40 cT values any idea how to lower it? - (Sep/05/2011 )
R18S analyzed simultaneously show ~10 cT values, but I'm getting 40 cT values for this particular gene which is supposed to be elevated (we are very sure). So we tried with 3 different primers (of the same gene of course), but still the best we could get was 36.. 36.. and 90% of it are still at 40.
I started with RNA from single population of 50,000 cells, had only around 25ng/ml RNA.
Did 2-step RT-qPCR. RT was 20ul volume, qPCR was 10ul / well.
Today I will try to use 4.5 ul of cDNA in the qPCR, but that should only reduce the cT value by.. Hmm 0.5, 1, 2, 4... around 3 cT values?
What are your thoughts? I know I could use a higher population of cells, these are from 24-well. But in another cell line, similar treatment give very good cT value, like 20. For this same gene that we're now having problems with.
For your high Ct values, the 18S reference is too abundant, if you don't want to use a different reference gene, that has comparable abundance, limit your 18S primers, 10Ct is way too low for quantification.
25ng/ml is really too little, did you mean 25ng/ul?
How much RNA do you put in your RT? How much RT reaction you put in qPCR?
Using more undilluted RT in qPCR reaction is not a good idea, it contains molecules that may inhibit PCR, usually 10% is the recommended maximum.
So you say you have different cultures with same amount of cells (and same Ct for 18S? but remember that Ct below 15 are not valid for quantification) and you get high Ct for one but not for other? That points out there is only a low abundance of your target gene there, I'm affraid.