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.

Multi-omic brain and behavioral correlates of cell-free fetal DNA methylation in macaque maternal obesity models

“…The lipid extraction protocol was performed as previously described. 90 Briefly, 30 mg of homogenized half brain samples were used. Upon obtaining Folch bottom layers, lipids were evaporated under nitrogen, reconstituted into 1.5mL chloroform (Fisher Scientific; Cat #C607-4): isopropyl alcohol (Fischer Scientific, Cat #464-1) (v/v; 2/1).…”

“…Metabolites were extracted from each of the brain tissues as previously described. 90 To 207 µL of either plasma filtrate or brain tissue extract, 23 µL of internal standard containing DSS-d6 was added and samples were placed in 3 mm Bruker nuclear magnetic resonance (NMR) tubes. Proton NMR spectra were acquired on each sample at 25 °C using the noesypr1d pulse sequence on a Bruker Avance 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA) and analyzed using Chenomx NMRSuite (version 8.1, Chenomx Inc) as previously described.…”

“…Proton NMR spectra were acquired on each sample at 25 °C using the noesypr1d pulse sequence on a Bruker Avance 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA) and analyzed using Chenomx NMRSuite (version 8.1, Chenomx Inc) as previously described. 90 Immunology A longitudinal analysis on the maternal cytokine/chemokine profile that included 22 analytes (GM-CSF, IFN-γ, IL-1b, IL-ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/23(p40), IL-13, IL-15, IL-17a, IL-18, MCP-1, MIP-1b, MIP-1a, sCD40L, TGFα, TNFα, and VEGF) was measured in plasma using a non-human primate multiplexing bead immunoassay (Millipore-Sigma, Burlington, MA) according to the manufacturer's protocol. The plates were read on a Bio-Plex 200 system (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using Bio-Plex Manager software (Bio-Rad Laboratories).…”

Simultaneous Polar Metabolite and N-Glycan Extraction Workflow for Joint-Omics Analysis: A Synergistic Approach for Novel Insights into Diseases

“…Instead of separate extractions, simultaneous extractions minimize sample consumption and could save sample preparation time, etc. In consequence, several simultaneous or one-pot extraction methods for a myriad of combinations (e.g., metabolite–lipid, metabolite–lipid–protein, DNA–RNA–miRNA–protein, DNA–RNA–protein–metabolites) have already been developed. − However, for an obvious choice of metabolite–glycan combination, there are few validated analytical workflows. Regardless, an endeavor to simultaneously isolate metabolites and protein-bound glycans may be highly feasible due to the large difference in physicochemical properties between metabolites and proteins.…”

Integrated Metabolomics and Transcriptomics Using an Optimised Dual Extraction Process to Study Human Brain Cancer Cells and Tissues

“…Three simultaneous RNA/metabolite extraction methods exist, optimised for plant tissue, microbes and human cell lines [15][16][17]. Recently, a new method has emerged, in which the non-polar metabolite (lipid) extraction is optimised for oxylipins [18]. The three methods used different ratios of methanol, chloroform and water, and different physical extraction methods varying from cryomilling to rotating or shaking the sample, to extract the metabolites.…”