Simultaneous DNA-RNA extraction from coastal sediments and quantification of 16S rRNA genes and transcripts by real-time PCR

Tatti, E., McKew, B. A., Whitby, C. and Smith, C. 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), e54067. (doi: 10.3791/54067) (PMCID:PMC4927785)

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: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Smith, Professor Cindy
• Authors: Tatti, E., McKew, B. A., Whitby, C., and Smith, C. J.
• College/School: College of Science and Engineering > School of Engineering > Infrastructure and Environment
• Journal Name: Journal of Visualized Experiments
• Publisher: JoVE
• ISSN: 1940-087X
• ISSN (Online): 1940-087X
• Published Online: 11 June 2016
• Copyright Holders: Copyright © 2016
• First Published: First published in Journal of Visualized Experiments 112:e54067
• Publisher Policy: Reproduced under a creative commons license