Standard Operating Procedure

RNA Isolation

Hazards:

Protection:

Wear gloves

Waste:

Spill Clean-Up:

Procedure:

Notes:

• All steps at RT, unless otherwise noted.
• Set rotor speed for homogenizer to 20.
• Wash homogenizer with RNase-free ETOH and then DEPC water between each sample.
• Elution with 2 x 50 µl water gave great yields.
• Use Invitrogen Micro-to-Midi Total RNA Purification System kit.

RNA Lysis Buffer

Add 10 µl 2-ME per ml of lysis buffer-make fresh each time.

RNA Isolation from cells

1. Spin cells and wash in cold PBS in eppendorf tube. Carefully remove all supernatant.
2. Add appropriate volume of lysis solution (see table) Add 2-ME (see above)!.

Number of cells	RNA Lysis solution (µl)
≤ 5 x10^6	300
5x10^6 to 5x10^7	600
5x10^7 to 1x10^8	1200

3. Homogenize for about 30 seconds. Move homogenizer up and down in the tube.
4. Spin tube, 12000 rpm, 5 minutes.
5. Transfer supernatant to clean 50 ml tube.
6. Add 600 µl 70% ETOH, homogenize 30 seconds.
7. Add 600 µl of sample to RNA spin cartridge tube.
8. Centrifuge 14000 rpm, 15 seconds. Discard the flow-thru.
9. Apply remainder of sample to RNA spin cartridge, spin, discard flow-thru.
10. Add 350 µl Wash Buffer I.
11. Centrifuge 14000 rpm, 15 seconds. Discard flow-thru.
12. Add 70 µl RDD buffer to 10 µl DNase and add to column.
13. Incubate 15 minutes, RT. (Be careful not to incubate longer!)
14. Add 350 µl Wash Buffer I and wait 3-5 minutes.
15. Centrifuge 14000 rpm, 15 seconds. Discard flow-thru.
16. Place spin cartridge into clean RNA wash tube.
17. Add 500 µl Wash Buffer II to cartridge.
18. Centrifuge 14000 rpm, 15 seconds. Discard flow-thru.
19. Centrifuge 1 minute to dry membrane, 14000 rpm.
20. Remove cartridge from tube and place it in RNA recovery tube.
21. Add RNase-free water (see tables below for amount), let stand 1 minute.
22. Centrifuge 14000 rpm, 2 minutes. Collect elutions in same tube.
23. Store RNA at –20ºC.

RNA yields from HeLa Cells
Number of Cells	RNA yield (µg)
1x10^5	1.4-1.6
1x10^6	19-20
1x10^7	144-179
5x10^7	513-729
1x10^8	900-1123

Expected RNA (µg)	Elution volume (µl)
≤30	1 x 30
30-60	2 x 30
60-300	2 x 40
300-600	2 x 50
≥600	2 x 100

Determine Optical Density (OD)

1. Add 3.75 µl RNA to 71.25 µl H₂0-to determine concentration of sample). (1:20 dilution)
2. Add 3.75 µl RNA to 71.25 µl 10mM Tris, pH 8.0-to determine purity of sample.

• (Conc. ug/ml) x (1 ml/1000 µl) x (n µl sample) = ug total RNA
• 260/280 ration of Tris-RNA should be between 1.7 and 2.0