I intend to carry out relative gene expression analysis for genes conferring antibiotic resistance using Taqman based qPCR assay. I want to quantify the mRNA level from E. coli and K. pneumoniae grown in presence and absence of the antibiotics.
Now, I have confusion in selecting reference gene. So kindly suggest me which gene from this bacteria will be best suited for this purpose?
I have gapA gene in my mind for serving the purpose.
There's no straight answer to that question. If you want to use gapA, first run gapA across all of your treatments to make sure there is no significant difference among the treatments.There is considerable evidence that in many cases, a perfect internal control gene doesn't exist, and multiples should be used.
Vandesompele et al (link below) developed GeNorm, to geometrically average the values for multiple genes to get more stable values. You can also use Reffinder for your candidate internal control genes and it will analyze them and advise the best combination of genes to use.
In short, use RefFinder, GeNorm, or something similar to determine which background genes are most stably expressed.
Dear Anne Laarman,
Thanks for your reply.
My relative gene expression study includes four target genes. I am unable to afford multiple reference genes due to lack of funds.
In this present situtation would you still stress upon multiple reference gene? Or is there any other way to address this issue?
Looking forward to your reply.
Please check this paper out for recommendation : http://www.biomedcentral.com/1471-2199/12/18
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