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5.2 Isolation of total RNA

Total RNA (including small RNA, e.g., miRNA) is isolated with the following protocol. 1

HomogenizationHomogenize tissue samples with a rotor-stator homogenizer or another mechanical disruption device using up to 100 mg of tissue per 1 mL NucleoZOL. For tissues with high DNA content (e.g., spleen) it is recommended to use 50 mg of tissue/mL reagent.

For simplicity, this protocol describes RNA isolation using 1 mL of NucleoZOL. For processing the sample in 1.5 mL or 2 mL microcentrifuge tubes, use an 880 μL aliquot of the homogenate (80 mg tissue + 800 μL NucleoZOL). Residual homogenate can be stored at -20 °C or -70 °C for at least one year for later use. to the culture disk (diameter 3.5 cm, 10 cm2). Ensure complete lysis by repeated pipetting. Calculate the amount of reagent based on culture dish area, not on cell number.

An insufficient volume of the reagent will lead to DNA contamination of the isolated RNA.

1 mL NucleoZOL per 107 cells and lyse cells by pipetting up and down several times.

Do not wash the cells before addition of NucleoZOL. Washing of cells might contribute to RNA degradation. Add 1 mL NucleoZOL per 400 μL liquid sample for homogenization and lysis. For processing sample volumes smaller than 400 μL, add 1 mL of NucleoZOL and add water to a final volume of 1.4 mL.Homogenize lipid-rich samples as described above. Centrifuge the samples for 5 min at 12,000 x g. After centrifugation, a fat layer appears on top of the sample. Pierce the upper layer with a syringe/pipette tip and transfer the supernatant into a new tube.