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

Total RNA was extracted from individual tissues by combined use of the BioMasher and an RNeasy Mini kit (Qiagen). Small pieces (approx. 30 mg) of tissue were immediately placed in the BioMasher after either of the following two treatments: soft tissues were cut into small pieces on ice, or fibrous (hard) tissues were crushed using a hammer after being frozen in liquid nitrogen. The BioMasher filter tube was set within a recovery tube, and tissues were manually squeezed using a masher equipped with an O-ring (the BioMasher Power-Plus was not used). Then, 200 µl RLT buffer (Qiagen) was immediately added to the filter tube, and the filter tube and recovery tube (including the masher) were centrifuged at 15,000 × g for 15 sec at 20°C. Next, the masher was removed, and 500 µl RLT buffer was added to the filter tube, still attached to the recovery tube, which was centrifuged again at 15,000 × g for 15 sec at 20°C. The filter tube was removed, and the recovery tube containing the resultant tissue lysate was centrifuged at 30,000 × g for 5 min to remove any insoluble matter. Total RNA was then purified from the supernatant using the RNeasy Mini kit, according to the manufacturer’s protocol.
To increase yield and purity, total RNA from the liver, lung, heart, small intestine, uterus, cerebrum, and cerebellum was extracted according to the following optional protocol: (1) 50% ethanol was added to the lysate instead of 70%ethanol; (2) the RNeasy spin column was incubated for 5 min, instead of no incubation, after the addition of RW1 buffer; and (3)
the RNeasy spin column was incubated for 10 min, instead of no incubation, after the addition of RNase-free water. The quality and yield of total RNA were analyzed with a RNA 6000 Nano LabChip using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, U.S.A.), according to the manufacturer’s protocol.

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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Mini Kit below.

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