ArticleJournal of visualized experiments : JoVE2014
Fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of RNases in vivo.
Article in Journal of visualized experiments : JoVE, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed, 16 citations in OpenAlex.
- Histidine transport is essential for the growth of Staphylococcus aureus at low pH.PLoS pathogens · 2024Article
- Transcriptional control of two distinct lactococcal plasmid-encoded conjugation systems.Current research in microbial sciences · 2024Article
- Hydrogen Peroxide Affects Growth ofFrontiers in immunology · 2021Article
- High-throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors.Molecular microbiology · 2020Article
- Genetic Adaptation of a Mevalonate Pathway Deficient Mutant inFrontiers in microbiology · 2018Article
- Daptomycin Tolerance in the Staphylococcus aureus pitA6 Mutant Is Due to Upregulation of the dlt Operon.Antimicrobial agents and chemotherapy · 2016Article
- A novel point mutation promotes growth phase-dependent daptomycin tolerance in Staphylococcus aureus.Antimicrobial agents and chemotherapy · 2015Article
- The MazEF Toxin-Antitoxin System Alters the β-Lactam Susceptibility of Staphylococcus aureus.PloS one · 2015Article
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Authors and funding
2 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5'-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure. In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5' ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.
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