I am working with embryos and oocytes, i am confused about how many an which internal controls to use for qPCR?
in literature, i have seen only one internal control has been used and study is published in reputed journal. but i am not getting clear idea.
Please suggest.
Thank you
Hello Riddhi Kirit Pandya,
First of all, what do you call "controls" for RT-qPCR? Housekeeping genes ?
Indeed, results (the relative expression level of each gene) need to be normalized by two housekeeping genes (and not usually only on one); as recommended by the MIQE guidelines (Bustin et al., 2009) with the use of geometric mean to generate an accurate normalization; available at http://www.rdml.org/miqe.php.
Then, be careful to evaluate RT-qPCR results visely (fold-change, relative expression, the 2^(-ΔΔCt), etc...). After computing your RT-qPCR slopes & curves data you obtain your 2^(-ΔΔCt) data. During this procedure, you have to normalise all your data on housekeeping genes (a couple) and on a "control experiment" (associated as the "1:1" value) to compare them as ratio-values: data are presented as relative expression of your genes. --Be careful to use geometric means at this step!--
If you want more more precise and detailed informations, please see Livak and Schmittgen (2001).
Dear community of RG, be free to add modifications & precisions to my Answer, especially on commonly used internal controls to study mice oocytes and embryos!
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