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

RNA was extracted using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. RNA was treated with DNase (Qiagen) on the spin columns. RNA quantity was assessed with a spectrophotometer (Nanodrop Technologies, Wilmington, DE). cDNA was synthesized by incubating 12.8 μl RNA solution with 7.2 μl cDNA mix containing 2'-deoxynucleoside 5'-triphosphate (final concentration of 0.5 mM), 2 U RT-AMV, 20 U rRNase inhibitor, 100 ng oligo-dT primers, 500 ng random primers, and 1′ reverse transcriptase buffer (all from Promega, Leiden, The Netherlands). Primer sets (Supplemental Table I) for qPCR were selected using Beacon Designer Software (version 7.02, Premier Biosoft International, Palo Alto, CA) and were obtained from Eurogentec (Liège, Belgium). PCR mixes contained 1 μM forward and reverse primers, 3 mM MgCl2, and 1× iQ SYBR Green Supermix (Bio-Rad, Veenendaal, The Netherlands). PCR was performed using an iCycler MyiQ (Bio-Rad). The PCR program consisted of 1 cycle of 10 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C, and was finalized with a melting curve analysis. Reactions were carried out in optical 96-well plates (Bio-Rad) covered with Microseal ‘B’ Film (Bio-Rad). The mean signal of the reference genes GAPDH and β-actin served as normalization factor.

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Manufacturer protocol

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

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