DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison.
作者： Michael Lebuhn , Jaqueline Derenkó , Antje Rademacher , Susanne Helbig , Bernhard Munk
DOI: 10.3390/BIOENGINEERING3010007
关键词:
摘要: 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 pilot scale fermenters. A kit format DNA extraction system on physical chemical lysis with excellent efficiency yielded highly reproducible results among the clearly outperformed a traditional CTAB/chloroform/isoamylalcohol method. Analytical purpose, sample texture, consistency upstream pretreatment steps determine modifications that should be applied to achieve maximum trade-off between extract purity recovery rate. was much more variable, destination determines method used. stabilization quaternary ammonium salts as satisfactory approach flash freezing liquid N₂. Due co-eluted impurities, spectrophotometry proved limited value qualification quantification extracts obtained kit, picoGreen® trustworthy. Absorbance at 230 nm can extremely high presence certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute by qPCR requires application reliable internal standard which correct Y-intercept values are important must reported.
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Stephen A. Bustin, Jim F. Huggett, Tania Nolan, MIQE: guidelines for the design and publication of a reliable real-time PCR assay Caister Academic Press. ,(2013)
Gemma Johnson, Afif Abdel Nour, Tania Nolan, Jim Huggett, Stephen Bustin, Minimum information necessary for quantitative real-time PCR experiments. Methods of Molecular Biology. ,vol. 1160, pp. 5- 17 ,(2014) , 10.1007/978-1-4939-0733-5_2
M. Lebuhn, M. Effenberger, A. Gronauer, P.A. Wilderer, S. Wuertz, Using quantitative real-time PCR to determine the hygienic status of cattle manure Water Science and Technology. ,vol. 48, pp. 97- 103 ,(2003) , 10.2166/WST.2003.0230
George L Mutter, David Zahrieh, Chunmei Liu, Donna Neuberg, David Finkelstein, Heather E Baker, Janet A Warrington, Comparison of Frozen and RNALater Solid Tissue Storage Methods for Use in RNA Expression Microarrays BMC Genomics. ,vol. 5, pp. 88- 88 ,(2004) , 10.1186/1471-2164-5-88
Mandy Schönberg, Bernd Linke, The influence of the temperature regime on the formation of methane in a two‐phase anaerobic digestion process Engineering in Life Sciences. ,vol. 12, pp. 279- 286 ,(2012) , 10.1002/ELSC.201100062
M. Lebuhn, M. Effenberger, G. Garcés, A. Gronauer, P.A. Wilderer, Evaluating real-time PCR for the quantification of distinct pathogens and indicator organisms in environmental samples. Water Science and Technology. ,vol. 50, pp. 263- 270 ,(2004) , 10.2166/WST.2004.0065
Andreas Schroeder, Odilo Mueller, Susanne Stocker, Ruediger Salowsky, Michael Leiber, Marcus Gassmann, Samar Lightfoot, Wolfram Menzel, Martin Granzow, Thomas Ragg, The RIN: an RNA integrity number for assigning integrity values to RNA measurements BMC Molecular Biology. ,vol. 7, pp. 3- 3 ,(2006) , 10.1186/1471-2199-7-3
Michael Lebuhn, Stefan Weiß, Bernhard Munk, Georg M. Guebitz, Microbiology and Molecular Biology Tools for Biogas Process Analysis, Diagnosis and Control Biogas Science and Technology. ,vol. 151, pp. 1- 40 ,(2015) , 10.1007/978-3-319-21993-6_1
ANA RIESGO, ALICIA R. PÉREZ‐PORRO, SUSANA CARMONA, SALLY P. LEYS, GONZALO GIRIBET, Optimization of preservation and storage time of sponge tissues to obtain quality mRNA for next-generation sequencing. Molecular Ecology Resources. ,vol. 12, pp. 312- 322 ,(2012) , 10.1111/J.1755-0998.2011.03097.X
Can Su, Liping Lei, Yanqing Duan, Ke-Qin Zhang, Jinkui Yang, Culture-independent methods for studying environmental microorganisms: methods, application, and perspective. Applied Microbiology and Biotechnology. ,vol. 93, pp. 993- 1003 ,(2012) , 10.1007/S00253-011-3800-7