I know GAPDH, beta-actin, tubulin, etc. for mice, but I want to study the expression of a gene in a thermoacidophilic archaea species. I can't seem to find papers anywhere of people doing this except for population studies using the 16s RNA gene. That gene is bound to be really highly expressed though, and I'm looking for something that should be reasonably constant under varied conditions. Any thoughts?
You would have to test a number of genes to find a good housekeeping gene, or genes. With out testing you will not not know if it as good control gene or not. It is very risky to assume a gene is stable under a certain condition if it has not been tested , just because it is stable under other conditions.
Maybe elongation factor-1 alpha. It changes a little with treatment, but it is highly abundant and highly conserved across a number of species.
You would have to test a number of genes to find a good housekeeping gene, or genes. With out testing you will not not know if it as good control gene or not. It is very risky to assume a gene is stable under a certain condition if it has not been tested , just because it is stable under other conditions.
Hi, I agree with Andrew. And you have to use 2-3 genes for housekeeping genes. Read therefore the MIQE guidelines. All the best, Nadine
I like that idea; I think I'll give it a try, thanks! I'm a little concerned that it will vary a lot depending on growth stage, but if so perhaps I will just normalize to total RNA.
Open to other ideas if anyone has any :).
16S rRNA gene is a often the first candidate people choose but a recent publication showed that its expression is growth phase dependant so it depends what you want to compare. It worked well in our hands for relative gene expression studies in Sulfolobus solfataricus P2 and 98/2. Alternatively or as second reference gene we use for exemple lacS which expression seems to not vary which different growth phase:
Growth phase-dependent gene regulation in vivo in Sulfolobus solfataricus
FEMS Microbiology Letters
Volume 321, Issue 2, August 2011, Pages: 92–99, Melissa DeYoung, Michelle Thayer, John van der Oost and Kenneth M. Stedman
It is difficult to find a gene or RNA species that does not change with treatment. However, as you continue performing QRT-PCR with your "house-keeper", normalizing to total RNA will eventually tell you how much it actually changes with various treatments. Once you have that information, it is possible to go back and correct your original data.
@David: That's the reason why you first of all test a lot of different genes and then choose two or three where the lowest difference in the treatment occurs.
If it varies with growth stage then it would not be a good gene for normalisation, which is why you have to test more than one gene . I think you will find that if you test a few genes to start with under the conditions that you are interested in then it will possible to find a "good" housekeeping gene
Hi Jason,
we are using since a few years the secY gene for qRT-PCRs in Sulfolobus solfataricus and S. acidocaldarius. It is part of the translocon and as far as we know not regulated and works very well in our hands ( we also published this ).
I agree with all the comments, it is hard to find out a houskeeping gene which its level of expression remain constant in all circumstances, but I recommend TATA Binding Protein, TBP. Give it a go.
rpS4, merA and pdhA might be helpful.
plz look at this article
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