An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene.

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

Enhanced detection of post-transcriptional modifications using a mass-exclusion list strategy for RNA modification mapping by LC-MS/MS.

There has been a renewed appreciation for the dynamic nature of ribonucleic acid (RNA) modifications and for the impact of modified RNAs on organism health resulting in an increased emphasis on developing analytical methods capable of detecting modifications within specific RNA sequence contexts. Here we demonstrate that a DNA-based exclusion list enhances data dependent liquid chromatography tandem mass spectrometry (LC-MS/MS) detection of post-transcriptionally modified nucleosides within specific RNA sequences. This approach is possible because all post-transcriptional modifications of RNA, except pseudouridine, result in a mass increase in the canonical nucleoside undergoing chemical modification. Thus, DNA-based sequences reflect the state of the RNA prior to or in the absence of modification. The utility of this exclusion list strategy is demonstrated through the RNA modification mapping of total tRNAs from the bacteria Escherichia coli, Lactococcus lactis, and Streptomyces griseus. Creation of a DNA-based exclusion list is shown to consistently enhance the number of detected modified ribonuclease (RNase) digestion products by ∼20%. All modified RNase digestion products that were detected during standard data dependent acquisition (DDA) LC-MS/MS were also detected when the DNA-based exclusion list was used. Consequently, the increase in detected modified RNase digestion products is attributed to new experimental information only obtained when using the exclusion list. This exclusion list strategy should be broadly applicable to any class of RNA and improves the utility of mass spectrometry approaches for discovery-based analyses of RNA modifications, such as are required for studies of the epitranscriptome.

Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism.

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of Salmonella enterica serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards. Salmonella cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.

Labeling of ribosomal ribonucleic acid with iodine 125I.

RNA was labeled with iodine in the reaction catalyzed with lactoperoxidase (LP). Labeling of RNA requires additional purification of LP on Sephadex G-100 and application of RNase inhibitors. Immobilized LP shows lower activity than a soluble enzyme but still it is sufficient to label RNA to 7% IC. Immobilized enzyme is easy to remove from the post-reaction mixture and can be used many times. Optimum conditions for enzymatic labeling were as follows: concentration ratio C/KI for the soluble enzyme equal to 7.5 (for the immobilized enzyme--12). The oxidizing agent (H2O2) used in the enzymatic method of labeling at the concentrations required in the reaction (3.5 X 10(-5) M) does not cause the degradation of nucleic acids as opposed to the oxidizing agent (TlCl3) used in the chemical method. The degree of RNA degradation increases with the amount of incorporated iodine. The RNA preparations of high degree of labeling (10% iodocytosine) by means of the chemical and enzymatic methods do not differ much. At lower labeling the enzymatic method produces less degraded preparations.