better primer for cDNA synthesis in real time pcr - (May/05/2008 )
I have just started with my real time experiments, and had synthesised cDNA using random hexamer primer. But on putting real time pcr with Beta Actin Taqman probe, I could not observe any amplification. On reapeating, the same hing happened. Can anyone help me figure out what could be the reasons for this? I am working on a mammalian cell line, and the probes designed are specific. Could it be because in case of Random hexamer, standardisation of rna to primer ratio is required, as well as that random hexamers take all rna population as the template. But everyone says it does not matter with Taqman gene expression assays. Whereas in case of oligo dT priming, only 3' poly A tails of rna are the template, and hence it could be more specific.
I can only agree with "everyone". I did the RT wiht random hexamers many times and never experienced such a problem. Thus, I do not believe that your problems is related to the choce of RT procedure.