The choice of reference (housekeeping) gene depends on the experimental setting. Some reference genes work very well in one setting but are utterly useless in others. As there are no perfect housekeeping genes, what you have to do is to evaluate a series of housekeeping genes and then pick 2 or 3 of the most stable ones. There are algorithms for doing that, like Normfinder (an excel add-on found at www.mdl.dk)

For more info on qPCR methodology, check out www.gene-quantification.com section on normalization - you'll find good advice and lots of references

good luck 

R

Hi Ladan,

Sure, you can use the B-actin gene (ACTB) for real-time normalization...depending on what you are trying to do! In theory, you can use any piece of the genome to normalize (even intergenic regions) if you are just trying to account for how much total DNA is in your reaction from a gDNA preparation.

However, there are certain considerations. You want to make sure that you have a good pair of primers to whatever region of the genome you are using as your control to give you an efficient amplification. Thus, standard PCR rules apply: Design primers that have the appropriate qualities such as melting temperature, GC%, no secondary structures, and no repeats of more than 4 bases at once. 

Also, if you are trying to actually obtain genome copy number, you cannot just use any piece of the genome. Certain genes, including ACTB, are highly conserved and contain sequences that are found in numerous other genes. One time, I tried to design a pair of primers that would only amplify ACTB and I failed because the actin sequence is found in lots of places throughout the human genome. My primers would actually amplify I think 9 different pieces of the genome! 

So a long answer short...it depends on what you are trying to do. 

If you tell us exactly what you are trying to accomplish that would be helpful to give a more clear answer.

Hope this helps, and best of luck!

The choice of reference (housekeeping) gene depends on the experimental setting. Some reference genes work very well in one setting but are utterly useless in others. As there are no perfect housekeeping genes, what you have to do is to evaluate a series of housekeeping genes and then pick 2 or 3 of the most stable ones. There are algorithms for doing that, like Normfinder (an excel add-on found at www.mdl.dk)

For more info on qPCR methodology, check out www.gene-quantification.com section on normalization - you'll find good advice and lots of references

good luck 

R

Dear Ladan

in short most of the time yes you can. In fact GapDH and b actin are probably the most commonly used housekeeping genes

Nevertheless, as mentioned by Rasmus, it does depend on expression system, i.e. tissue and the way you treat your cells; In particular:

1.  Make sure that the housekeeper(s) you elect to use is not affected by treatment

2. Make sure the housekeeper you use is stably expressed in the context of your tissues/cells and treatment

To evaluate that as mentioned by Rasmus there are standard algorithms you can use like normfinder and genenorm which are add ons to excel spread sheets

In terms of how you go about selecting initial housekeepers to evaluate using these programs I would look for precedents in the literature for your system

Failing that there are a number you can evaluate that tend to be ubiquitous but the exact ones which are optimal for your context need to be determined (as described and made ref to by Ramus), e.g.

GapDH

B actin

B tubulin

B2 Micro globulin

TATA box binding protein

18s rRNA  (or what ever ubiquitous ribosomal RNA pertains to your system)

Screen then pick the best 2-3 for actual normalisation purposes: It is not becoming standard practice to use 2-3 and compute the over geometric mean for normalisation purposes. There is a lot of literature out there so Google my description

For starters however see attached  

 thanks all for really good advice and information

i work on drug design on wnt pathway on cancer cell.  i want to compare cancer  cell lines after treatment by drug and with out it. for determine that drug is affected wnt pathway on cancer cell lines i should assay b-catenin as a final gene that express in this pathway by real time pcr and approved it by western blot. in my articles in this field  that i read use b-catenin as a target gene and GAPDH as a housekeeping gene for real time pcr  vis a vis they use b-catenin as a target gene and b-actin as a housekeeping gene for western blot. i don not know why they use two different  house keeping gene for two different test. could you help me is it important or not?

Dear Ladan

for Western blotting you tend to use structural genes like B actin and B tubulin as housekeeping normalisers. That is part is due to the availability of excellent antibodies to different proteins

In short: as long as your housekeeper is stably and universally expressed at high levels it is acceptable to use one set of housekeepers for qPCR and another for Western blotting

Dear Laurence 

thanks a lot for helping me

Best Rigards

In general you can use, but it also depends upon the species you are using. For eg. in zebrafish I found papers suggesting not to use GAPDH, because its expression level may alter with stressful conditions provided during disease induction. So I suggest you should look for the species you are using. again as you told that the same paper suggested b- actin for western blot which indicates that both the genes can be used as house keeping.

hello

I study GSTs enzyme in red palm weevil one of my objective is to evaluate the gene expression of GSTs enzyme in different tissues of the RPW.so used RT-qPCR

I used B-actin as housekeeping gene for my study . It gave me different ct value for each tissue . the values are 14 in fat ,15 in muscles,18 in brain and 16 for gut.

Is it normal( acceptable ) to have these result? ,same concentration of RNA used for cDNA .samples are fresh from the field.Can you please help me

. Laurence Stuart Dawkins-Hall Rasmus Goll

What is the best internal control for serum mRNA for real time PCR ?