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 RNA was isolated from PC12 cells with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Firststrand cDNA was synthesized from 1.0 μg of total RNA with RT-&GO Mastermix (MP Biomedicals, Solon, USA) and random nanomer (Takara, Tokyo, Japan) according to the manufacturer’s instruction. Real-time RT-PCR was performed with Mx3005P QPCR systems (Agilent Technologies, Forster city, USA) to quantify the mRNA levels of APP, BACE1, and β-actin. Each PCR reaction contained 2 μl cDNA, 10 μl of SYBR Premix Ex TaqTM (TaKaRa, Tokyo, Japan), and 125-500 nM of the forward and reverse primers (Table 1). After an initial denaturation at 95℃ for 30 sec, the amplification program consisted of 40 cycles of denaturation at 95℃ for 5 sec, annealing at 55℃ for 30 sec, and extension at 72℃ for 30 sec. Semi-nested RT-PCR method was used to quantify the mRNA expression of NEP. The relative differences in mRNA expression in the Hg- or MeHg-treated cells were calculated and expressed as a relative increase, setting the control at 100%.

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Manufacturer protocol

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

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