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 pancreatic tissue using the RNeasy® mini kit (Qiagen, Inc., Valencia, CA, USA) and the concentration was determined using a SmartSpec Plus Spectrophotometer (BioRad, Hercules, CA, USA). First-strand cDNA was synthesized using 1 μg of total RNA in a 20 μl reverse transcriptase reaction mixture using iScript™ cDNA synthesis kit (BioRad) according to the manufacturer’s instructions. The region of tumor necrosis factor-α (Tnfa), interleukin-6 (Il6), monocyte chemoattractant protein-1 (Mcp1), vascular adhesion molecule 1 (Vcam1), sonic hedgehog (Shh) and Gli transcription factor (Gli) mRNA were amplified using a real-time PCR approach according to our previous published protocol (17, 18). All real-time PCR reactions and quantitations were performed using the MiniOpticon Real-Time PCR System (BioRad). Data of each mRNA expression are shown as the relative fold change normalized by that of glyceraldehyde 3-phosphate dehydrogenase (Gapdh).

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Manufacturer protocol

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

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