Standardizing cDNA concentration before doing PCR - (May/05/2015 )

Hi all,

I was told that I should standardize my cDNA concentration before going onto PCR, so as to ensure consistent results.

I have not done this so far, and my PCR results have been pretty good. But should I as a good habit/scientific practice?

Thank you!

Sakumi

I don't think this is necessary. Often cDNA concentrations are quite low, but that is fine for use as a PCR template.

I know this is late, but thank you for your reply!

I have recently noticed that I am getting very faint bands when my RNA concentration before RT is high (250-350 ng/uL). Otherwise, the PCR bands are pretty good when my RNA concentration before RT is about 100ng/uL. In the future, should I dilute the RNA to around 100ng/uL before RT to ensure good cDNA synthesis and PCR?

Thank you.

Sakumi 

Extracted DNA and RNA often has ontaminants that inhibit reactions. Often, diluting before reactions. or adding smaller amounts, will make a reaction succeed which would otherwise fail. Many or most reactions need smaller amounts of template than expected.

I have heard the same thing from our lab tech, thanks!

It's funny, my RNA extraction is suddenly yielding quite low concentration of RNA, going from ~100-200ng/uL to ~30-60ng/uL. I have not done anything different, and am using freshly autoclaved tubes and tips. It could be that I am extracting RNA at a different time point, where gene expression may be low, but I didn't even get this low with my naive animals... Any ideas why?