RNA Extraction from Sediment

Contact: Slil Siripong

N.B. All solutions must be made with RNase-free water. Glassware has to be baked at 180-200°C for 8 hours. Plasticware should be unopened. Gloves should be worn all the time, and changed frequently.

Organic waste: phenol, chloroform, and tubes or tips that have been in contact with them should be put in the plastic jars provided by Research Safety.

Materials

2-mL conical screw-cap tubes, with smooth caps
2-mL Eppendorf tubes
1-mm zirconium beads (for bacteria), baked at 200°C overnight in conical flask with metal spatula
Bio101 Beadbeater
1X and 5X low-pH buffer (from 10X stock)
- 10X low-pH buffer
- 500 mM sodium acetate 41.0 g/L
- 100 mM EDTA 37.2 g/L
- adjust pH to 5.1 by adding concentrated HCl
20% (w/v) SDS
Phenol, with 0.1% 8-hydroxyquinoline, pH 5.1
- Use amber bottle or clean bottle covered with aluminum foil.
- Warm 500 mL redistilled phenol in 60°C water bath.
- Keep bottle cap loose except when shaking.
- Add 0.5 g 8-hydroxyquinoline (antioxidant, makes phenol yellow).
- Add 500 mL 5X low-pH buffer, shake vigorously, allow phases to separate.
- Check pH of buffer with pH paper.
- Pipette off buffer, replace with 1X low-pH buffer, shake, check pH.
- Repeat until pH of buffer remains ~5.1.
- Store in the dark and keep refrigerated.
- I use phenol, saturated with buffer, pH 4.3 [Fisher: BP1751I-400] instead.
Phenol: Chloroform: Isoamyl alcohol 25:24:1, pH 5.2 [Fisher: BP1753I-100]
Chloroform: Isoamyl alcohol 24:1 [Sigma: C-0549]
5 M ammonium acetate
100% isopropanol (or 100% ethanol; advantage of isopropanol is that only 0.7 sample volume required, where as for ethanol, 2 sample volumes required)
Ice bucket

Method

For each sample (~0.2 g), weight 0.5 g zirconium beads into a 2-mL screw-cap tube. For most sediments, need at least 8 tubes per sample.
Add: 500 μL equilibrated phenol, pH 5.1
35 μL (w/v) SDS
300 μL 5X low-pH buffer
Label four 2-mL Eppendorf tubes per sample – one each for phenol extraction, phenol-chloroform extraction, chloroform extraction, and isopropanol precipitation. (The isopropanol tube will be for storage, so label it completely.)
Bead beat at speed 5 for 30 sec. Incubate on ice for 1 min. Bead beat again at speed 5 for 30 sec.
1. Centrifuge at 14,000 rpm for 5 min at 4°C to separate phases.
2. Transfer the top aqueous phase to fresh Eppendorf tube.
3. Add 100 μL 1X low-pH buffer to the remaining sample.
4. Bead beat, as above.
5. Centrifuge, combine top aqueous phase with the first one.
Add equal volume of phenol, pH 5.1 to the sample, vortex 20 sec, centrifuge for 5 min. Transfer aqueous phase to fresh Eppendorf tube without disturbing the interface. (N.B. If there is a visible layer at the organic/aqueous interface following phenol, phenol-chloroform, and/or chloroform extraction, repeat the extraction until the interface is clear.)
Add equal volume of Phenol: Chloroform: Isoamyl alcohol 25:24:1, pH 5.2 . Vortex and centrifuge as before. Transfer aqueous phase to fresh Eppendorf tube.
Add equal volume of chloroform. Vortex and centrifuge.
Transfer final aqueous phase to fresh tube.
Add:    7.5 M NH₄Ac to 2.5 M (i.e., 0.5 volume sample)
            0.7 volume isopropanol
            [MgCl₂ to 2 mM may help with low RNA samples]
            Vortex to mix. Incubate at -20°C overnight.
Centrifuge for 30 min at 4°C.
1. Carefully decant isopropanol.
2. Wash with 80% (v/v) ethanol.
3. Centrifuge to collect the pellet.
4. Dry pellet using vacuum or air dry.
5. Resuspend pellet in RNase-free water.
6. Store at -80°C.

Reference

Lin, C. and D. A. Stahl. 1995. Taxon-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations. Appl. Environ. Microbiol. 61: 1348-1351.

MacGregor, B. J., D. P. Moser, E. W. Alm, K. H. Nealson, and D. A. Stahl. 1997. Crenarchaeota in Lake Michigan sediment. Appl. Environ. Microbiol. 63: 178-1181.

With contributions by Stefan Mützel, Kerstin Sahm, Laura Sappelsa, Richard Sharp, and Cherie Ziemer.