Green Chemistry Preservation and Extraction of Biospecimens for Multi-omic Analyses

The Environmental Protection Agency's definition of "Green Chemistry" is "the design of chemical products and processes that reduces or eliminates the use or generation of hazardous substances. Green chemistry applies across the life cycle of a chemical product, including its design, manufacture, use, and ultimate disposal." Conventional omic tissue extraction procedures use solvents that are toxic and carcinogenic, such as chloroform and methyl-tert-butyl ether for lipidomics, or caustic chaotropic solutions for genomics and transcriptomics, such as guanidine or urea. A common preservation solution for pathology is formaldehyde, which is a carcinogen. Use of acetonitrile as a universal biospecimen preservation and extraction solvent will reduce these hazardous wastes, because it is less toxic and more environmentally friendly than the conventional solvents used in biorepository and biospecimen research. A new extraction method never applied to multi-omic, system biology research, called cold-induced phase separation (CIPS), uses freezing point temperatures to induce a phase separation of acetonitrile-water mixtures. Also, the CO₂ exposure during CIPS will acidify the water precipitating DNA out of aqueous phase. The resulting phase separation brings hydrophobic lipids to the top acetonitrile fraction that is easily decanted from the bottom aqueous fraction, especially when the water is frozen. This CIPS acetonitrile extract contains the lipidome (lipids), the bottom aqueous fraction is sampled to obtain the transcriptome (RNA) fraction, and the remaining water and pellet is extracted with 60% acetonitrile to isolate the metabolome (<1 kD polar molecules). Finally, steps 4 and 5 use a TRIzol™ liquid-liquid extraction SOP of the pellet to isolate the genome (DNA) and proteome (proteins). This chapter details the multi-omic sequential extraction SOP and potential problems associated with each of the 5 steps, with steps 2, 4, and 5 still requiring validation. The metabolomic and lipidomic extraction efficiencies using the CIPS SOP is compared to conventional solvent extraction SOPs and is analyzed by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS), respectively. Acetonitrile biospecimen preservation combined with the CIPS multi-omic extraction SOP is green chemistry technology that will eliminate the generation of the hazardous substances associated with biospecimen processing and permits separation and safe disposal of acetonitrile avoiding environmental contamination.