Stéphanie M. Swarbreck, Erika A. Lindquist, David D. Ackerly, and Gary L. Andersen.
Earth Sciences Division, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, CA 94720, USA.
Department of Integrative Biology, 3060 Valley Life Sciences Building, University of California, Berkeley, CA 94720, USA.
Department of Energy Joint Genome Institute, 2800 Mitchell Dr, Walnut Creek, CA 94598, USA.

Overview

  • Key words: Avena barbata and A. Barbata, climate change, ESTs, root
  • Aim of the study: Generation of a large amount of cDNA sequence data for transcriptomic studies in Avena barbata and A. barbata.
  • Application: Transcriptome analysis by Sanger sequencing & pyrosequencing
  • Sample name: Avena barbata and A. barbata
  • Sample type: Root
  • Material: FastPrep-24™ instrument
  • Buffer: Modified CTAB (CetylTrimethylAmmonium Bromide) buffer: 50 ml of 0.1 M of aluminum ammonium sulfate and 0.5 ml of phenol: chloroform: isoamyl alcohol (25: 24: 1)

Protocols & Parameters

Total RNA was extracted from 200 mg of roots using a modified CTAB (cetyltrimethylammoniumbromide) method.

  1. 0.5 ml of modified CTAB buffer was added to the samples
  2. Samples were bead beaten for 30 s at 5.5 m/s in a FastPrep-24™ instrument
  3. Samples were centrifuged at 16,000 x g for 5 min at 4°C.
  4. A second extraction with the modified CTAB buffer was conducted
  5. A 1 ml aliquot of chloroform was then added to the aqueous supernatant followed by a centrifugation at 12,000 x g for 5 min at 4°C.
  6. 2 vols. of 30% (w/v) polyethylene glycol 6,000 in 1.6 M NaCl solution and 1 ml of linear acrylamide were added to the aqueous supernatant to precipitate the nucleic acids.
  7. The RNA/DNA pellet was subsequently washed with 60% ice-cold ethanol and resuspended in diethylpyrocarbonate (DEPC)-treated water..

Conclusion

  • The results show that the FastPrep-24™ instrument extraction method generates a good-quality RNA for sequencing.
  • The combined use of pyrosequencing and Sanger sequencing was successful in generating a high number of expressed sequence tags (ESTs).