I have been differentiating multiple iPSC lines into NPCs and then into astrocytes. I would like to do some qPCR to further characterise my iPSCs and astrocytes but was wondering how best to choose a housekeeping gene. I've tried 3 different ones for all cell types from all lines (GAPDH, YWHAZ and b-actin). Should I try and find the least variability across cell type, or across cell lines? Or is it okay to use different housekeeping genes for different cell types?
Sorry if I've explained this very badly! I'd appreciate any advice. :)
hi Marianne King
All these housekeeping genes are good in nature for iPSCs work. To be specific if you provide the Ct values for housekeeping genes then members can give better suggestions. Usually between the range of 15 to 22 ct depending on the cell types. and on input cDNA, you have taken as a sample for the reaction. As always suggested that within technical replicates, the difference should not be more than 0.5 cycles and for biological replicate the value should be more than 0.5-1 cycle between assays for the sample.
Regards,
Saurabh
Though not specifically for IPSC or cell cultures, I think you would profit from reading the article:
Rocha-Martins M, Njaine B, Silveira MS. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development. PLoS One. 2012;7(8):e43028.
Saurabh Mandal Thank you for your reply, I'll definitely have a look at the Ct values to see how many cycles they differ by. I'm quite new to qPCR so I'm trying to learn as much as I can!
I recommend you the database "Housekeeping and Reference Transcript Atlas" (www.housekeeping.unicamp.br). It offers candidate reference transcripts stable across multiple tissue types.
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