Dataset Information

Optimization of a Method for the Simultaneous Extraction of Polar and Non-Polar Oxylipin Metabolites, DNA, RNA, Small RNA, and Protein from a Single Small Tissue Sample.

ABSTRACT: A more comprehensive picture of tissue biology can be obtained through the application and integration of multiple omic technologies. However, the common challenge in working with a precious sample is having a sample too small to separately extract analytes of interest for each experiment. Considering the high heterogeneity that can be present in a single tissue sample, extracting all biomolecules from a single and undivided tissue is preferable because it allows direct comparison of results. Here, we combined a modified Folch extraction method with DNA, RNA, small RNA, and protein extraction using two commercial kits, which allowed us to extract polar metabolites and non-polar oxylipin metabolites, DNA, RNA, small RNA, and protein simultaneously from a small tissue sample. The method was validated in terms of quantity and quality of analytes for downstream analyses.

SUBMITTER: Hasegawa Y

PROVIDER: S-EPMC7564281 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Optimization of a Method for the Simultaneous Extraction of Polar and Non-Polar Oxylipin Metabolites, DNA, RNA, Small RNA, and Protein from a Single Small Tissue Sample.

Hasegawa Yu Y Otoki Yurika Y McClorry Shannon S Coates Laurynne C LC Lombardi Rachel L RL Taha Ameer Y AY Slupsky Carolyn M CM

Methods and protocols 20200826 3

A more comprehensive picture of tissue biology can be obtained through the application and integration of multiple omic technologies. However, the common challenge in working with a precious sample is having a sample too small to separately extract analytes of interest for each experiment. Considering the high heterogeneity that can be present in a single tissue sample, extracting all biomolecules from a single and undivided tissue is preferable because it allows direct comparison of results. He ...[more]

PMID: 32859006

Similar Datasets

Project description:A key requirement for 13C Metabolic flux analysis (13C-MFA), a widely used technique to estimate intracellular metabolic fluxes, is an efficient method for the extraction of intermediate metabolites for analysis via liquid chromatography mass spectrometry (LC/MS). The 13C isotopic labeling results in further distribution of an already sparse pool of intermediate metabolites into isotopologues, each appearing as a separate chromatographic feature. We examined some of the reported solvent systems for the extraction of polar intracellular metabolites from three strains of cyanobacteria of the genus Synechococcus, viz., Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 7942, and a newly isolated Synechococcus elongatus PCC 11801 (manuscript under review). High resolution-LC/MS was used to assess the relative abundance of the extracted metabolites. The different solvent systems used for extraction led to statistically significant changes in the extraction efficiency for a large number of metabolites. While a few hundred m/z features or potential metabolites were detected with different solvent systems, the abundance of over a quarter of all metabolites varied significantly from one solvent system to another. Further, the extraction methods were evaluated for a targeted set of metabolites that are important in 13C-MFA studies of photosynthetic organisms. While for the strain PCC 7002, the reported method using methanol-chloroform-water system gave satisfactory results, a mild base in the form of NH4OH had to be used in place of water to achieve adequate levels of extraction for PCC 7942 and PCC 11801. While minor changes in extraction solvent resulted in dramatic changes in the extraction efficiency of a number of compounds, certain metabolites such as amino acids and organic acids were adequately extracted in all the solvent systems tested. Overall, we present a new improved method for extraction using a methanol-chloroform-NH4OH system. Our method improves the extraction of polar compounds such as sugar phosphates, bisphosphates, that are central to 13C-MFA studies.

Dataset Information

Optimization of a Method for the Simultaneous Extraction of Polar and Non-Polar Oxylipin Metabolites, DNA, RNA, Small RNA, and Protein from a Single Small Tissue Sample.

Publications

Optimization of a Method for the Simultaneous Extraction of Polar and Non-Polar Oxylipin Metabolites, DNA, RNA, Small RNA, and Protein from a Single Small Tissue Sample.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets