Difference between revisions of "Preparation of RNA Samples from Mouse Tissues"

Revision as of 18:45, 13 October 2010

Materials

• RNeasy Kit (Invitrogen)
• Mouse Tissue (20-30 mg, about a 3mm cube)
• Label tubes, for each sample need 2 eppies, one RNeasy

Protocol

1. Cut tissue and weigh in a fresh tube to ensure a sample of up to 30 mg tissue
2. Prepare buffer RLT by adding 10 uL B-ME per mL of RLT in a clean 15 mL falcon tube. Need 600 uL per sample
3. Using dounce homogenizer, homogenize tissue 10x and transfer to a clean tube
4. Centrifuge 3 min at room temperature at 14 000 RPM
5. Remove centrifuge to a clean tube
6. Add 600 uL of 70% ethanol and mix immediately by pipetting
7. Remove 700 uL of mixture (a precipitate may have formed) and add to a RNeasy spin column in a collection tube
8. Spin 15s at 14 000 RPM. Discard flow through
9. Add 700 uL RW1 to spin colum
10. Per sample, combine 10 uL DNAse I (in Enzymes Box) with 70 uL RDD (in Molecular Biology Box) and mix
11. Add this mixture (80 uL per sample) to spin columns and sit on bench for 15 min
12. Add 350 uL RW1 to spin column
13. Spin 15s at 14 000 RPM. Discard flow through
14. Add 500 uL RPE
15. Spin 2 min at 14 000 RPM
16. Remove spin column to a new eppie and spin again to remove residual RPE
17. Add 50 uL RNAse free water and spin 1 min to elute RNA