Choice Of Extraction Method Research Articles

Quantitative Evaluation of Intracellular Metabolite Extraction Techniques for Yeast Metabolomics

Accurate determination of intracellular metabolite levels requires well-validated procedures for sampling and sample treatment. Several methods exist for metabolite extraction, but the literature is contradictory regarding the adequacy and performance of each technique. Using a strictly quantitative approach, we have re-evaluated five methods (hot water, HW; boiling ethanol, BE; chloroform-methanol, CM; freezing-thawing in methanol, FTM; acidic acetonitrile-methanol, AANM) for the extraction of 44 intracellular metabolites (phosphorylated intermediates, amino acids, organic acids, nucleotides) from S. cerevisiae cells. Two culture modes were investigated (batch and chemostat) to check for growth condition dependency, and three targeted platforms were employed (two LC-MS and one GC/MS) to exclude analytical bias. Additionally, for the determination of metabolite recoveries, we applied a novel approach based on addition of (13)C-labeled internal standards at different stages of sample processing. We found that the choice of extraction method can drastically affect measured metabolite levels, to an extent that for some metabolites even the direction of changes between growth conditions can be inverted. The best performances, in terms of efficacy and metabolite recoveries, were achieved with BE and CM, which yielded nearly identical levels for the metabolites analyzed. According to our results, AANM performs poorly in yeast and FTM cannot be considered adequate as an extraction method, as it does not ensure inactivation of enzymatic activity.

Influence of cocoa components on the PCR detection of soy lecithin DNA

The labelling of genetically modified organisms (GMOs) is mandatory in the EU. To comply with this regulation, the International Standardisation Organisation recently published standards for the extraction of DNA and PCR detection of GMOs. The PCR detection of (GM) soy lecithin in chocolate however is not an easy assignment. For the most part this is due to inhibitors such as polyphenols resulting from the cocoa mass and the low level of soy lecithin present. Many factors however, such as the choice of extraction method, sample size and PCR protocol influence the PCR end result. Therefore, preconditions regarding the quality, quantity and purity of the extracted DNA should be carefully determined to guarantee a successful GMO analysis.

Ester-linked polar lipid fatty acid profiles of soil microbial communities: a comparison of extraction methods and evaluation of interference from humic acids

Analyses of polar lipid fatty acids isolated from soil are frequently used for characterization of microbial communities, and any interference from fatty acids derived from dead organic material is assumed to be negligible. We studied the initial extraction of lipid material from eight different soils and from purified humic acids using four different combinations of solvent (chloroform or dichloromethane), methanol and buffer (potassium phosphate, pH 7.4 or sodium citrate, pH 4). The quantitative yields of polar lipid fatty acids (PLFA) and PLFA composition of soils and humic acids were compared with absorbance spectra (200–850 nm) of lipid extracts for evaluation of extraction efficiency and potential interference. Chloroform + citrate buffer generally gave the highest, and dichloromethane + phosphate buffer the lowest PLFA yields, and it was estimated that <20% of the yield difference between extraction methods could be explained by interference from humic acids. Principal component analyses of PLFA composition suggested an effect of extraction method for several soils, but when all soils were analyzed together the differences between soils were much more important than the choice of extraction method. Co-extraction of lipids from living cells during preparation of humic acids was quantified and, correcting for this, it was estimated that the interference from non-microbial sources in PLFA analyses was probably not more than 5–10% with the extraction methods employed.