Lebuhn Michael, Derenkó Jaqueline, Rademacher Antje, Helbig Susanne, Munk Bernhard, Pechtl Alexander, Stolze Yvonne, Prowe Steffen, Schwarz Wolfgang H, Schlüter Andreas, Liebl Wolfgang, Klocke Michael
Bavarian State Research Center for Agriculture, Department for Quality Assurance and Analytics, Lange Point 6, 85354 Freising, Germany.
Leibniz Institute for Agricultural Engineering Potsdam-Bornim, Department Bioengineering, Max-Eyth-Allee 100, 14469 Potsdam, Germany.
Bioengineering (Basel). 2016 Jan 13;3(1):7. doi: 10.3390/bioengineering3010007.
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.
五个机构合作伙伴参与了一项实验室间比对，该比对涉及从不同青草青贮消化实验室规模和中试规模发酵罐获得的沼气生物群落的核酸提取、RNA保存以及基于定量实时PCR（qPCR）的检测。一种基于物理和化学裂解的试剂盒形式的DNA提取系统，具有出色的提取效率，在合作伙伴之间产生了高度可重复的结果，并且明显优于传统的基于CTAB/氯仿/异戊醇的方法。分析目的、样品质地、稠度和上游预处理步骤决定了为在提取物纯度和核酸回收率之间的权衡中实现最大效率而应采用的修改方法。RNA提取的变化更大，提取物的用途决定了所使用的方法。用季铵盐进行RNA稳定化与在液氮中速冻一样是一种令人满意的方法。由于共洗脱的杂质，分光光度法被证明对于用试剂盒获得的提取物中的核酸鉴定和定量价值有限，基于皮考绿的定量更值得信赖。在某些离液胍盐存在的情况下，230nm处的吸光度可能极高，但异硫氰酸胍不影响（q）PCR。通过qPCR进行绝对定量需要应用可靠的内标，其正确的PCR效率和Y轴截距值很重要且必须报告。
