Sample Preparation

• Adherent cells: Wash the culture dish with 1X PBS and detach cells using a cell spatula. Add 1 ml of RNA Extraction Reagent per 10 cm³ culture dish and lyse cells by pipetting up and down. Collect the lysate in a microcentrifuge tube and allow to stand at room temperature for 5 min.
• Suspension cells: Collect the suspension cells in a microcentrifuge tube and centrifuge at 8000 × g at 2-8 °C for 2 min. Discard the supernatant and add 1 ml of RNA Extraction Reagent per 10⁷ cells. Pipette up and down to remove any precipitates from lysate and allow to stand at room temperature for 5 min.
• Tissue homogenates: Weigh the tissue sample and grind completely to make a tissue powder using liquid nitrogen. Collect the completely grinded tissue powder in a microcentrifuge tube and for each 50-100 mg tissue, add 1 ml of RNA Extraction Reagent. Homogenize using a homogenizer and repeatedly pipette up and down. Allow the tissue homogenate to stand at room temperature for 5 min.

Assay Procedure

1. Add 0.2 ml of Chloroform per ml of RNA Extraction Reagent used. Shake the tube vigorously for 30 sec and allow to stand at room temperature for 3 min.
2. Centrifuge the tube at 10000 × g at 2-8 °C for 15 min. The solution thus obtained seperates into three different layers: lower pink organic phase, an interphase and a colorless upper aqueous phase which contains the RNA.
3. Collect the colorless upper phase layer containing the RNA in a RNase-free tube. 
4. Add 0.5 ml Isopropanol for per ml of RNA Extraction Reagent used. Mix thoroughly by inverting the tube and allow to stand a room temperature for 10 min.
5. Centrifuge at 10000 × g at 2-8 °C for 10 min. Discard the supernatant.
6. Add 1 ml of 75% ethanol (prepared with RNase-free water) per 1 ml of RNA Extraction Reagent used. Vortex vigorously.
7. Centrifuge at 7500 × g at 2-8 °C for 5 min. Discard the supernatant and air dry the RNA pellet for 5 min.
8. Dissolve the RNA pellet in 50-100 µl of RNA Dissolving Solution and incubate at 55-60 °C for 10 min or at -70 °C for long term storage.

Note

• Ensure the complete extraction by sufficiently shaking the tube after adding Chloroform.
• All other reagents and consumbles like Isopropanol, 75% Ethanol, which are not provided, should be RNase-free.