RNA Extraction Protocol

RNA Extraction Protocol

RNeasy Plant Mini Kit

Crushed & frozen (-80) plant tissue

Bench diaper

RNA filter tips (P10, P200, P1000)

Micropipettes (P10, P200, P1000)

RNAse-free EtOH3

Centrifuge tubes (1.5mL)

Solution of 10mL Buffer RLC (from kit) + 100 μl of 2-mercaphtoethanol (found in chemical hood)

1.    Prepare your bench with the lab diaper and all the things you’ll need. It is a good idea to do multiple samples (4+) because RNA is super tricky – no matter how careful you try to be there is a significant chance you will lose all your RNA.

2.    If none already, create Solution of 10mL Buffer RLC (from kit) + 100 μl of 2-mercaphtoethanol. Store for future use

3.    Clearly label enough centrifuge tubes

4.    Put 450 μl Buffer RLT or Buffer RLC in each 1.5 mL centrifuge tube

5.    Weigh each tube + buffer solution and write it down

6.    Add approx'''. 100mg of frozen tissue''', working inside the -80. DO NOT LET THE PLANT TISSUE THAW OUT. As quickly as possible shake the tubes so that the tissue is suspended in the buffer. Vortex if needed.

7.    Weigh each tube again, making sure that you have taken approx. the right amount of tissue. Write down weight of tissue added. Vortex all samples vigorously.

8.    Transfer lysate to a QIAshredder spin column (lilac) placed in a 2mL collection tube.

9.    Centrifuge lilac columns for 2 min at full speed.

10. Transfer supernatant of the flow-through to a 1.5 mL micro-centrifuge tube without disturbing the pellet.

11. Add 0.5 volume of ethanol (250 μl) to each tube. Mix by pipetting up and down vigorously. A good measure of when to stop mixing is once your thumb starts getting really sore.

12. Let incubate in the deli case for at least one hour, and up to 24 hours.

13. Transfer the sample (usually 650 μl) with any precipitate to an RNeasy Mini spin column (pink) in a 2mL collection tube (supplied).

14. Close the lid, centrifuge for 15s. Discard flow-through.

15. Add 600-650 μl Buffer RW1 to the RNeasy spin column.

16. Close the lid, centrifuge for 15s. Discard flow-through.

17. Add 500 μl Buffer RPE to RNeasy spin column.

18. Close the lid, centrifuge for 15s. Discard flow-through.

19. Add 500 μl of Buffer RPE to RNeasy spin column.

20. Centrifuge for 2 minutes. Discard flow through.

21. Centrifuge again for 1 min to dry the membrane.

22. Place the RNeasy spin column in a new, clearly labeled, 1.5 mL collection tube (supplied).

23. Add 10 μl RNase-free H20 directly to spin column membrane (but without touching it with the pipette tip) in order to elute RNA.

24. Centrifuge for 1 min. Discard spin column and put RNA on ice.

25. Use nano-drop to measure RNA concentration of product.

Good RNA concentration ≈ >80 ng/μl

Good RNA OD (260/280) ≈ 1.8-2.1