Hi,
I've designed qPCR primers to target my gene of interest in HeLa cell line. I designed them such that it will encompass an exon-exon junction and it the product is 100-200bp long. Apparently, the first set of primers did not amplify, so I re-designed new ones until I reached 5 different sets and still, all of them could not amplify anything. I'm starting to think that maybe the gene has very low expression, thus the results of my qPCR reactions.
Any thoughts on this one? Does this happen in your experiments? Thanks in advance for your help.
Best,
Carla
Hello, Madam Fatima,
Since you have tried with almost 6 different sets of primers for the gene of your interest, I believe you must have validated your gene sequence of your interest with the primers. I would like to suggest you, check its melting temperature and GC content as well. If everything is fine, you can run a gradient PCR rather than the qPCR, in order to validate the TM. Once u get a specific desired band in a particular TM; Run your qPCR supplemented with 5% DMSO. The actual function of DMSO in PCR is: it binds to the DNA at the Cytosine residue and changes its conformation which makes the DNA more labile for heat denaturation. As most of the primers are instructed to have a rich GC content. So, in that case, DMSO can indirectly facilitate the annealing of the primers to your desired template which can ultimately enhance the amplification of the gene of your interest.
I hope it will help you.
How are you generating your primers? I usually use a software on-line rather than by hand. It may be worthwhile to run your sequence through a primer designing program and see what you get. Most of primer designing programs should give you several different primer sets. If one of those covers the junction, I would use that primer set. Also, you may want to go ahead and order different set that gives you the size you want and test it out, even though the set may not cover the junction. At least that will tell you if your assay is working.
I'd recommend using a primer design software, as Dr. Kobayashi suggested. IDT's Real Time PCR Tool (linked) is generally rather effective. Have you confirmed that a loading control is working (e.g. GAPD, Beta Tubulin, Beta Actin)? Do you have another RNA source that might be useful as a positive control (e.g. another cell line or organism tissue?).
https://www.idtdna.com/scitools/Applications/RealTimePCR/
Hi Carla,
First, this is not due to low copy of genes, as PCR can amplify genes in genome with only two copies.
Please use primer design software, like primer premier (http://www.premierbiosoft.com/primerdesign/), the primer should not be two long, and also try different Annealing temperatures.
If still does not work, try a nest PCR, that is first design primers for a large range, then second round with narrow down range.
At last, you shall also control your procedure of genome DNA preparation, so you can PCR another easier gene with avaliable primers as control.
Good luck!
/Hui
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