I have been doing rt pcr from different mice tissue for b actin for 3 times now. Every time, certain tissues are amplified and others are not. everytime I do new cDNA. I quantified my dnase free RNA and its conc. and quality are good. What can I do to resolve this issue? Thank you.
Are they all the same strain of mice with different treatments? Sometimes, treatments can affect the expression of housekeeping genes. For example, ethanol changes the expression of Gapdh. You should probably pick a new (and a few) housekeeping genes and perform normalization using GeNorm, which will tell you which genes' expression are most stable across your tissues or treatments. Usually normalizing to 3 genes is sufficient. Look at this paper for guidelines on qPCR (http://www.rdml.org/clinchem.2008.112797v1.pdf).
Another possible issue is when you make a standard curve, you should make it from the same tissue in which your samples are being assayed. If b-actin is super abundant in cerebellum, for example, and you make your standard curve out of cerebellum tissue and b-actin is not nearly as abundant in nucleus accumbens, then it may not fall within your high and low standards on your standard curve. In this case, it is a bad housekeeping gene across tissues anyway.
If you are looking at b-actin in two different strains of mice, remember to check for SNPs under primer binding sites which may be present in one strain and not the other, thus leading to amplification in one and not the other.
Have you checked if your housekeeping gene (B-Actin) was influenced by the stimulation factors with which you are working?
Usually I run a screen test RT-PCR to see if there are significant changes in expression of my HKG (B-Actin, etc) in my samples after treatment (virus,etc.) vs. my control to make sure there is no differentiation, as Jo Lynne has stated.
For the most part, I've found 18S rRNA to be stable and a reliable target for RT-PCR housekeeping gene use.
Thank you all for your answers. Those are black 6 wild type mice with no treatments.
18SrRNA is also a good housekeeping gene. I use more than one housekeeping gene in all experiments to avoid such problems.
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