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

Total RNAs were isolated from cultured cells using RNeasy plus Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s manual. The RNA pellet obtained in the final step was dissolved in 30 μl of sterile diethylpyro-carbonate (DEPC)-treated water, and its concentration was determined using a UV spectrophotometer at 260 nm. RNA was kept in DEPC-treated water at −70°C until use. Reverse transcription was performed using High Capacity RNA-to-cDNA Kit (AP, Foster, CA, USA). PCR amplifications were then carried out with the primers. The PCR primers used were 5′-ACAAGCCTGTAGCCCATGTT-3′ and 5′-AAAGATGACCTGCCCAGACT-3′ for TNF alpha, 5′-ACCAATGCCACAAAGGAAC-3′ and 5′-CTG CAATTGAAGCACTGGAA-3′ for the human TNF receptor 1, 5′-CTCAGGAGCATG GG G-ATAAA-3′ and 5′-AGCCAGCCAGTCTGACATCT-3′ for the human TNF receptor-2, 5′-ATGGCGATGGCTGCGTGTCCTG-3′ and 5′-AGCGCCTCCTGGGTCTCGGGGTAG-3′ for the human DR3, 5′-ACTTTGGTTGTTCCGTTGCTG TTG-3′ and 5′-GGCTTTCCATTTGCTGCTCA-3′ for the human DR 4, and 5′-CAAAGCCCATTTTTCTTCCA-3′ and 5′-GACAAAGCCACCCCAAGTTA-3′ for human FAS, 5′-TCG CAGAAGTGC A CCTAA AG-3′ and 5′-AGCCTTCCCCTCATCAAAGT-3′ for human ApoL, 5′-CAGCTCTTCCACCTACAG AAG G-3′ and 5′-AAGATTGAACACTGCCCCCAGG-3′ for FasL, 5′-AGACCTGCGTGCTGATCGTG-3′ and 5′-TTATTTTGCGGCCCAGAGCC-3′ for human TRAIL, 5′-GAAGGTGAAGGTCGGAGT-3′ and 5′-CTTCTACCACTACCCTAAAG-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. The reaction mixture containing 10 μl premix, 1 μl of each forward and reverse primers and 1 μl of cDNA are made upto 20 μl. The reverse transcription PCR is carried out in three steps. Step1: 95°C-3 min, Step2: 95°C-30 sec, 55–65°C (ambient temp)-30 sec and 70°C-1 min and Step3: 72°C-10 min. The Step 2 is repeated for 30 cycles and lid temp-105°C. The samples are loaded into horizontal gel electrophoresis and detected under UV light using MyImage software.

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Manufacturer protocol

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

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