How were your brains initially frozen? If the freezing method was slow (eg, putting whole brains into a freezer) then the RNA may already be degraded within the frozen brain tissue and nothing you do now will improve the RIN very much. If they were snap frozen (dry ice or liquid nitrogen), it is much more likely that the RNA within the brains is intact, and the quality can be improved by optimising handling and extraction.

If your brain tissue has been snap-frozen, then getting high RIN should be possible with a bit of optimisation - we routinely get 8.5 to 9.5 from snap-frozen mouse brain tissue. Because tissue that has been frozen no longer has cellular integrity, the RNA will degrade REALLY fast once it thaws - much much faster compared to intact fresh tissue at the same temperature. So to get RINs this high, it's critical that the frozen tissue is homogenised in chaotropic buffer as it's thawing, and spends basically no time thawed. Are your brain slices still frozen while you are taking the micro punches? If not, can you think of any way to do this without ever allowing them to thaw? I've seen someone do it by transferring the slices off the cryostat directly onto a stainless steel dissecting tray that is resting on dry ice. The shorter you can get the interval between leaving -80C storage and being homogenised, the better.

It's also possible that your extraction and purification protocol is not ideal. Have you tried extracting RNA from fresh brain tissue (never frozen) using the same protocol? This would help determine if the problem is with the sectioning and micropunch steps, or with the RNA extraction.

@Laura Leighton

Thanks for your thorough answer to Hannah question as you actually answered many of my questions as well. Do you think extracting RNA from fresh brain tissue is a good idea? Because while dissecting, it seems that due to release of active RNAse enzymes there is a high risk of losing many of RNA molecules, which subsequently can affect the accuracy of results.

I know this question is old, but I'll answer for future readers! My experiments involve extracting RNA from a specific brain region of X.laevis frogs. The protocol I use is:

1. remove brain

2. immediately place whole brain in RNAlater (invitrogen) & store at 4C overnight

3. replace RNAlater with fresh & move the brain sample to -20C or -80C (stored for 2 weeks - not usually longer if I can help it)

when processing for RNA

4. Thaw the brain to RT

5. Slice the brain with a vibratome filled with RNAlater(I reuse the RNAlater for the vibratome bath because it is expensive).

6. Pin brain region "slab" to a small dish with fresh RNAlater & go to dissecting scope. Use blunt needle filled with Homogenization Buffer plus Thiol to "punch" the region desired.

7. Store the brain punches at -80C until ready to prep for RNA

Note: I use a promega kit to prep the RNA, but have also used Zymo with success. The RINs were all between 9-10.

I hope this is helpful for someone!

cheers,

Valerie