I want to carry out a qPCR for a human gene of interest and I need to decide on a suitable reference gene. For the past few years I have been using Actg1 for my murine studies and therefore I decided to design primers for the human ortholog. However, although the primers seem to amplify one product with high efficiency, the Ct values of the qPCR with human PBMCs are 28 cycles which makes me sceptical.
The principle behind reference genes lies on the low variability in expression between several conditions and samples. Hence, so far everything is alright. But they should also be ubiquitous and abundant which is why I worry. 28 cycles does not reflect low expression but I would be more confident if that was 22-23.
Can ppl share their thoughts?
Hi Theodore
The most important thing about a reference gene (or genes - many people use more than one now…) is that it does not vary under the conditions being tested fro your GOI. So, for example, if your RefGene is stable in freshly-isolated PBMCs and you want to test IL-4 expression under Th2 conditions, then you need to check that your RegGene is not regulated by those same Th2 conditions.
Since the purpose of the RefGene is to provide a comparator for your GOI that compensates for different amounts of cDNA, you need first to perform a checking experiment where you select several candidate RefGenes and run only them, on material that has been prepared mimicking your actual experiment - for example on CD4+ve T-cells that have been put through a Th2-induction protocol, rested and stimulated with PMA/Iono.
Preferably, you don't want an intronless gene as RefGene, or a duplicated gene, or one where there may be a cross-reacting pseudogene, but on the other hand, you should never let these precautions prevent you from doing "no-RT" and "no Template" controls on your plate.
Most likely you'll have three or four candidates after this (and don't be surprised if you *do* see some candidates varying with the treatment), so how do you choose the ideal one? Think about the Ct value you're expecting to see for your GOI (in our example, IL-4, IL-13 etc) then look for a candidate that is "reasonably" close to the Ct-GOI - within 8-10 Cts, for example is better than 20. Remember that you can't go off Cts alone - if your machine allows, check the melting curves.
Then of course, you'll need to be sure (as in *sure*), that the efficiencies of your reactions for RefGene and GOI are similar - 90-100% is the range we use for *all* our qRT-PCRs, and after all this, you'll be good to test your reagents in a real experiment.
Good Luck!
G
Hi Theodore
The most important thing about a reference gene (or genes - many people use more than one now…) is that it does not vary under the conditions being tested fro your GOI. So, for example, if your RefGene is stable in freshly-isolated PBMCs and you want to test IL-4 expression under Th2 conditions, then you need to check that your RegGene is not regulated by those same Th2 conditions.
Since the purpose of the RefGene is to provide a comparator for your GOI that compensates for different amounts of cDNA, you need first to perform a checking experiment where you select several candidate RefGenes and run only them, on material that has been prepared mimicking your actual experiment - for example on CD4+ve T-cells that have been put through a Th2-induction protocol, rested and stimulated with PMA/Iono.
Preferably, you don't want an intronless gene as RefGene, or a duplicated gene, or one where there may be a cross-reacting pseudogene, but on the other hand, you should never let these precautions prevent you from doing "no-RT" and "no Template" controls on your plate.
Most likely you'll have three or four candidates after this (and don't be surprised if you *do* see some candidates varying with the treatment), so how do you choose the ideal one? Think about the Ct value you're expecting to see for your GOI (in our example, IL-4, IL-13 etc) then look for a candidate that is "reasonably" close to the Ct-GOI - within 8-10 Cts, for example is better than 20. Remember that you can't go off Cts alone - if your machine allows, check the melting curves.
Then of course, you'll need to be sure (as in *sure*), that the efficiencies of your reactions for RefGene and GOI are similar - 90-100% is the range we use for *all* our qRT-PCRs, and after all this, you'll be good to test your reagents in a real experiment.
Good Luck!
G
Dear please go through attached article it will definitely help you out.
I have used ACT- B and GAPDH for pbmc's. it gives a good Ct range between (17-23) but it basically depend the your cDNA concentration (25 - 75 ng).
good luck
Perhaps this link will help
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0000898
Have a look at the following paper: http://dx.doi.org/10.1016/j.tig.2013.05.010
It could be usefull for you.
http://dx.doi.org/10.1016/j.tig.2013.05.010
To Andrey V and Theodore,
Part of the conclusion section of that paper:
These included most of the stable housekeeping genes reported based on microarray data, ( ref 30)
ref 30= plos one paper 2007: Evidence Based Selection of Housekeeping Genes
I would recommend you to use GAPDH, ACTB, B2M, TBP. You must not forget to take atleast 3-4 house keeping genes to get a good fold change. Sometimes, you may get variations within genes, so to remove this variations you must keep 3-4 HKGs, take average of it. Try reading MIQE guidelines, before starting
Cheers
Prit Benny
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