RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- 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.

Protocol tips
- To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
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

Purification of total RNA from liver and kidney

We used RNAeasy kit (Qiagen) for purification of total RNA from animal tissues. The RNeasy procedure represents a microspin column-based technology for RNA purification, using the selective binding properties of a silica-based membrane and successive centrifugations. The RNAlaterstabilized tissue (approx. 20 mg) was disrupted in 600 μl Buffer RLT (contains guanidine thiocyanate and β- mercaptoethanol) and homogenized using Ultraturax homogenizer. The homogenate was centrifuged at 10,000 rpm for 3 min and the supernatant was carefully removed and transferred to a new microcentrifuge tube. An equal volume of 70% ethanol was added to the clear supernatant and mixed immediately by pipetting. Ethanol helps in creating conditions that promote selective binding of RNA to the RNeasy membrane. Up to 700 μl of the above solution was transferred to an RNeasy spin column placed in a 2 ml collection tube and centrifuged at 10,000 rpm for 15 s. After discarding the flowthrough, 700 μl of Buffer RW1 were added to the RNeasy spin column and centrifuged at 10,000 rpm for 15 s to wash the spin column membrane. After discarding the flow-through, 500 μl of Buffer RPE were added to the RNeasy spin column and centrifuged at 10,000 rpm for 15 s to wash the spin column membrane. RPE washing was repeated with the same volume of buffer but increased centrifugation time (2 min). The RNeasy spin column was removed and placed in a new 1.5 ml collection tube. After adding 30 μl RNase-free water to the spin column membrane, the tube was centrifuged at 10,000 rpm for 1 min to elute the RNA.

Determination of RNA yield and purity

The RNA yield and purity were determined spectrophotometrically using a Nanodrop spectrophotometer (Thermo Fisher). RNA is known to have a maximum absorption (λmax) at 260 nm. An absorbance at 260 nm (A260) reading of 1.0 is equivalent to about 40 μg/ml of RNA, was used to determine the RNA concentration in the solution. To assess the purity of RNA preparation, the ratio of the absorbance at 260 and 280 nm was used. Pure RNA has an A260/A280 of around 2.0.

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Manufacturer protocol

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