We are designing a set of qPCR experiments and would ideally like them to be MIQE-compliant so just ran through the checklist. The only one we are having trouble with is the "if frozen - how and how quickly?"
This is an "essential" item so we have to attempt to address it, but I'm assuming "stick it in a -20/-80 freezer" isn't sufficient. We could say what volume of sample we are freezing and what freezer model we are using, but although those would be sufficient to closely reproduce the conditions experienced by the nucleic acid in our sample they do not strictly speaking answer the question.
If you design MIQE-compliant qPCR experiments, how do you address this requirement?
In all honesty, what's really required for good quality RNA is that it's either prepared fresh as possible, or prepared from samples frozen FAST.
Putting tissue into a -20 will freeze it relatively slowly, allowing time for cell death, cell lysis and significant amounts of RNAse activity.
Putting tissue into a -80 will freeze it faster, but is still not ideal.
"Snap frozen" is generally the approach you should be aiming for, so "tissue collected quickly, placed in tube, tube into liquid nitrogen", for instance. And I would describe this as "snap-frozen under liquid nitrogen", which should be sufficient for MIQE standard.
Fantastic - thank you very much (both for the advice and the explanation). If liquid nitrogen is a problem, would something like RNAlater be better than normal freezing?
I've never tried RNAlater, so I can't really comment on that (it sounds amazing on paper, but these things always do).
If liquid nitrogen is a problem, sticking samples directly onto dry ice might be a viable alternative, provided you're not freezing very thick tissue samples (admittedly, if you're freezing very thick tissue samples I'd recommend cutting them up beforehand anyway).
Pelleted cells in eppendorfs, for instance, I would trust to freeze rapidly enough on dry ice to qualify as snap frozen.
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