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 CAOV-3 cells by using RNeasy Mini Kit (Qiagen, Germany). The final concentration of total RNA and its purity were measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific). Conversion from RNA to cDNA was performed using the Two-Step qRT-PCR Kit (Applied Biosystems, USA). A total of 1 μg/mL of cDNA (1 μL) was used in the genes expression assay with seven TaqMan specific primers and probe (β-actin, Bax, Bcl-2, survivin, as well as caspases 3, 8 and 9) genes were added in the assay (Applied Biosystem, USA). The gene expression levels were then evaluated using the StepOne Plus Real-time PCR System (Applied Biosystems, USA); each cycle consisted of 2 min of reverse transcriptase at 50°C, 20 sec of polymerase activation at 95°C, 1 sec of denaturation at 95°C and 20 sec of annealing at 60°C. The process was completed after 40 cycles of reading. Data were then calculated based on the comparative Ct (2-ΔΔCt) standard method described in the study by Wong and Medrano (2005)

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Manufacturer protocol

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

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