Reverse transcription - (Aug/05/2005 )
Sometimes when I perform reverse transcription it works well. But most of time reverse transcription doesn't work. I tried to improve it with different Mg concentrations, lowering extension temperatures and increasing the amount of RNA template, but I havent been able to standarized o good reverse transcription. I am believing that it could be present some kind of inhibitor in the solution in which is dissolved RNA (I mean guinidinium thiocianate or alcohols). How reasonable is that? is there another most likely cause?
I would just try a new isolation of RNA, the quality of your template can also be bad this time. Also, multiple freeze-thaw cycles decrease quality of the template, so increasing template after several freeze thaw cycles won't help too much. Try making separate aliquots per 2 or 3 RT-reactions.
You may also want to try a different RT enzyme. I switched from Superscript II (basically MMLV) to AMV and voila! RT-PCR was consistent. (I had to increase the temperature for RT since there was secondary structure in my RNA that stopped the reaction).
Reducing the amount of template may also help to dilute out any inhibitors (try 1:10 or 1:25 or greater). You can test for inhibitors by spiking in RNA of a known concentration into your extract. It depends what sort of a lab you have, but you could borrow some RNA from a colleague (who knows that RT-PCR is working) to spike into your sample, then use their primers to amplify. If you get a product then it's your RNA, if you don't then it's an inhibitor (of course use all the controls, such as none of your extract, etc). You can also check if it is a PCR inhibition by spiking in plasmid DNA to your PCR.
BTW here's what I mean by the term "spiking":
-Take 5ul of your extract (or however much you use) and add (spike) 1ul of RNA that you know will work in RT-PCR (it has worked for a colleague).
-Set up a negative control (your extract minus the spiked RNA) and a positive cotrol (no extract, 1ul spiked RNA only)
-Do RT-PCR as per normal, using your colleagues RT-PCR primers.
-> you should get:
No band in negative (extract) control,
One band in positive (RNA only) control.
For your sample:
-If you get a band in your extract plus RNA control, then there is no inhibition of the reaction - the problem is likely to be your primers.
-If you don't get a band then you have inhibition of your RT or PCR. Then you can try diluting out the inhibitors, cleaning up the RNA more, etc. etc.
I routinely use TMV (tobacco mosaic virus) RNA as a positive control as described in this reference (though don't use it as a duplex - I do two reactions): Torok, V. A. & Randles, J. W. (2001). Tobacco mosaic virus RNA as an internal control for duplex RT-PCR assay of pea germplasm. Australasian Plant Pathology 30, 227-230.
Let me know if you have a hard time finding this reference if you want it - I should have a copy somewhere ...