Chen, F., J.M. González, W.A. Dustman, M. A. Moran, and R. E. Hodson. 1997. In situ reverse transcriptase: an approach to characterize genetic diversity and activity of prokaryotes. Appl. Environ. Microbiol. 63:4907-4913.
         Reverse transcription of RNA molecules inside intact bacterial cells
    was carried out using reverse transcriptase with a single oligonucleotide
    complementary to specific 16S rRNA or mRNA sequences. Fluorescently-labeled
    nucleotides were incoporated into each transcribed cDNA inside cells.  This
    protocol is termed in situ reverse transcription (ISRT).  In this
    study, using species-specific primers targeting unique regions of the 16S
    rRNA sequences, ISRT was used successfully to detect and enumerate the two
    lignin degrading bacteria Microbulbifer hydrolyticus IRE-31 and
    Sagittula stellata E-37 in culture mixtures and complex enrichment
    communities selected for lignin degradation.  Image analysis revealed that
    M. hydrolyticus IRE-31 and S. stellata E-37 accounted for
    approximately 30% and 2%, respectively, of the total bacterial cells in
    lignin enrichment communities. Populations estimated by ISRT were comparable
    with those estimated by in situ hybridization (ISH) techniques and
    with those estimated by hybridizations against extracted community DNA. ISRT
    was also successfully used to detect Pseudomonas putida F1 expressing
    the todC1 gene, in seawater exposed to toluene vapor. ISRT provided a
    higher signal intensity compared to ISH, especially when targeting mRNA. The
    calculated pixel intensities resulted from ISRT were up to 4.2 times more
    intensive than those from ISH.  It suggests that multiple incorporation of
    fluorescently-labeled nucleotides into cDNA provided a high sensitivity for
    phylogenetic identification of bacterial populations as well as detection of
    cells expressing a specific gene within complex bacterial communities.