Isolation of cellular RNA Sample Clauses

Isolation of cellular RNA. Extraction of RNA from snap-frozen cell pellets was performed using the Isolate RNA Mini Kit (Bioline, London, UK) as per manufacturer’s instructions, with all steps undertaken within the confines of a PCR hood (SLEE, Mainz, Germany). Briefly, the pellet was resuspended via the addition of 400 µL Lysis Buffer R onto the pellet and pipetting up and down 10 times. The lysate was incubated at room temperature for 3 minutes. Spin Column R1 was placed into a collection tube and the lysate was pipetted into it. The Spin Column was centrifuged for 2 minutes at 10,000 x g, after which 400 µL of 70% ethanol was added to the filtrate and thoroughly mixed by pipetting up and down 10 times. The entire volume was then transferred into Spin Column R2, placed inside a new collection tube, and centrifuged at 10,000 x g for 2 minutes to bind RNA. Five hundred µL of Wash Buffer AR was added to the Spin Column which was then centrifuged at 10,000 x g for 1 minute. Seven hundred µL of Wash Buffer BR was added to the Spin Column which was again centrifuged at 10,000 x g for 1 minute. Using a fresh collection tube, the Spin Column was spun at 10,000 x g for 3 minutes to remove all traces of wash buffer. Finally, Spin Column R2 was placed in an Elution Tube, and 30 µL of RNase-free water was applied directly to the Spin Column membrane and incubated for 1 minute at room temperature, before RNA was eluted by centrifugation at 6,000 x g for 1 minute.
Sample 1
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