RNeasy Plus Mini Kit

Protocol tips

Upstream tips	Protocol tips	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.

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

Calu-3 cells were treated with ricin, TRAIL, the combination of ricin and TRAIL, or medium alone (negative control) for 24 h. RNA was extracted from treated or nontreated cells using an RNeasy Plus minikit with additional on-column DNase digestion with an RNase-Free DNase set (Qiagen Hilden, Germany). Protocols were followed according to the manufacturer's instructions. Extracted RNA samples were stored at −80°C until use. Upon assay, RNA integrity was verified by agarose gel electrophoresis. RNA quality and concentrations were measured using an Agilent 2100 Bioanalyzer (Life Technologies, Carlsbad, CA). RNA (100 ng) was hybridized with a predesigned nCounter human immunology panel that included 594 target genes and 15 internal reference genes. The geNorm algorithm was used to select the most stable of the reference genes (GAPDH [glyceraldehyde-3-phosphate dehydrogenase], PPIA, G6PD, EEF1G, GUSB, HPRT1, SDHA, and RPL19) used for normalization. The experimental procedures were carried out on a NanoString preparation station and digital analyzer according to the manufacturer's instructions. Two biological replicates were selected from each of the four groups for analysis.

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Manufacturer protocol

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

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