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

Human granulosa cell tumor-derived cancer cells KGN and COV434, endometrial ECC1, HEC-1A, HEC-1B, Ishikawa, RL95-2 and AN3CA, testicular Ntera-2D, breast MCF-7 and MDA-231, prostate PC3, DU145 and LNCap, and colon SW480 and WIDR cell lines were cultured in medium supplemented with 10% fetal calf serum (SAFC Biosciences, KS), 2mM L-glutamine, 100µg/ml streptomycin/100 IU/ml penicillin (both from Gibco, Carlsbad, CA). All cell lines were cultured in DMEM, except for HEC-1A in McCoy's, and RL95-2 cells in DMEM supplemented with 10µg/ml bovine insulin (Actrapid, Novo Nordisk, Denmark). RNA was isolated using RNeasy Minikit (Qiagen GmbH, Hilden, Germany) and ribonuclease-free deoxyribonuclease-1 (DNA-free kit, Ambion, TX). Total RNA from human breast, prostate and testes was from Ambion (Life Technologies). Approval was obtained from the Human Research Ethics Committee at Monash Medical Centre, Melbourne to isolate total RNA from ovarian, colon and endometrial tissues.

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Manufacturer protocol

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

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