Diluting your cDNA for qRT-PCR? - (Oct/13/2006 )

... do you guys dilute it just prior to running a real-time [i.e. take 5ul and mix it with 45ul] or do you dilute the whole batch as it comes out of the thermal cycler... i only ask as i find that if i'm running a small number of samples i might not be getting a representative sample of cDNA by pipetting only 4-5ul

I always dilute it immediately after the RT reaction is finished. I find I get more uniform sampling every time this way. Good luck!

thats what i was thinking, my sampling can be quite variable... makes sense really... anyone think this is a bad idea???

I have some bad feeling about using gene of interest [GoI] Ct value lower than housekeeping gene for this argument: What you are measuring [GoI] is not a variable, where as your housekeeping gene may become variable IF AND ONLY IF at some point your GoI has higher Ct that the housekeeping gene. In this case, it is rather advisable to reduce the expression of GoI so that it is 2 fold lower than the housekeeping gene. This is especially true if your system is a eukaryotic expression system.

This past week, I was doing some efficiency testing. On Day 1, I made 5 serial dilutions of cDNA from 20 ng/µL to .002 ng/µL. I retested these diluted samples on Days 2 and 3, and found that my efficiency decreased by about 10-15% each day. On Day 3, I also did an efficiency test making fresh serial dilutions from the stock cDNA sample, stored at 100 ng/µL, and got high efficiencies similar to those on Day 1. I concluded from this that diluted cDNA is less stable than concentrated. Anyone find similar results?

absolutely sol, i used some diluted cDNA which had been frozen down for 2 weeks and it gave me Ct values about 4 less than those freshly made up!