Hello everyone, I am working with qPCR and relative gene expression study of some candidate miRNAs. How many methods are there which can be used to normalize the PCR data? I know most famous is by normalizing with reference endogenous control, but if someone experience with some other methods then, kindly let me know.
Thank you and looking forward for your answers.
Hello,
I strongly suggest you search for a few review articles in e.g. google scholar. You will find excellent reviews by M. Kubista, M.W. Pfaffl and many others. Once you have gathered a background knowledge and have a specific question, please do come back to ask and I'm sure you will get a lot of answers.
Also, I really recommend you to read reviews on experimental design first, related to your species of interest, your experimental conditions and the miRNAs you are testing, so you design a proper experiment that you can normalise.
The TATAA Biocenter can be a great starting point:
http://www.tataa.com/research/publications/
You will notice that qPCR approaches for miRNAs are not the general ones you can find for mRNAs, and the articles you find in the above link will prove quite useful.
Best,
Ruben
I agree with Ruben, there are some excellent articles out there on normalisation for qPCR for mRNA and miRNA.
One article that I can definitely recommend is: Article Normalization strategies for microRNA profiling experiments:...
In my experience, it is worthwhile using a panel of endogenous reference genes as some typical reference genes (e.g. GAPDH or ACTB) may be affected by your treatment conditions.
Thank you very much Dr. Ruben for sharing your experience and suggesting valuable review articles. I will go through this.
Thank you Regina for recommendation, I have used some endogenous control for normalizing my target tissue miRNA expression (FFPE sample) but unfortunately didn't get significant P-value, that is why I am searching for some other possibilities too.
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