RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- To yield hign 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. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT). - 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 hign 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. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT). - 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 the RNeasyMini Kit (Qiagen) and cDNA synthesis was performed with First Strand cDNA synthesis kit (Fermentas) according to the manufacturer’s instructions. USP52 real-time assays were performed using PerfeCtaTM SYBR
Green fast mix (Quanta Biosciences) at 95 ◦C for 10 min, and 45 cycles of 15 s at 95◦C and 1 min at 58◦C. All other real-time RT– PCR was performed using probe-based Solaris assays (Thermo Scientific) using the manufacturer’s recommended reactions and
cycling conditions.

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Manufacturer protocol

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

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