is it wrong that my reference gene expression is lower than my target gene? i used CPR gene for my references, does it make problem with my analysis?
the my reference gene CT is greater than my target gene?
i used several internal gene but had problem such primer dimer.
the CPR was good but is that ok to use it as referense gene?
thanks for your helps
1. May be the efficiency of amplification is low. You need to optimize to temperature that gives best Ct values and rules out primer dimer formation.
2. It is no problem if reference gene expression is lower than target (I hope this is decided based on ct values comparison), you just need to ensure that reference gene expression is uniform in all samples/replicates.
If the expression of your reference gene is not significantly different for all your samples then you do not need to worry about this. It is not necessary that the reference gene has the highest expression of all your analyzed genes.
It is not mandatory to your reference genes be up or down -regulated compared to target ones. What you must pay close attention is your primer/reaction efficiency (the closer to 2, better) and if the values are constant across same samples and same conditions. Set a treshold to your SD among your replicates, such as +/- 0.5 SD. Thus, if you get values outside this range, than you must check your primers and experiment setup.
I agree, Reference gene expression may be lower than your gene of interest provided it's expression is stable. Your assay is well characterized and validated for all your assay conditions.
I agree with the rest of the replies. No matter how abundant is the transcript of your reference gene, the crucial fact is that its expression has to be stable throughout all your samples and treatments, under your experimental setting.
Low expression of reference genes is not a problem but the reference gene expression must same level expression in all of your samples.
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