Simultaneous isolation of DNA, RNA, and protein from <i>Medicago truncatula</i> L.

Xiong, Ji; Yang, Qingchuan; Sun, Yan; Zhang, Tiejun; Margaret, Gruber; Wang, Ding

doi:10.1002/elps.201000425

Cited by 39 publications

(31 citation statements)

References 22 publications

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“…For example, there is an increased number of proteins with high molecular masses in gels from phenol-extracted proteins, whereas TCA/acetone precipitation is more efficient for the extraction of proteins with molecular masses lower than 25 kDa [31,41]. In addition, phenol is able to selectively dissolve proteins and lipids [56], allowing the separation of proteins from polysaccharides and nucleic acids, contributing to yield of highly clean protein extracts [31,41]. Therefore, although phenol extraction may lead to better quality of protein extractions in some situations, the two procedures should be considered as complementary rather than mutually exclusive methods [46].…”

Section: Phenol Extraction Of Proteinsmentioning

confidence: 99%

Proteomic studies of arbuscular mycorrhizal associations

Couto¹,

Lovato²,

Wipf³

et al. 2013

ABC

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Arbuscular mycorrhizal (AM) fungi are soil-borne microorganisms forming mutualistic associations with the vast majority of land plants, including most agricultural relevant crops. In this association the plant provides the fungus with plant photosynthates allowing it to complete its life cycle, while the fungus provides the plant with mineral nutrients, mainly phosphorus and can also help the plant to tolerate biotic and abiotic stresses. In regard to these benefits there is growing interest on the use of AM fungi to improve productivity and sustainability in agricultural systems. AM fungi and their interactions with plants have been extensively studied using proteomic techniques, but some difficulties have been faced. 1) Little is known about the AM fungal typical protein repertoire because it is currently impossible to grow AM fungi in pure axenic cultures; 2) Plant tissues often contain high amounts of interfering substances that make protein extraction for the study of AM interactions a difficult procedure; 3) Most nutrient exchanges between AM fungi and their host plants involve participation of membrane proteins, still poorly resolved in most separation techniques. Finally, 4) the formation of the arbuscule is an asynchronous process, making it difficult to distinguish which proteins are essential in the early or late stages of AM associations. In this review we present a historical summary of how these difficulties have been overcome by technological advances in proteomics and we discuss current and future trends in the study of the proteins involved in AM interactions.

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Section: Phenol Extraction Of Proteinsmentioning

confidence: 99%

Proteomic studies of arbuscular mycorrhizal associations

Couto¹,

Lovato²,

Wipf³

et al. 2013

ABC

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show abstract

“…A well-established method for the isolation of concomitant biomacromolecules only is based around the simultaneous addition of a monophasic mixture of phenol and guanidine isothiocyanate, commercially available as TRIzol and TRI Reagent (TR), and chloroform to biological samples to obtain an aqueous phase containing primarily RNA, an interphase containing DNA and an organic phase containing proteins (Chomczynski, 1993; Hummon et al , 2007; Chey et al , 2011; Xiong et al , 2011). Individual biomacromolecular fractions are purified from the respective phases and may be subjected to specialized downstream analyses.…”

Section: Introductionmentioning

confidence: 99%

A biomolecular isolation framework for eco-systems biology

et al. 2012

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Mixed microbial communities are complex, dynamic and heterogeneous. It is therefore essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single samples to facilitate meaningful data integration, analysis and modeling. We have developed a new methodological framework for the reproducible isolation of high-quality genomic DNA, large and small RNA, proteins, and polar and non-polar metabolites from single unique mixed microbial community samples. The methodology is based around reproducible cryogenic sample preservation and cell lysis. Metabolites are extracted first using organic solvents, followed by the sequential isolation of nucleic acids and proteins using chromatographic spin columns. The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and biomedical research interest. The developed methodological framework lays the foundation for standardized molecular eco-systematic studies on a range of different microbial communities in the future.

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“…Root samples were ground to a fine powder in liquid nitrogen. Total protein extracts were prepared from the ground root samples using an optimized TRIzol method (Xiong et al, 2011). The final protein pellets were washed three times in 1 ml ethanol and resuspended in 1 ml lysis buffer (8 M urea, 4% w/v CHAPS, and 2% w/v DTT).…”

Section: Sample Preparation and 2-dementioning

confidence: 99%

Comparative proteomic analysis reveals differential root proteins in Medicago sativa and Medicago truncatula in response to salt stress

Long¹,

Li²,

Zhang³

et al. 2019

The Model Legume Medicago Truncatula

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Salt stress is an important abiotic stress that causes decreased crop yields. Root growth and plant activities are affected by salt stress through the actions of specific genes that help roots adapt to adverse environmental conditions. For a more comprehensive understanding of proteins affected by salinity, we used two-dimensional gel electrophoresis and mass spectrometry to characterize the proteome-level changes associated with salt stress response in Medicago sativa cv. Zhongmu-1 and Medicago truncatula cv. Jemalong A17 roots. Our physiological and phenotypic observations indicated that Zhongmu-1 was more salt tolerant than Jemalong A17. We identified 93 and 30 proteins whose abundance was significantly affected by salt stress in Zhongmu-1 and Jemalong A17 roots, respectively. The tandem mass spectrometry analysis of the differentially accumulated proteins resulted in the identification of 60 and 26 proteins in Zhongmu-1 and Jemalong A17 roots, respectively. Function analyses indicated molecule binding and catalytic activity were the two primary functional categories. These proteins have known functions in various molecular processes, including defense against oxidative stress, metabolism, photosynthesis, protein synthesis and processing, and signal transduction. The transcript levels of four identified proteins were determined by quantitative reverse transcription polymerase chain reaction. Our results indicate that some of the identified proteins may play key roles in salt stress tolerance.

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Simultaneous isolation of DNA, RNA, and protein from Medicago truncatula L.

Cited by 39 publications

References 22 publications

Proteomic studies of arbuscular mycorrhizal associations

Proteomic studies of arbuscular mycorrhizal associations

A biomolecular isolation framework for eco-systems biology

Comparative proteomic analysis reveals differential root proteins in Medicago sativa and Medicago truncatula in response to salt stress

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