RNeasy Plus Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Publication protocol

We employed the same method for RNA isolation for these different tissues as was used for our previously-published brain RNA isolation study, using the RNA EasyPlus Mini Kit (Qiagen, Valencia, CA) (Birdsill et al. 2011). Each tissue sample was dispersed in RNA-lysis buffer (containing 0.1 M β-mercaptoethanol) by mild sonication (10–15 s), and then processed according to the manufacturer’s instructions. In brief, extracted tissue is passed through a prefilter to remove DNA, adjusted with an equal volume of 70 % ethanol and applied to the RNA column. After washes with kit buffers, RNA is eluted from the column with 100 μl of water. A point to note was that for highest yield and integrity, sonication of tissue sample should be carried out for as brief a time as necessary to disrupt the tissue; excessive sonication significantly reduced the yield of RNA from tissues. Some tissues contained insoluble connective tissue or fat that did not dissolve in RNA lysis buffer, but were removed on the DNA prefilters prior to RNA isolation. This same method was used for all cerebellum brain samples.

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Manufacturer protocol

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