Using phylogenetic probes for quantification of stable isotope labeling and microbial community analysis

摘要

Herein is described methods for a high-sensitivity means to measure the incorporation of stable isotope labeled substrates into RNA following stable isotope probing experiments (SIP). RNA is hybridized to a set of probes such as phylogenetic microarrays and isotope incorporation is quantified such as by secondary ion mass spectrometer imaging (NanoSIMS).

主权项

1. A method for determination of stable isotope incorporation in a community of organisms comprising the steps of:
a) supplying said community of organisms with at least two substrates simultaneously for a defined period of time, wherein each of the at least two substrates is labeled with a different stable isotope; b) extracting RNA from the organisms; c) fragmenting said RNA to provide fragmented RNA; d) labeling a fraction of the fragmented RNA with a detectable label to provide labeled fragmented RNA; e) hybridizing the labeled fragmented RNA to a set of oligonucleotide probes, wherein the set of oligonucleotide probes is an array of oligonucleotide probes attached to a substrate; f) detecting hybridization signal strength of labeled fragmented RNA hybridized to the oligonucleotide probes to determine the community organism composition; g) identifying a responsive set of oligonucleotide probes based on the hybridization signal strength in step f); h) hybridizing a fraction of unlabeled fragmented RNA to a second array of oligonucleotide probes, wherein the second array comprises the responsive set of oligonucleotide probes attached to a conductive substrate; i) detecting the unlabeled fragmented RNA hybridized to the responsive set of probes to determine the stable isotope incorporation into the community of organisms using imaging mass spectrometry or spectroscopy.