I have b-actin as my reference gene and 9 other genes as targets. B-actin primers have a annealing temperature of 52 degree and few genes have got annealing temperature (Ta) 55 and rest have 58.
Hi Charles,
I guess you want to run all PCRs with the same temperature protocol. To do so, you need to extend the lenth of your b-actin primers to match the annealing temperature of your other PCRs. Successively add one or two bases at the 5' terminus of your actin primers and then calculate the annealing temperature until they match. By adding bases at the 5' end you do not alter the specificty of your primers, usually. However, you need to check on this by blasting, and you may want to check for primer dimers and secondary structures as well.
Alternatively run the actin PCR separately.
An important issue in comparing reference gene expression with target gene expression in RTD-PCR is to consider PCR-efficiency. You need to correct for differing PCR-efficiencies for all PCRs of your set. Hope this will be of help.
Cheers Jörg
I read that all most all primers designed for real time PCR will have same temperature and it will be around 60 degree Celsius, Is it true?
@Jorg Berg: If my problem was different annealing temperatures for reference gene primer and target gene primer, I should have done as you suggested. But in my case I have two different annealing temperatures (55 and 58) in target genes also. Can I put temperature gradient in my Real time PCR?
It's common to make qPCR primers have Tms of 58-60, yes. And generally amplifying only very short (80-200bp) sequences. Primer3 is a very useful resource for generating suitable qPCR primers to a region of interest.
http://primer3.ut.ee/
But aside from redesigning primers, why not simply run three different plates? One for all your samples with the reference gene, one for all your samples with target gene 1, one with all your samples with target gene 2? Run to run variation is usually low, but if you do it this way it's also irrelevant: you're comparing expression of any given gene between all your samples at each point.
It doesn't matter if you can't necessarily confirm that a GAPDH Ct of 19 in one run corresponds exactly with a GOI Ct of 24 in another run, because that's not a comparison you're actually interested in anyway. You're comparing GAPDH values of all your samples with each other, and then using THOSE relative values to normalise the GOI values of your test genes.
As long as all the data for a given gene comes from a single run, there's no issue with using multiple runs for different genes. And to be honest, you can even use multiple runs for a single gene as long as you include enough duplicate samples in each run to allow you to cross-compare.
When you prepare all your plates with cDNA (so no primer mix) cover them with special foil and freeze them. You can thaw a plate when you want to analyze a gene. And one plate you use for your reference gene. All other plates with genes can be compared to this one reference gene because all plates with cDNA have undergone the same freeze-thaw cycle.
I am using the software from Biorad for analyzing qPCR runs. It keeps in mind the different efficiencies.
The site of Roche also has a primer design tool. Just enter gene of interest and you get appropriate primer sequences.
Charles
For the 58 house keeping genes, I suggest to redesign them to be around 52 degrees.
Thank you all for your valuable answers. I'll try to do the experiment in three plates, each for different temperature. I am even trying for a gradient temperature setting in the qPCR machine. Hope these will work. Thanks again.
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