quantifying cDNA for Real Time PCR - (Feb/24/2006 )
How are users determining the amount of cDNA that you have for your real time PCR?
We take an RNA estimation after DNase treatment by UV , calculate the ng RNA/ul, and then calculate how many ng of RNA is needed to make 500 ng in 10 ul, and then do an RT reaction. We then purifiy the cDNA with a Qiagen kit, and re-estimated the cDNA. From this value, we calculate for 10 ng/ul for the Real Time PCR. Our CT values have been high, so we are wondering if the amount of cDNA we think is going into the reaction is actually less than 10 ng/ul.
I think perhaps there are many different methods, based on what I have heard from colleagues and from what I have read here that others have posted.
what works for me:
1. quantify RNA (using a spec).
2. adjust volume so each sample is exactly the same concentration.
3. RT each sample using same specific volume of adjusted RNA
4. use aliquot of cDNA for real-time (the same volume for each sample; with triplicates and ROX used to normalize as much as possible)
my housekeeper generally gives pretty tight Ct values across all samples
I never do quantify my cDNA directly. so tell me please why do you purify your cDNA after the RT step, then quantify, then go to realtime? that seems like a few additional steps, where perhaps sample can be lost?
Thanks. We purified our cDNA based on advice from a colleague who found that this step
lowered the CT value by removing the excess dNTP's etc from the cDNA synthesis. We do
lose alot of sample though when we do this.
If you are basing the amount of cDNA that you have from your RNA estimate, does that mean that
the efficiency of the cDNA synthesis is close to 100%?
from all my reading / questioning others / pestering tech support prior to starting real-time, it is difficult-to-impossible to control the exact efficiency of the RT reaction from one batch of samples to the next. that is one reason why I don't standardize based on the amount of cDNA obtained, but instead use the amount of input total RNA. another reason would be the potential for sample loss.
I typically use a very small amount of the cDNA for each set of replicates, and supposedly my system doesn't leave much in the way of interfering substances.
there are several others here who do real-time. I would perhaps check here , here , andthere
pBluescript and Mistic Matt always seem to have excellent advice, and they may be able to add more here for you?