Hi,
My reference gene (HKG, RPO) is stably expressed (Ct= 21) across different samples however the Ct value is kind of similar to the Ct value of my target genes (varying from 20-25). the difference is very less and in some case it is zero. Can this be used for normalization?
what should be the cycle difference between reference gene and GOI?
Thanks.
Hi,
The only thing that is important is that your reference gene is stably expressed among the samples/conditions you are analyzing. If the deltaCT is close to zero it just means that your gene of interest is expressed at roughly the same level as your reference gene. Ideally you would use more than one reference gene (see guidelines in http://clinchem.aaccjnls.org/content/55/4/611.long), although it is sometimes hard to find more than one.
If you are using the ddCT method for relative quantification, it is very important that all primer sets have the same efficiency as those for your reference gene. So make sure you do dilution curves to check this before yuo analyze your experiments.
bw
Rob
Thanks Rob.
It is clear now and as you mentioned the primer efficiency was already checked before the sample analysis.
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