NTC's in RT or just qPCR? - (Jun/21/2006 )
I know I should know this by now, but in my readings it isn't clear to me..
I have been preparing my NTC's so that they undergo RT with all the RT components, but no RNA. Then, I use a dilution of this sample similar to the dilutions I use for real samples, and add it to a qPCR Master Mix.
However, in a recent collaboration with a different lab, they told me that their NTC's are created with qPCR Master Mix and no cDNA; their NTC's do not go through the RT reaction.
Which is the better way to do include an NTC?
hmmm good question
my NTC has H2O instead of cDNA with the master mix; it is a test of the qPCR components for contamination
my NAC has RNA instead of cDNA with the master mix; it is a test for gDNA contamination of my RNA prep
I have never done it the other way, but it makes sense for sure; tests for contamination of RT componenets
I have always two NTCs: one has water instead of cDNA to test for contamination and one without cDNA and without primers (I always check for primer dimers). I think it doesn't hurt to do it the way you do. But it doesn't hurt either to do it with water instead of cDNA as well. Maybe you could do both?
Thanks for your replies, it seems like either way is acceptable. I'll probably stick with my current method for now, then.