Critical Factors Influencing the Recovery and Integrity of rRNA Extracted From Environmental Samples: Use of an Optimized Protocol to Measure Depth-Related Biomass Distribution in Freshwater Sediments

J Microbiol Methods. 2000 Apr;40(2):153-62. doi: 10.1016/s0167-7012(00)00120-2.

Abstract

A protocol was developed for the efficient recovery of intact, high molecular weight rRNA from different environmental matrices. Critical variables were identified in sample processing that influenced yield and integrity of recovered nucleic acid. Most notably, the order of addition and the buffer to sample volume ratio profoundly influenced the efficiency of nucleic acid recovery from sediment material when utilizing a guanidine thiocyanate-beta-mercaptoethaol extraction buffer. Addition of one sample volume to five buffer volumes contributed to an order of magnitude increase in recovery relative to reverse order of addition (buffer addition to sample). An optimized extraction protocol was used to evaluate rRNA yield by seeding samples with whole cells and radiolabeled nucleic acid. Recovery of intact rRNA was confirmed by polyacrylamide gel electrophoresis, which was also used to provide another estimate of quantity. This optimized protocol was used to measure depth-related changes in biomass distribution in Lake Michigan deep-water sediments. This revealed a biomodal biomass distribution; a maximum near the water/sediment interface and a secondary peak associated with the oxic/suboxic boundary. A significant portion of the community at the oxic/suboxic boundary was composed of non-methanogenic Archaea.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biomass*
  • Buffers
  • Electrophoresis, Polyacrylamide Gel
  • Environmental Microbiology*
  • Escherichia coli / genetics
  • Fresh Water / microbiology*
  • Genetic Techniques
  • Geologic Sediments / microbiology*
  • Great Lakes Region
  • Guanidines
  • Mercaptoethanol
  • Nucleic Acid Hybridization
  • RNA, Ribosomal / analysis
  • RNA, Ribosomal / isolation & purification*
  • Seawater / microbiology
  • Thiocyanates

Substances

  • Buffers
  • Guanidines
  • RNA, Ribosomal
  • Thiocyanates
  • guanidine thiocyanate
  • Mercaptoethanol