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Molecular Biology Protocols |
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3B. Purification of Total RNA from Animal Tissues: Filter-Based Procedure
Refer to the introduction to Chapter 3A for precautions. Wear disposable gloves throughout and change frequently. Use only nuclease-free tips and tubes.
PROMEGA SV TOTAL RNA ISOLATION SYSTEM
Prepare solutions according to Promega technical manual 048:
1 ml β-mercaptoethanol to 50 ml lysis solution. Store at 4oC.
Nuclease-free water to DNAse as indicated. Freeze in aliquots of 12 μl.
Ethanol to wash solution and DNAse stop solution as indicated.
1. Collect tissues into RNAlater (Ambion), a solution that will protect against RNAse-induced degradation of RNA. Use at least 5 volumes of RNAlater for normal tissues, more for RNAse-rich tissues such as hepatopancreas. Store at 4oC overnight, then at -20oC indefinitely. Tissue is stable in RNAlater for days at room temperature, weeks at 4oC, and probably years at -20oC.
2. Prepare homogenizer with RNAse-Zap, rinsing thoroughly with nuclease-free water. Rinse with a small amount of lysis solution briefly before use.
3. Add 1 ml of SV RNA Lysis Buffer (with β-ME) to a 10-ml nuclease-free tube and weigh.
4. Remove gill tissue from RNAlater with clean dissecting instruments and blot on clean Kimwipe. Add 175 to 350 mg gill tissue to Lysis Buffer and homogenize as quickly as possible at high speed until no visible tissue fragments remain (about 30 secs).
5. Weigh the tube to calculate tissue mass to be sure that no more than 350 mg tissue were added.
6. Transfer 175 μl of the tissue lysate to a clean 1.5-ml microcentrifuge tube. Freeze remaining lysate in a 2-ml microcentrifuge tube if desired.
7. Add 350 μl SV RNA Dilution Buffer (blue) and mix by inverting 3-4 times.
8. Place in heating block at 70oC for 3 minutes maximum, then on ice for 4-5 minutes.
9. Centrifuge for 10 minutes at 12,000-14,000 x g.
10. Transfer the supernatant to a fresh microcentrifuge tube by pipetting. Avoid disturbing the pelleted debris.
11. Add 200 μl 95% ethanol to the cleared lysate and mix by pipetting 3-4 times.
12. Transfer mixture to a Spin Column Assembly and centrifuge at top speed for one minute.
13. Remove the Spin Basket from the Spin Column Assembly and discard liquid in the collection tube. Put the Spin Basket back into the collection tube. Add 600 μl SV RNA Wash Solution to the Spin Column Assembly and centrifuge at top speed for one minute.
14. Empty the collection tube. Prepare a DNAse incubation mix by combining 40 μl Yellow Core Buffer, 5 μl 0.09 M MnCl2 and 5 μl DNAse enzyme solution (in this order). Mix by gentle pipetting – do NOT vortex. Keep the thawed DNAse enzyme on ice.
15. Apply 50 μl of freshly prepared DNAse incubation mix directly to the membrane inside the Spin Basket, thoroughly covering the membrane. Incubate for 15 minutes at room temperature.
16. Add 200 μl SV DNAse Stop Solution to the Spin Basket and centrifuge at maximum speed for one minute.
17. Add 600 μl SV RNA Wash Solution and centrifuge at maximum speed for one minute.
18. Empty the collection tube and add 250 μl SV RNA Wash Solution. Centrifuge at maximum speed for two minutes.
19. Remove the cap from the Spin Basket by using a twisting motion.
20. Remove one capped 1.5-ml Elution Tube from the packaging and tape the package shut. Transfer the Spin Basket to the Elution Tube and add 50 μl nuclease-free water to the membrane, completely covering the membrane. Wait one minute, then centrifuge at maximum speed for one minute. (Protocol specifies 100 μl with no wait.)
21. Remove the Spin Basket and discard. Cap the Elution Tube containing the purified RNA and store at -70oC (or -20oC).
(22. If a more concentrated RNA is required, it can be vacuum-dried and resuspended in a smaller volume of water.)