I am studying Unfolded protein response in yeast and I am checking Hac-1 mRNA splicing through semi RT-PCR followed by PCR. But I did not found any similar pattern of Hac-1 splicing in at least any two technical repeats of two biological repeats. I perform 3-4 technical repeats of both biological repeats. I take 350 ng of RNA for semi-RT-PCR.
so, my question is should I increase the amount of RNA semi-RT-PCR reaction volume 5 ul to 10 ul in semi-RT-PCR or anything else I am missing?
The model system is saccharomyces cerevisiae.
Actually that is what I am trying to say. Maybe you can prepare known and graded concentrations of that mRNA sample and see if there are differences between a sample with low volume from those with higher volumes so you can really confirm the right amount that fits your experiment... There are reports where a researcher cites a particular amount or volume that works or has worked well with a previous experiment but do not expect that this holds true to your experiment. Expect some differences and be ready for some adjustments...
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