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

Total RNA was extracted from 25 mg of either fresh NEC and non-NEC ileum using the RNeasy Mini Kit or from six 10-micron sections of formalin-fixed, paraffin-embedded tissue pieces using the RNeasy FFPE Kit (Qiagen Valencia, CA). Total RNA was reverse transcribed using the RT2 First Strand Kit (Qiagen) per manufacturer's instructions. The cDNA-containing reaction mixture was added to each well of a 96-well-plate PCR array for quantitative real-time (RT) PCR (RORC: Th17 for Autoimmunity and Inflammation PCR Array, occludin: cat no. PPH02571B RT2 Profiler PCR Array; Qiagen). PCR cycles were performed according to the manufacturer's instructions. Expression levels of cytokine genes were quantified using quantitative RT-PCR analysis based on intercalation of SYBR® Green on an ABI 7300 Real-Time PCR system (Life Technologies, Carlsbad, CA). The relative level of mRNA expression for each gene in each sample was normalized to the expression level of reference gene GAPDH and the data were analyzed using the ΔΔCt method

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Manufacturer protocol

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

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