I want to do SYBR real time PCR for relative quantification of TGFB1. Gel electrophoresis of real time PCR product showed two bands in around about 80 bp (79 oC melting peak) and 180 bp (84 oC melting peak) products but I expected to see a 125 bp band. Blast of primers did not show any other probable PCR products. I am totally confused because I cannot see the true product. I want to know that anybody knows about another spliced form of this mRNA? These are our designed primers: 5-ACTACGCCAAAGAAGTCACC-3 and 5-CACTGCTTCCCGAATGTCT-3
Hi. One thing you may want to consider is having a positive control for your TGFb1 experiment. I assumed you were doing an in vivo experiment but you could assess your primers in vitro. Using other type of cells are fine but you need to know the positive control for TGFb1 expression.
I had some marker expression experience before. You could try adding IL6 or any other inflammatory-inducing substances-I could not remember exactly what I added to my primary cells to induce TGFb1 expression, you could look it out from literature--. Bottom line is, have a positive control in your experiment sometimes is a more practical way to solve problems.
Another option is to design other primer pairs and see whether you still get 125 bp product. If you were to get the same product, then it would be worth your time to fully investigate other spliced form of your mRNA of interest, as what you had suspected where the problem came from.
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