Which housekeeping genes for qRT-PCR control-Real-Time
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I am wondering about the choice of housekeeping genes for my assay. If the gene of interest is a nuclear gene, is it better/ worse to use a mitochondrial gene as a housekeeping one. Are there any major pros/cons of the most popular housekeeping genes, like 18 S rRNA or GAPDH?
thanks
I use beta-actin
for our application, it seemed the most widely-used.
I would encourage you to read this plug for 18srRNA :
...it makes some good points
good luck; it's a tough choice and there are many different opinions out there
I use GAPDH...even though there is a number of articles arguing that GAPDH is a bad choice for a housekeeping gene. I look for changes in rat toxicological related genes and have done the time consuming validation of GAPDH against my tox. targets.... in my case it works well with this reference.
The point is to validate I suppose..you might have to do some trial/error before you come across the right gene for you
hi i use beta actin as as positive control for my qRT PCR . i think this is one of the most popular positive control
I dont think there is a real answer to this question. I would use a panel of genes and then use the free genorm software to calculate which are the best for your particular experiment.
heres a good reference about normalisation in realtime:
I agree with Mark, I have used also several genes and normalized with the genorm.
I think that the idea of a perfect house keeping gene, what is expressed at the same levels in all tissues and conditions, is not realistic, but theoretical.
Only if the stability of a reference gene for certain assay conditions and type of samples has been validated, then the use of only one hk is ok. But never with a heterogenous sample set, eg. a set of different tissues.
I think that the problem is that the use of only one hk gene has been applied for many years in many works, that is why we still think that it is ok. But nowadays we have good normalization tools, which I really think we should get advantage of. Yeah, it requires a little bit of extra work when you run your samples for several genes, but it is worth it when you are sure that you can rely on your results.
coco
I use beta actin. I guess it is pretty popular.
Thanks everyone for you thoughts.
I have decided to try a few different housekeeping genes. I have downloaded genorm and we'll see how it goes.
I use rRNA because I work with bacteria. Don't really understand why you'd use anything other than rRNA or Beta-actin.
-Matt
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!
I'm about to try GAPDH with eels undergoing a sexual maturation, will let you know how I go.
Our lab use cyclophilinA. But I didn't get very good results lately. maybe I'll give it a go with GAPDH next week.
GAPDH with my eel cDNA was not good. Three sets of inrton spanning primers and I was getting wierd amplification everytime. I got poor amplification with my first set, and then I was getting two products shown as two peaks on my dissociation curve (81 and 83C), no primer dimer on the gel and one single band at where I expext my product. We reckon on some sort of pseudogene/isoform which is pretty common for eels (and other teleosts which have undergone genome duplication events). So we thiink we were getting two very similar products, with a very slight ly different Tm.
We are now trying RPL-P0, (aka ARP-P0) a ribosomal protein which has introns. Will update in due course.......
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