Any advice, regarding which internal control ( housekeeping gene) like GAPDH, HPRT to be used for macrophages, ( PBMCs derived macrophages) ?
I am assuming you are looking for a gene to normalize your qPCR data. The gene of your choice should be stable (same/similar expression) under your experimental conditions. Also its expression level should be similar to your gene of interest. HPRT is good for genes with lower expression, GAPDH for genes with higher expression. However, you should test it first.
Thank you for the reply. I was wondering whether its a big change when in one stimulation if the GAPDH Ct value is around 18.9 and in other stimulation Ct value is like 19.8 ?
As a raugh estimate if its 1 fold change its not much of change. But preferentially test 2-3 HKGs and pick the best one. Best one would mean showing less variation among duplicates and samples. You can also run a statistical test, for ex Grubbs outlier test, http://graphpad.com/quickcalcs/Grubbs1.cfm, to see if any HKG Ct value from all wells, including experimental and control samples, is an outlier, if so only this can be excluded. Generally this does not happen to be the case but to be on the safe side, one can run this test to cross check.
I would also recommend to test Bactin as a housekeeping gene.
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