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. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

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. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Publication protocol

For the extraction of total cellular RNA, the medium was removed from the culture flasks and the cell layer was washed with PBS prior to adding trypsin. The trypsinized cells were resuspended in medium and counted. Cells (1.5x106) were centrifuged at 1,000 x g for 10 min and the cell pellet was subsequently used for RNA purification with RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentration was determined photometrically on an Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany). RNA (1 μg) was used for cDNA synthesis using the iScript cDNA Synthesis kit (Bio-Rad, Munich, Germany) following the manufacturer’s instructions.

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Manufacturer protocol

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

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