A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms

Abstract

SUMMARYHere, we describe a method for the combined metabolomic, proteomic, transcriptomic and genomic analysis from one single sample as a major step for multilevel data integration strategies in systems biology. While extracting proteins and DNA, this protocol also allows the separation of metabolites into polar and lipid fractions, as well as RNA fractionation into long and small RNAs, thus allowing a broad range of transcriptional studies. The isolated biomolecules are suitable for analysis with different methods that range from electrophoresis and blotting to state-of-the-art procedures based on mass spectrometry (accurate metabolite profiling, shot-gun proteomics) or massive sequencing technologies (transcript analysis). The low amount of starting tissue, its cost-efficiency compared with the utilization of commercial kits, and its performance over a wide range of plant, microbial, and algal species such as Chlamydomonas, Arabidopsis, Populus, or Pinus, makes this method a universal alternative for multiple molecular isolation from plant tissues.

Exploring natural variation of Pinus pinaster Aiton using metabolomics: Is it possible to identify the region of origin of a pine from its metabolites?

“…Metabolite extraction was performed according to Valledor et al . (). Briefly, 600 μL of cold (4 °C) metabolite extraction solution [methanol:chloroform:H 2 O (2.5:1:0.5)] was added to each tube and strongly vortexed.…”

“…About 30 mg (fresh weight) of each type of tissue under study (needles and apical and basal sections of bud) was ground in liquid nitrogen. Metabolite extraction was performed according to Valledor et al (2014). Briefly, 600 lL of cold (4°C) metabolite extraction solution [methanol:chloroform:H 2 O (2.5:1:0.5)] was added to each tube and strongly vortexed.…”

“…The identification of several components of multigene regulatory networks that regulate complex processes, such as adaptation, is difficult using traditional genetic screening, which typically only identifies single regulatory elements. Recent new approaches using natural variation in plants, such as genomewide association studies (GWAS), have become an obvious choice for studying the genetics of natural variation and traits of agricultural importance Meij on et al 2014).…”

Field-omics—understanding large-scale molecular data from field crops

“…As such, there is a need for new sampling strategies to be developed. For example, it is important to arrange that the same material can be split up in a representative fashion for different types of analysis or to analyse metabolites, proteins and transcripts from the same sample (Weckwerth et al, 2004; Valledor et al, 2014). In field conditions, light variation, temperature and relative humidity need to be recorded (and normalized to the extent possible) at least for sampling events and ideally over the entire growth season in order to capture accumulative effects.…”

Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers from a single sample

“…Here the phase separation of the homogeneous, one phase chloroform–methanol mixture, was achieved only after a centrifugation and the separation of the solid and the liquid phase in an independent step [18–21]. This two-step approach partially overcomes the problem of the interphase between the two liquid phases but it is more time consuming, since the phase separation has to be achieved in an independent step [22]. Additionally, unwanted phase separation could occur if the water content of the extracted samples are too high.…”