of genes with the new cDNA.
2. do RT from the same patient and mix the two together solutions together (old and new RT)
what do you suggest? Do you think that it will compromise my data?
Thanks a lot!
You can remake the cDNa and compare it to your previous run. Just make sure you use a control and that you keep your conditions consistent. If you are worried, run a couple of different normalizes during each qPCR and this will help convince you that your cDNA is consistent.
Another option for next time..
As I understand, you already did some of the genes with the first batch, but you didn't specify how many and what have you done and what volume was left.
If you run just few (one) genes and given you will have Cts low enough, then you would have enough cDNA to dilute further, to make all genes from the new dilution and discard the ones you run before. But sometimes the Cts are high enough already and further dilution would increase variability too much.
Otherwise, in relative quantification, you always need to use the same cDNA dilution/batch... for the target gene(s) and their respective reference gene(s). So you could run half of the genes with a reference and then make a new cDNA and run the rest of the genes again with reference, and compare old batch genes with old reference and new batch genes with new reference. There will be only one risk, that the original RNA got more selectively degraded, on the second batch, but..
Of you could join both and redo all genes with references. But certainly you can't run half of the genes and reference, and then with the new cDNA run the rest of target genes, and use the old reference values for normalization.
Just always keep in same conditions, what you need to compare together.