RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT). - 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 15-20 min. - 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
- For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT). - 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 15-20 min. - 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

Gene silencing by siRNA transfection in Vero cells was confirmed for the top ten genes. Cells were transfected in triplicate with individual siRNAs as previously described. Forty-eight hours post-transfection, cells were harvested for RNA isolation using RNeasy Mini Kit (Qiagen) following manufacturer instructions. qRT-PCR was performed in triplicate to measure mRNA levels of targeted genes using 1 ug RNA (SYBR Green RT Kit; ThermoFisher). The cycle threshold (CT) values were normalized to the house keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or 18s ribosomal RNA. Values are presented as the mean of the triplicates±s.e.m.

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Manufacturer protocol

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

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