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. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

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. - For purifying RNA add either 10 μl β-mercapto ethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Publication protocol

Total RNA obtained from cultures using RNeasy mini kit (Qiagen, Hilden, Germany) was reverse transcribed (SuperScript, Invitrogen, Karlsruhe, Germany) with oligo-dT and gene specific primers for hTRPM8 (5′-cttgggcaaaacacacaatg-3′). Real-time PCR was performed on the template using the TaqMan system in an AbiPrism 7700 Sequence Detector (Applied Biosystems, Foster City, CA) or a LightCycler 480 (Roche, Mannheim, Germany). The following fragments were amplified: nt 2910–3058 from sequence NM_024080.4 was detected with the hTRPM8 probe (5′-FAM-tcgccatgtttggctacacggtgggcacc-TAMRA-3′) and nt 1632–1732 from sequence NM_003234 was detected with the hTFR probe (5′-JOE-tgaatggctagagggatacctttcgtccc-(6-TAMRA)-3′). The human transferrin receptor was used as a control template for RNA integrity and PCR performance. Relative quantification was performed using the REST software (Relative Expression Software Tool

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Manufacturer protocol

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

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