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housekeeping gene has not uniform pattern - (Mar/07/2012 )

I use Beta actin as house keeping gene in my Reverse transcription PCR experiments. It doesn't have a uniform pattern expression. share youre ideas.


I take this to mean that you performed reverse transcription on equal ng quantities of RNA from multiple samples and you expected beta-actin levels to be equal when you performed qPCR on these RTs. A certain amount of variability is expected due to efficiency of the RT reaction and the quality of the starting RNA. This is why you assess housekeeping gene levels. You can now assess the expression of other genes and report gene levels per beta-actin level. If you don't think beta-actin is a good housekeeping gene in your system, you can assess some other standard housekeeping genes.


That's not unusual. We have compared adult stem vs. differentiated cells (single cell multiplex with spiked-in external mRNA), and found 10 to 100 fold lower expression of ActB, ActG, GapDH and Aldoa in stem cells. Most stable were ribosomal proteins. To determine your most stable HKG, you can use the approch by Vandesompele et al.

and there:

But this applies only, if your genes are "randomly" controlled, not by a global down regulation of mRNA transcription like in adult stem cells.


I recommend to abandon commonly used housekeeping genes like GAPDH, beta-actin and so on since they got debilitated by this publication: de Jonge HJM, Fehrmann RSN, de Bont ESJM, Hofstra RMW, Gerbens F, et al (2007) Evidence Based Selection of Housekeeping Genes. PLoS ONE 2(9): e898. doi:10.1371/journal.pone.0000898

De Jonge et al. furthermore tested a wide range of possible genes in order to use them as housekeeping genes and found quite a few more suitible ones like RPS13, RPL27, etc. I tested them for my experiments and they worked quite well!