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Forum for discussion about GenEx and qPCR (quantitative real-time pcr)
Here we discuss aelection and normalization of reference genes
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I am fairly new to the technique quantitative RT-PCR so I have a question I hope you guys can help me with.
In my experiments I have tested the expression of my receptor gene in different tissue and developmental stages in the honey bee. My reference gene is Actin.
My data show, that my receptor is highly expressed in the brain of the honey bee, as I would expect. So far so good.
My problem is, that my data show, that there is nearly twice as much of my receptors mRNA in the brain than Actin mRNA. I would not have expected this as Actin is abundantly expressed in nearly every cell, so I am not sure this is realistic.
So my question is: Can I trust my QPCR data? and what could be the reason for my data showing nearly twice as much mRNA compared to actin mRNA.
You cannot compare Cq values of two assays because they have different sensitivities unless you calibrate them (i.e., performing absolute quantification). Hence, you do not know the relative expression of you receptor and Actin in your experiment.
When comparing two genes in different tissues one has to be careful with interpretation. Certainly, it is a relative expression measurement, but what do you expect from Actin expression when you classify it reference gene in the context of comparing tissues? Surely, Actin expression levels will be different in different cell types. When comparing tissues other means of normalization are usually preferred, such as normalization to cell count (or DNA), total amount of RNA, average of multiple genes etc.
Comparing developmental stages is also tricky; there may be no gene with constant expression throughout development. More pragmatic approach is then to compare expression profiles, see:
M. Kubista, B. Sjögren, A. Forootan, R. Sindelka, J. Jonak, JM. Andrade. Real-time PCR gene expression profiling. European Pharmaceutical Reviews (2007), Vol. 1.
(available on www.tataa.com)
2 posts • Page 1 of 1
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