What is the best positive expression control for your experiments? - Which is better? (Jun/03/2006 )

Hi All,

I have been doing some qRT-PCR and would like to know which expression controls do people use and swear by. Just to be safe I have recently used three positive expression controls, to be sure to be sure.

Nick

I wouldn't bet my life or my work on any of them, to be honest. I think that is the inherent flaw with real-time. c'est la vie, non?...I use actin and do lots of reps, and don't ever consider my data to be more than a good approximation of trends

When I first started with realtime a few years ago I did lots of reading and digging and worried about the same issues...there is all sorts of arguments over which are best, both anecdotal and in the literature, and sometimes I think it depends on your cell treatments and lines as well. I tested both GAPDH and b-actin when I started out, and I got the most consistent results with all the initial set-up and optimization steps with b-actin. But, this could certainly be due to my primer design and to the way that I set it up

I think it's a little scary how much room for error exists in this technology!

anyways, perhaps not very helpful, but that is my take on HKG's

Hi Nick,

check out this thread...
http://www.protocol-online.org/forums/inde...hl=housekeeping

good luck !

Also depends on what samples you are working with and conditions. Here are some examples:

1. For example 18S only has one exon and therefore genomic DNA contamination will affect the 18S readout. If your samples have not been DNase treated then 18S is not the way to go.

2. If you are performing experiments related to hypoxia the GAPDH may be affected.

3. The pronosis of some patients suffering from hematological malignancies can sometimes be predicted by B2M levels so therefore B2M cannot be used as an endogenous control.

Therefore it is best to look carefully at what you are studying before deciding on an endogenous control.

thanks smurry,

JPstewart,

we are looking at cell line expression levels or candidaate genes which can be affect by drugs inhibiting chromatin modifications and DNA methylation, which I would think in turn affect the expression levels of both the candidate genes and the positive control genes. Is this a safe assumption? can i go further to say that if the housekeeping gene haas been affected by the drug treatment, the effect would be to the same extent as with the candidate gene.

Nick

Hi guys,

I am analysing differentially regulated genes during the process of sexual maturation in salmon. I am finding it impossible to find a good housekeeper. Its seems sex mat is such a huge change that all the genes I have used are differential when I compare immature to mature. Have tried actin, B-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophilin but no joy! Does anyone know of any suitable control genes for this process. Im at my wits end!