Hi everyone, I want to set up PCR for two genes (GPR120 and PPAR-γ). I twice designed two specific primer for each of this two genes by online primer design sites, but unfortunatly none of two designed primers do not respond. The housekeeping gene (GAPDH), as a positive control, always responds along with my tests. I use ready to use PCR master mix, 2X for PCR reactions. The expected size of my PCR products is about 94-121bp, but there are only primer dimer bands. My samples were venous blood, and total RNA has been extracted by column method. cDNA synthesis has been performed by takara kit. The designed primers are very specific, but I do not know why are my primers not responding? What is the problem?
Please help and guide me.
Thank you.
Can we see a picture of a gel please?
Do you get your small band in the no sample negative control
Are the primers designed in the exons of the gene?
What percentage agarose gel are you running.
If this does turn out to be primer dimer can you borrow some hot start taq to amplify your product as this ofeen stops primer dimer but if not you might try using much less primer to cut down the primer dimer...try 1/8 the amount you are using now
Hi dear Paul Rutland, Ok sure.
Yes, for each of the genes, I designed one of the primers in exon-exon junction. I used 2 percent agarose gel for running of samples. I tested 0.6 and 1μL amounts of the primers, but none of them did not respond (total volume of reaction=25μL). The used ladder is 100bp-3K. I used GAPDH (product size: 107bp) as a positive control (It is clear in GPR120 gel picture: wells 1 and 11).
Gel images are attached.
Thank you.
Hi dear Dirk Schmidt, thanks a lot for your comment.
I've done gradient PCR for each of the genes (50-60 centigrade degree for GPR120 gene and 50-58 for PPAR-γ gene), and the relevant gel pictures were sent above.
Thank you.
Hi Ramin,
can I clarify that both primers in each set are designed either in 2 exons completely or to cover two consecutive exons/ Also are the large bands in the top picture the correct size for genomic dna amplifying correctly for these primers. I agree your small bands are primer dimer but if even with p-d you can amplify a larger band then the smaller band should amplify if the cDNA has been correctly produced
Is your gene alternatively spliced and could this effect the primer design between exons I wonder?
Dear Paul Rutland, Thank you very much for your excellent guidance.
Yes, Both genes (GPR120 and PPAR-γ) are spliced alternatively. GPR120 has two isoforms (short and long), and PPAR-γ has 6 isoforms (10 variants). For each gene, I designed one of the primers (forward or reverse) in exon-exon junction.
The cDNA is correctly produced because positive control (GAPDH) has the expected band (107bp: It is clear in GPR120 gel picture: wells 1 and 11).
What advice do you have for me? What should I do?
Thank you.
I agree about the cDNA being ok so my concerns are more with whether the primers can work. Obviously primers designed in introns cannot work. Primers designed between 2 exon junctions which have been spliced out will not work and primers with their 3' ends sitting over a mismatched polymorphism are unlikely to work.,Any mismatches in the primer sequences will lower the annealing temperature so the primers will not work if the annealing temperature is too high. I would try designing a primer pair from within 2 exons separated by an intron in the part of each molecule common to all spliceoforms and without known snps under the primers , My feeling is that if you can amplify from genomic dna then there is a mismatch between the cdna sequence and the primer sequence so either this mismatch must be sorted out or the simpler strategy of pcr from mid exon to mid next exon where the sequence is clearer should be tried.
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