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Bioengineering 2016, 3(1), 7; doi:10.3390/bioengineering3010007

DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

1
Bavarian State Research Center for Agriculture, Department for Quality Assurance and Analytics, Lange Point 6, 85354 Freising, Germany
2
Leibniz Institute for Agricultural Engineering Potsdam-Bornim, Department Bioengineering, Max-Eyth-Allee 100, 14469 Potsdam, Germany
3
Beuth University of Applied Sciences, Department of Life Sciences and Technology, Luxemburger Strasse 10, 13353 Berlin, Germany
4
Department of Microbiology, Technische Universität München, Emil-Ramann-Str. 4, D-85354 Freising-Weihenstephan, Germany
5
Institute for Genome Research and Systems Biology, CeBiTec, Bielefeld University, Bielefeld, Germany
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Ali Khademhosseini
Received: 15 July 2015 / Accepted: 24 December 2015 / Published: 13 January 2016
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Abstract

Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported. View Full-Text
Keywords: ring-trial; fermenter sludge; DNA purity; PCR suitability; extraction efficiency; RNA preservation; reverse transcription; methanogens; Bacteria; absolute quantification ring-trial; fermenter sludge; DNA purity; PCR suitability; extraction efficiency; RNA preservation; reverse transcription; methanogens; Bacteria; absolute quantification
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Lebuhn, M.; Derenkó, J.; Rademacher, A.; Helbig, S.; Munk, B.; Pechtl, A.; Stolze, Y.; Prowe, S.; Schwarz, W.H.; Schlüter, A.; Liebl, W.; Klocke, M. DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison. Bioengineering 2016, 3, 7.

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