Housekeeping gene not affected by HGF treatment - (Mar/28/2008 )
I am seeking your help because I need to analyse the expression of several genes with or without HGF treatment in MDCK. So far I've tried two housekeeping genes : GAPDH and ALAS1...Unfortunately, the mRNA of these two genes is elevated when my cells are treated with HGF. The levels are increased by a factor of about 2 whereas my GOI are increased by a factor of about 10. Thus, when normalized, the increase of my GOI seems to be lower that it actually is.
Has anybody of you been through that situation ? Would you a suggestion for a good HKG suitable for my system ?
Thanks in advance
There are plenty housekeeps you can find via Google in HGF treatments - beta-actin, beta-2-microglobulin, some even used GAPDH. The best way to do it, is to find a several genes, that are used most, run them all and then feed the results to some software like geNorm to determine the best one/best combination to normalize to.
I'm using Human acidic ribosomal protein (HuPO) for my infection models as this is pretty stable in any condition.
http://www.gene-quantification.de/dheda-bustin-hkg-2004.pdf might be worth a look.
"The most stable housekeeping gene in whole blood was HuPO, with an average fold change of <2 and a maximal
variability of <5-fold. There was considerably greater variability for GAPDH, β-actin, and hypoxanthinephosphoribosyltransferase (HPRT) (maximum variability of 20- to 25-fold)."
You won't find any gene that is 100% stable I fear. If you do, tell me.