how to determine the amount of cDNA for qPCR - (Feb/23/2011 )

Since i only measure the conc of RNA, how do i know how much of RNA should i put so that the product cDNA is in the range of acceptable template conc?

We just put the same amount of RNA to RT reaction, usualy 1 ug (and use 2ul of 20ul reaction as a template). But important thing is abundance of your target gene(s), that may be too much or too little, you can't tell unless you try that.

We pretty much go with a 100% conversion efficiency for RT. If you put 1 ug/ul of RNA in, you'll get 1 ug/ul of cDNA. It's not exact but it works. 1ug is usually more than enough.

-RT

Trof on Wed Feb 23 18:12:29 2011 said:

Meaning use 1ug of RNA for RT in 20ul, then 2ul of cDNA from the rxn?
My extractions yield very low conc of RNA: 20-40 ug/ml. The max amount of RNA that i can use for a 25ul rxn is only 0.2ug.

hianghao on Thu Feb 24 08:04:14 2011 said:

Yes.
Well then you have to stick with 200ng (it could be enough, as I said, depends on the gene) or try other ways how to increase RNA yield. Or use TaqMan PreAmp Master Mix from ABI, to pre-amplificate your cDNA (but I have no experience with it).

Trof on Sun Feb 27 08:19:16 2011 said:

I see! Thanks for the information!