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Bead Beating: A Primer (continued)

APPENDIX C

Isolation of Total RNA from Animal Tissues

RNA can be relatively stable within cells, especially tissues, but once released into a homogenate, it can be enzymatically degraded by RNases quickly.  RNases are ubiquitous and must be rendered inactive to protect RNA during sample processing.  This is accomplished by disrupting samples under conditions that inactivate the RNases (either cryogenically or with chemical denaturation) and then removing the RNases using organic extractions.  Phenol is used to remove protein, including RNases, while the chloroform/isoamyl alcohol removes residual phenol from the RNA solution.  As this process requires the use of phenol (which can cause serious chemical burns) and chloroform (carcinogenic), use extreme caution when performing this protocol.  It is best to do the extractions in a chemical hood.

Homogenization Buffer:  4 M Guanidine thiocyanate, 42 mM citrate pH 4.3, 0.1% N-lauroylsarcosine, 0.1 M 2¬≠mercaptoethanol

Isolation Reagents: Phenol saturated (stored under) 42 mM citrate buffer, pH 4.3, chloroform/isoamyl alcohol (ratio of 24:1), 3 M sodium acetate, pH 5.2, 95% ethanol (store at -20°C), 70% ethanol (store at -20°C),  water (molecular biology grade).

Procedure

1. Place the sample, homogenization buffer and grinding media in a grinding vessel and homogenize by bead beating.  Alternatively, tissue can first be pulverized cryogenically, with homogenization buffer added to the powdered tissue subsequently.  In either case, centrifuge the homogenate to pellet insoluble cellular debris and transfer the supernatant to a clean polypropylene tube.
2. Add an equal volume of buffer saturated phenol, vortex for 30 sec., than add an equal volume of chloroform/isoamyl alcohol.  Vortex for 30 seconds, and centrifuge for 5 min. to separate the phases.
3. Transfer the upper aqueous phase containing the RNA to a clean tube.
4. Repeat the phenol/chloroform extraction again and transfer the supernatant to a clean tube.
5. Add 1/10 volume of 3 M sodium acetate, pH 5.2.  Mix and add 2.5 volumes of 95% ethanol and mix.  Incubate at ¬≠20°C for 30 minutes to overnight.
6. Centrifuge for 15 minutes (12,000 g or on high in microfuge) to pellet the RNA.
7. Decant and wash the pellet with 70% ethanol.  Centrifuge for 1 minute, decant, and air dry pellet.
8. Resuspend the RNA in molecular biology grade water and store at ¬≠80°C.