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 using RNeasy Midi Kit (Qiagen, cat. #75144) as described in the following. Mice were euthanized and lungs, liver, and lymph nodes were isolated and snap frozen in liquid nitrogen before being stored at −80°C. For cell cultures, cells were harvested in a good growth phase and 107 cells were pelleted. Organs or cell pellets were mixed with RTL buffer and homogenized using an IKA T10 basic ULTRA-TURRAX homogenizer (Bie & Berntsen). The blender head was washed with 3× PBS, 1× TAE buffer, 1× 70% EtOH, and 1× 96% EtOH between every sample. Samples were then centrifuged for 5 min at 6000 rpm and the supernatant was isolated. For homogenized organs, this step was repeated. Equal volumes of 70% EtOH were added to the supernatant, samples were vortexed, and half of the volume of the samples was added to RNeasy Midi spin columns and centrifuged for 10 min at 5000 rpm. The step was repeated with the last half of the samples. The columns were then washed once in RW1 buffer followed by centrifugation at 5000 rpm for 5 min, and twice in RPE buffer succeeded by centrifugation at 5000 rpm for 2 and 5 min, respectively. The columns were then transferred to new tubes and RNAse free H2O was added to the spin columns. The samples were left to stand for 1 min and then centrifuged at 5000 rpm for 3 min. This step was repeated and total RNA was collected and stored at −80°C. Subsequently, total RNA concentration and purity was measured using a NanoDrop 2000c Spectrophotometer (Thermo Scientific) and the associated computer program NanoDrop 2000.

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Manufacturer protocol

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

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