RNA Visualization in Bacteria by Fluorescence In Situ Hybridization

Abstract

Detecting localized RNA in bacteria is difficult due to the properties of RNA and the small size of the cell. Fluorescence in situ hybridization (FISH) has been an invaluable method for detecting and imaging RNA. In FISH, RNA is fixed in its native subcellular position through chemical cross-linking. An oligonucleotide probe conjugated to a fluorophore is annealed to the target RNA, and the target RNA/probe hybrid is visualized using fluorescence microscopy. This chapter describes the use of FISH to visualize tmRNA, a regulatory RNA required for trans-translation. The method can be adapted to visualize the localization of other regulatory and messenger RNAs as well.

Contamination in Low Microbial Biomass Microbiome Studies: Issues and Recommendations

“…We would instead recommend the use of more nuanced filtering approaches that have been developed to help in situations where cross-contamination is high or when taxa closely related to common DNA contaminants are thought to be present in biological samples [70][71][72][73]. Finally, should contaminant taxa still be driving biological signal after filtering, they should be verified using a different approach such as an effectively used and validated Fluorescent In-Situ Hybridization (FISH) assay [74,75] (3) Bioinformatic modeling has been developed to estimate the source and proportions of contaminant taxa within biological samples. For example, SourceTracker analysis uses Bayesian modeling to estimate the proportion of potential contaminant taxa from a data set [76].…”

Current ocular microbiome investigations limit reproducibility and reliability: Critical review and opportunities

“…Samples with very low read counts should be excluded from further analysis. It should also be noted that additional methods are available should subtractive filtering fail to successfully remove suspected contaminant taxa 51 …”