RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	C. neoformans might require extra effort for lysis (bead beating, see reference) 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
C. neoformans might require extra effort for lysis (bead beating, see reference) 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

Yeast RNA was isolated using RNeasy mini kit (Qiagen, Valencia, CA) according to the manual instruction with minor modifications. Fresh yeast cultures at approximately 109 cells were prepared and washed with cold water and harvested at 1000 ×g, 4°C for 5 minutes. The pellet was resuspended in 350 μl of lysis buffer, and distributed into two 1.5-ml microcentrifuge tubes. Then, 200 μl of 0.5 mm diameter glass beads (MoBio Laboratories, Carlsbad, CA) were added and the cells were disrupted mechanically with one cycle of agitation at 6800 rpm for 40 seconds using a homogenizer (Precellyse 24 lysis, Berlin technology, France). Samples were incubated on ice for 5 minutes and centrifuged in a microcentrifuge (Hermle, Germany) for 2 minutes at 14,000 rpm. The supernatants were transferred to a new microcentrifuge tube, mixed with 1 volume of 70% ethanol before filtering through RNeasy spin column. Column was washed and RNA was eluted using 30 μl RNase free water. The quality and quantity of the RNA were analyzed using a bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). All samples showed RNA integrity number (RIN)>7. For each sample, two biological replicates (rep1 and rep2) were prepared from two independent yeast cultures for microarray analysis.

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Manufacturer protocol

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

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