title: Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.
abstract: Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.
authors.affiliation: Microbiology, School of Natural Sciences, National University of Ireland Galway.
authors.forename: Enrico
authors.initials: E
authors.lastname: Tatti
authors.affiliation: School of Biological Sciences, University of Essex.
authors.forename: Boyd A
authors.initials: BA
authors.lastname: McKew
authors.affiliation: School of Biological Sciences, University of Essex.
authors.forename: Corrine
authors.initials: C
authors.lastname: Whitby
authors.affiliation: Microbiology, School of Natural Sciences, National University of Ireland Galway; cindy.smith@nuigalway.ie.
authors.forename: Cindy J
authors.initials: CJ
authors.lastname: Smith
mesh_terms: D004247:DNA; D019015:Geologic Sediments; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012336:RNA, Ribosomal, 16S; D004247:DNA