qPCR standard for lipase genes - (Mar/02/2015 )

Dear all

I have already extracted my RNA and converted them into cDNA. I havent done any cloning into expression plasmids. Is it compulsory to clone the bacteria into these expression plasmids?

How to make standards from cDNA? Can anyone offer some guidelines.

Absolute or relative quantification?

You are asking for standard, so probably absolute, but question is if you really need it.

RNA standards (expressed from plasmids) are rarely used for mRNA quantification, only when no other option exists (i.e. precise quantification of viral RNA), mostly cDNA standards in a linearized vector mixed with some dummy NA. Or most often quantification of genes is done by relatively quantifing it in comparison with control sample.

But if you need the absolute quantity, you need a standard.

Hi currently I'm using a absolute quantification, hence do I need to clone the gene into a vector? Or is it possible to use the purified PCR product and dilute it for standard?