Hi, I want to conduct real-time PCR and I am looking for housekeeping genes.
Should I use ubiquitin (ubq-1)? Should I use more than 1? Should I try the kits from the market to select housekeeping genes?
Thanks.
you could at least choose ama-1, act-1, gpd-1 or gpd-2
(pls refer to different stage) as your starting point.
Obtain better confidence/convince yourself to go ahead
to next deeper /further experiment if they give you
consistent result if you pick 2 or 3 as reference
genes( the housekeeping gene might be a OLD name, I,d
like to call it as REFERENCE gene). Good luck!
When I was doing qRT-PCR I tried multiple control genes. act-4 is bad, it varies across stages. rpl-26 and F41H10.11 seem pretty good across stages. I also sometimes use 5s if I need a non-pol II gene.
I also found this paper
Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans
David Hoogewijs,1 Koen Houthoofd,1 Filip Matthijssens,1 Jo Vandesompele,2 and Jacques R Vanfleteren
I haven’t used these genes though.
You should indeed use more than one reference gene. There are some nice algorithms freely available to test which genes are best fit for your experiment.
I usually use geNorm (Vandesompele et al, 2002), sometimes combined with NormFinder (Andersen et al, 2004). They both have advantages and drawbacks, but to me, geNorm is easier when it comes to the benchtop work, since it doesn’t require identical cDNA input for all samples.
The testing of at least six candidate genes is recommended (generally 6-12), out of which the best performing (read: most stable) genes are chosen. You can test the genes easily yourself, commercially available kits are not needed. Be sure to select genes involved in different biological processes, it reduces the chance on co-regulation.
I usually test rpb-12, gpd-2 (this one is mostly the least well-scoring) and 4 of the Hoogewijs-genes: cdc-42, pmp-3, tba-1 and Y45F10D.4 . They score extremely well, probably because they were selected as being stable out of the Kim microarray database. I am currently considering to include ama-1 in the test, and perhaps drop rpb-12 (because it is also pol II), if ama-1 generally scores better.