RNA extraction from tissues

Procedures for RNA extraction from tissues:

1. pre-weigh an empty cryovial
2. take the tissue specimen out from the cryovial and place it on a 100mm petri dish
3. use a scapel and sterican (like fork and knife) to cut the tissue into desired size
4. transfer the tissue into the pre-weigh cryovial and weigh again (this will give the exact weigh of the tissue being process)
5. transfer the surplus tissue into the original cryovial and keep in the nitrogen tank
6. add 650µl of RLT buffer into the cryovial
7. pound the tissue using a pipette tip until the solution turn orange in colour.
8. transfer the solution into QIA shredder column and centrifuge at 14 000rpm for 4minutes
9. transfer the flow through into a new eppendorf tube and add in 550µl of 70% ethanol
10. centrifuge the eppendorf tube at 14 000rpm for 3 minutes
11. transfer the solution into Rneasy column and centrifuge at 14 000rpm for 15 seconds
12. discard the flow through
13. add 700µl RW1 buffer into the Rneasy column and centrifuge at 14 000rpm for 15 seconds
14. discard the flow through
15. add 500µl RPE buffer into the Rneasy column and centrifuge at 14 000rpm for 15 seconds
16. discard the flow through
17. centrifuge the Rneasy column at 14 000rpm for 1 minute
18. transfer the Rneasy column into a new sterilised round bottom tube
19. add 30µl Rnase-free water and centrifuge at 14 000rpm for 1 minute
20. transfer the flow into the column and centrifuge at 14 000rpm for 1 minute
21. the flow through is the RNA extracted

Thing to note
- after taking the cryovial of tissue out from the nitrogen tank, keep it in liquid nitrogen.

Yeo Sok Kian Jocelyn

0703359J