I am starting RT-qPCR to study the activity of the promoter of a gene present on a recombinant plasmid in E. coli. This is known to be a stationary-phase promoter. Samples would be taken from different steady states from a chemostat, each corresponding to a particular growth rate. So, I expect the target promoter should be expressed high at low growth rates (mimicking starvation). My problem is that no matter which reference gene I choose for normalisation, I think it is impossible to expect its expression to be steady over different growth rates since growth rate is a parameter with such global effects on the transcriptome. What is your take on this? Also, the effects of growth rate on the plasmid copy number and hence available copies of the target gene for transcription must also be taken into account during normalisation. Should I check multiple candidate reference genes from genome and plasmid?
Hi,
read some literature from Paffl, Vandesompele, Bustin etc. And read the MIQE guidelines.
http://www.rdml.org/clinchem.2008.112797v1.pdf
Cheers, Nadine
You are thinking in right direction. Although there is not definite answer to your question but at least you can try checking stability of various candidate reference genes in your samples with different growth rates. A good approach is to look at the literature from tumour research for some candidate reference genes. Always geometric mean of multiple reference genes is the best for normalization of gene(s) of interest since there are no universally stable single reference gene. Nadine suggested you exactly those research papers which elaborate this phenomenon. Good luck
thank you Nadine and Muhammad. I have already gone through the literature that you have mentioned. I was still interested to know if it is theoretically possible to expect any gene to be stable in its expression over different growth rates. Anyway, I will try with multiple genes..
Some candidate reference genes include ribosomal proteins, histones, 18S rRNA, but there is no magic bullet for all. You will need to examine the type of the cells you are working on and figure out which genes exhibit most stable expression level, which can be evaluated using GeNorm or other similar software packages.
As long as you can empirically demonstrate, in your own experiment, that your chosen reference genes are indeed housekeeping, all good!!!.
That’s why you should first run a previous experiment to test several housekeeping, and then, with those reference genes that you choose, run your real experiment.
How can you perform that previous experiment?, well you can use several programs available, but, if you know how to work with statistics, like nested ANOVA, I recommend you do it by your way. You will know better than any program what’s happening actually in your pilot experiment!!.
Good Luck!!!
Agree with the answers above. I've seen that often used housekeeping genes did differ in my samples and that less used genes worked perfectly as control! Choose a few, but be careful they don't belong to the same group of genes. For instance, ribosomal genes might be influenced by food intake and I would also guess they might change with growth state since protein synthesis plays a role in that. Dig in some literature, check which genes they used, pick some and do your pilot experiment. For the real experiment, I would still recommend you to use two references instead of one. In case something goes wrong, it's well worth the extra spot! Good luck.
In developing mammalian tissues we have found that few, if any, of the usual housekeeping genes exhibit constant levels of expression.
Using microarrays we have been able to find alternative genes which are suitable. Otherwise I would think the best approach would be to test several "housekeeping" genes using QPCR.
thank you all for your valuable responses. So, I guess the general consensus in this area is to test multiple candidate reference genes for every particular experiment.
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