I have been using cDNA from 1ug of total RNA isolated with trizol. Also taking care with RNAse
An original answer to this would be: "I can't - there must be black magic afoot" ;] But - that probably won't fly - since black magic is hard to prove by most statistical methods (i've read anyway).
The real possibilities might be:
Freeze-thawing sometimes gives you better Cq values since some inhibitors are freeze-thaw labile (mysteriously lose their inhibitory capacity after freeze-thawing a few times).
And/or, cDNA may suffer some from freeze-thawing as well - giving you worse Cq values over time. You also want to be sure that DNase isn't still actively degrading your cDNAs. If not properly inactivated, the DNase could perhaps still be chewing on your cDNA before use.
Also there is a "cusp dilution" you may be dealing with... the same sample, same dilution will give you a different Cq value depending on the influence of the inhibitor(s) after a different number of freeze-thaws. This will happen if you are using the cDNA too concentrated in your qPCR. If you are on the cusp, the Cq can be very schizophrenic. If you have inhibitors - they will of course be most concentrated in your most concentrated (least-diluted) cDNA samples. Too much cDNA is an inhibitor as well (template inhibition it has been called).
Do a dilution series of this cDNA sample (serial 1:4 diluion series) and see the shape of the standard curve that results. If there is inhibition, it will rear its head with the most concentrated dilutions (Cq values will be higher than they should be), then, inhibition will let up after a certain dilution point. (Actually, best would be to do serial 1:2 dilutions to see all of this). The point at which the phenomena disappears, is the point at and after which you should always work (standard curve and sample-dilution-wise for your particular sample cDNA preparations).
The other possibilities are: different master mix was used (different age or batch or assembly), machine issues, white magic, lab-mate messing with your pipets after 3 a.m. just to get back at you for last week (hopefully not).
On pipets: be sure to weigh water to prove that you are pipetting the amounts you think you are. I.e. if you are adding 1 uL of sample to a 20 uL reaction, and you actually added 1.2 or 1.5 uL of sample, you will get a different Cq value from the same sample... 1 uL should weight 0.001 g on a good analytical scale. Hopefully you're not dealing with amounts like that though. (I'm always adding 6 uL sample per 25 uL reaction -- amounts pre-checked by weighing water first; each time).
I've exhausted my bag of tricks for now... if I think of any more... I'll leave my witch-doctor's house immediately and run straight to the computer.
Hope some of this helps/seriously
Thank you! There are lot of possibilities, I also think thant the machine is not working propperly, because Ct varies from 10 to 30 in one week with the same sample, same standards, same genes....I am confused but will try again with other machines. Thank you!
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