i've been struggling with rt-pcr for a while now, and my latest problem really threw me. just when i've got my assay working consistently, my extractions start coming up wrong.
my last two extractions turned up with a missing gene. the message has always been present in the controls, but it isn't there this time. the other messages, like gap and my other gene of interest, are present. in the past i've had to put a lot more template in my reactions to get a signal from this gene, but after increasing the template 3x, i still wasn't getting signal. i tried putting in a much much higher level of template, 15x, and still didn't get a signal.
i'm taking tissue samples from 3 day old chicken embryos, extracting rna using quiagen's rneasy plus kit, and then using quiagen's one-step rt-pcr kit. the goal is to examine the relative expression levels of a few different genes after treatment with drugs or microsurgery. at first i was extracting somites to test, but then i moved on to just cutting out a chunk of tissue with the somites of interest. it's simpler, the genes of interest are only really expressed in the somites in that chunk of tissue, and i figured it would make the samples more consistent. i've gotten the assay giving results that look consistent with in situ and antibody labels.
any thoughts or suggestions?
thank you, matthew