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Real Time PCR internal reference (housekeeping) gene in E. coli


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#1 mnqcljsm

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Posted 27 January 2010 - 05:29 AM

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

#2 k_undertoe

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Posted 01 February 2010 - 08:49 AM

...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,

...

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


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.




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