Real Time PCR internal reference (housekeeping) gene in E. coli - is rrsB suitable (Jan/27/2010 )

Dear all,

Has anyone experinece of gene expression in E. coli +/- heavy metals? I have some good data for differentially regulated genes based on normalisation with the housekeeping gene, rrsB, encoding 16s rRNA.

I worry that i have no sure way of knowing that its expression is constant in control and experimental samples and therefore using it as a reference is based only on assumption,

however, as this gene has been used elsewhere to similar effects do i simply put it down to a drawback of the technique and present my data anyway?

should i use multiple - eg. gapA and accD (metabolic and structural roles) aswell?

Aaaaaaargh...why is the issue of reference genes for relative quantification so difficult?

thankyou in advance,

best wishes

mnqcljsm on Jan 27 2010, 05:29 AM said:

Whenever you are in doubt about the validity of a housekeeping gene, you need to run a panel of housekeepers and then decide which one is the best one by comparing the reference sample to the experimental sample. If there is a difference of greater than 2 SD, your housekeeper is not valid as a housekeeper. I'm sure there is a paper out there somewhere discussing how to select and validate a housekeeping gene.