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

A combination of TRIzol method and RNeasy Mini Kit (QIAGEN, Sollentuna, Sweden) were used to isolate RNA from bone samples (Reno et al., 1997). Frozen bone samples were pulverized by tungsten balls, inside vials cooled by liquid Nitrogen, in a Retsch mixer mill MM 200 (Retsch, Haan, Germany). Samples were kept frozen during the whole homogenization procedure. Immediately after pulverization, TRIzol (Invitrogen) was added to samples and left to thaw at room temperature. Next, chloroform wad added followed by centrifugation and the aqueous phase was then transferred to new tubes and mixed gently with 70% ethanol. RNA samples were purified according to the RNeasy Mini Kit instructions. RNase-free DNase Set (QIAGEN, Sollentuna, Sweden) was used to exclude possible DNA contamination. Quality of RNA samples including concentration and RIN (RNA Integrity Number) value were checked by Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE) and Agilent RNA 6000 Nano Kit (Agilent Technologies, Böblingen, Germany). Samples were kept at − 80 °C before microarray analysis.

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Manufacturer protocol

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

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