Preparation of RNA Sample from Cell Culture

Materials

• PureLink RNA Mini Kit (Invitrogen cat#12183-018A)
• Mouse Tissue (50-100 mg, about a 3mm cube)
• TRIZol (Invitrogen cat# 12183-555)
• Chloroform (in solvent cabinet)
• Label tubes, for each sample need 3 eppies, at least one of which is 2 mL and one of which is a recovery tube from the PureLink kit.
• 70% Ethanol make with RNAase free water and 100% Ethanol

Protocol

1. Add 1 mL TRIzol reagent to each well of 6 well plate
2. Scrape the cells into the TRIzol using an upside down pipet tip
3. Pipette the TRIzol plus cells into a 1.5 vial.
4. Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex.
5. Incubate at room temperature for 2-3 minutes.
6. Centrifuge in a cold eppendorf centrifuge (4C) on maximum for 15 minutes. Sample will separate into a lower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA.
7. Add 400 uL of 70% ethanol to a fresh tube.
8. Transfer 400 uL of the upper phase to the ethanol tube and mix by vortexing. The remaining chloroform in the previous vial should be disposed of in the phenol waste container under the hood.
9. Remove 700 uL of mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
10. Spin 15s on max. Discard flow through. Add remaining sample, respin and discard flow through.
11. Add 700 uL Wash Buffer I to spin column.
12. Spin 15s on max. Discard flow through the collection tube.
13. Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
14. Spin 15s on max. Discard the flow through and replace the collection tube.
15. Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
16. Spin 15s on max. Discard the flow through and keep the same collection tube.
17. Spin 1 min on max to dry the cartridge. Discard the collection tube and place into a clean recovery tube. Add 100 uL RNAase free water to the center of each tube. This can be adjusted to between 30 and 300 uL elution buffer if necessary.
18. Incubate at room temperature for 1min.
19. Spin 2 min at 12500 rpm to get purified RNA.
20. Quantify the RNA using the nanodrop.