Tatti, Enrico and McKew, Boyd A and Whitby, Corrine and Smith, Cindy J (2016) 'Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.' Journal of Visualized Experiments (112). ISSN 1940-087X

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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.

Item Type:	Article
Subjects:	Q Science > QH Natural history > QH301 Biology
Divisions:	Faculty of Science and Health > Biological Sciences, School of
Depositing User:	Jim Jamieson
Date Deposited:	27 Jun 2016 14:50
Last Modified:	06 Dec 2018 17:15
URI:	http://repository.essex.ac.uk/id/eprint/17068

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