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

A protocol was developed for the efficient recovery of intact, high molecul ar weight rRNA from different environmental matrices. Critical variables we re identified in sample processing that influenced yield and integrity of r ecovered nucleic acid. Most notably, the order of addition and the buffer t o 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 bu ffer volumes contributed to an order of magnitude increase in recovery rela tive to reverse order of addition (buffer addition to sample). An optimized extraction protocol was used to evaluate rRNA yield by seeding samples wit h whole cells and radiolabeled nucleic acid. Recovery of intact rRNA was co nfirmed by polyacrylamide gel electrophoresis, which was also used to provi de another estimate of quantity. This optimized protocol was used to measur e depth-related changes in biomass distribution in Lake Michigan deep-water sediments. This revealed a biomodal biomass distribution; a maximum near t he water/sediment interface and a secondary peak associated with the oxic/s uboxic boundary. A significant portion df the community at the oxic/suboxic boundary was composed of non-methanogenic Archaea. (C) 2000 Published by E lsevier Science B.V. All rights reserved.