housekeeping or not housekeeping? - (Jun/25/2007 )

Hi everybody

I'm new in the forum and absolutely new in real-time PCR and full of doubts, maybe anybody can help me...
I'd like to compare HSP gene expression beetwen treated and untreated samples setting gene expression in controls as 100% and relating gene expression in treated samples to it. Do you think I can do that but overall can I do that without housekeeping gene?
Thanks in advance for your help,
cheers
Raffaella

yes, you can do that, but also no, you can't do it sensibly without a housekeeper to normalise on....

mike

In case you don`t want to use a housekeeping gene you have to normalize your samples against a unit mass (cell number used for RNA isolation or µg nucleic acid), which means accurate quantification of your starting material. And even if you set up your reverse transription reaction with identical amounts of RNA for treated and untreated samples, efficiency of cDNA synthesis can differ from one sample to another, leading to different amounts of cDNA after reverse transcription and therefore in your PCR reaction.
So in your case you can`t be sure that there are identical amounts of cDNA in your treated and untreated samples and therefore you cannot compare them.
So it`s much easier to normalize your samples against a housekeeping gene, compensating for different amounts of cDNA input and no need for accurate quantification of your starting material.

ok
thanks to both of you...

Raffaella