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Lipidomic Analysis of Caenorhabditis elegans Embryos

秀丽隐杆线虫胚胎的脂质组学分析

HY Hung-Chi Yang
CH Cheng-Yu Hung
YP Yi-Yun Pan
SL Szecheng J Lo
DC Daniel Tsun-Yee Chiu

发布: 2017年09月20日第7卷第18期 DOI: 10.21769/BioProtoc.2554 浏览次数: 11627

评审: Neelanjan BoseLu HanTanxi Cai

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Abstract

Metabolomic is an emerging field of system biology. Lipidomic, a branch of metabolomic, aims to characterize lipophilic metabolites in biological systems. Caenorhabditis elegans (C. elegans) is a genetically tractable and versatile animal model for novel discovery of lipid metabolism. In addition, C. elegans embryo is simple and homogeneous. Here, we demonstrate detailed procedures of C. elegans culture, embryo isolation, lipid extraction and metabolomic data analysis.

Keywords: C. elegans (秀丽隐杆线虫) search Embryo (胚胎) search Lipid (脂质) search LC-MS (LC-MS) search Untargeted metabolomic (非靶向代谢组学) search

Background

The metazoan model Caenorhabditis elegans (C. elegans) offer a unique platform to discover novel functions and biological roles of metabolic enzymes. Current studies of genomic, transcriptomic and proteomic have advanced our understanding to appreciate the complexity of metabolic networks in C. elegans (Watson et al., 2015). Metabolomic is an emerging tool of system biology aiming to determine small molecule metabolites within biological systems. A number of C. elegans studies have used different metabolomic approaches, including nuclear magnetic resonance (NMR) spectroscopy, gas/liquid chromatography coupled mass spectrometry (GC/LC-MS), to dissect the metabolic networks in whole worm (Atherton et al., 2008; Hughes et al., 2009; Castro et al., 2012; Patti et al., 2014; Morgan et al., 2015; Wang et al., 2015; Wan et al., 2017). Currently, there is no metabolomic-based approach to characterize the lipid metabolites in C. elegans embryos. In this study, LC-MS-based untargeted lipidomic method is chosen for several reasons. First, LC-MS is sufficiently sensitive to analyze small quantity of C. elegans embryos. Second, untargeted metabolomic provides an unbiased view of detectable metabolites, which is crucial to generate a large amount of information. Subsequently, a hypothesis can be formulated based on the global metabolomic analysis. Last but not least, C. elegans embryo is considered simple and homogeneous compared to the whole worm. Taken together, these technical advantages provide opportunities for in-depth analysis of lipid metabolism in developing C. elegans embryos.

Materials and Reagents

  1. C. elegans culture
    1. Aluminum foil
    2. Autoclave tape (Fisher Scientific, catalog number: 15904 )
    3. 90 x 15 mm disposable plastic Petri dishes (China biotech corporation)
    4. 14 ml polypropylene round-bottom tubes (Corning, Falcon®, catalog number: 352059 )
    5. 15 ml centrifuge tubes (Corning, catalog number: 430791 )
    6. 50 ml centrifuge tubes (Corning, catalog number: 430829 )
    7. 250 ml NalgeneTM PPCO centrifuge bottles (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3120-0250 )
    8. Microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
    9. Glass slide
    10. 0.3 mm diameter platinum/iridium wire (Shineteh Instruments)
    11. 0.22 μm pore size syringe filter unit (EMD Millipore, catalog number: SLGP033RB )
    12. C. elegans N2 (wild type strain)
    13. C. elegans control-RNAi (Mock) and G6PD-RNAi (Gi) adults and embryos
    14. E. coli OP50 (University of Minnesota, C. elegans Genetics Center)
    15. E. coli HT115(DE3) harboring control-RNAi (Mock) and G6PD-RNAi (Gi) plasmids
      Note: The details of these plasmids, including design and construction, are described in a previous report (Yang et al., 2013).
    16. Ultrawater
    17. Bleach (NaOCl) (Clorox)
    18. Sodium hydroxide (NaOH) (Merck, catalog number: 1064980500 )
    19. M9 buffer
    20. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434-5KG-R )
    21. Agar (BioShop, catalog number: AGR001.1 )
    22. Peptone (Oxoid, catalog number: LP0037 )
    23. Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 1051041000 )
    24. Potassium phosphate monobasic (KH2PO4) (Avantor Performance Materials, J.T. Baker, catalog number: 3246-05 )
    25. Cholesterol (Sigma-Aldrich, catalog number: C8667-5G )
    26. Ethanol (Sigma-Aldrich, catalog number: 32221-2.5L )
      Note: This product has been discontinued.
    27. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M2643-500G )
    28. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3881-500G )
    29. Tetracyclin-hydrochloride (Boehringer Mannheim)
    30. Carbenicillin disodium salt (Sigma-Aldrich, catalog number: C1389-5G )
    31. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-25G )
    32. Isopropyl-b-D-thiogalactopyranoside (IPTG) (BioShop, catalog number: IPT001.50 )
    33. LB broth (BD, DifcoTM, catalog number: 244610 )
    34. Nematode growth medium (NGM) (see Recipes)
    35. 1 M KPI buffer pH 6.0 (see Recipes)
    36. 5 mg/ml cholesterol (see Recipes)
    37. 1 M MgSO4 (see Recipes)
    38. 1 M CaCl2 (see Recipes)
    39. 10 mg/ml tetracycline-hydrochloride (see Recipes)
    40. 25 mg/ml carbenicillin (see Recipes)
    41. 200 mg/ml ampicillin (see Recipes)
    42. 1 M IPTG (see Recipes)

  2. C. elegans sample preparation
    1. Falcon cell-strainer cap (12 x 75 mm) (Corning, Falcon®, catalog number: 352235 )
    2. Pyrex glass tube (20 x 125 mm) (Corning, PYREX®, catalog number: 9826-20 )
    3. Glass Pasteur pipettes (Kimble Chase Life Science, catalog number: 63A54 )
    4. Glass organic solvent-resistant pipette tips (HBG, catalog number: 1010-19 )
    5. Organic solvent-resistant polypropylene tip (Gilson, catalog number: F161110 )
    6. Organic solvent-resistant polypropylene microcentrifuge tube (STARLAB INTERNATIONAL, catalog number: S1615-5500 )
    7. HPLC vial (WATERS, catalog number: 186000272C )
    8. Chloroform (LC grade) (Merck, catalog number: 1024444000 )
    9. Methanol (HPLC grade) (Avantor Performance Materials, J.T. Baker, catalog number: 9093 )
    10. Chromasolv grade water (H2O) (Sigma-Aldrich, Fluka ,catalog number: 39253-1L-R )
    11. Chromasolv grade acetonitrile (ACN) (Avantor Performance Materials, J.T. Baker, catalog number: 9829-03 )
    12. Chromasolv grade isopropanol (Sigma-Aldrich, Fluka, catalog number: 34965-2.5L )
    13. Ammonium formate (Fluka, catalog number: 70221 )
    14. Formic acid (Sigma-Aldrich, Fluka, catalog number: 56302 )

Equipment

  1. Pyrex narrow mouth Erlenmeyer flasks (Corning, PYREX®, catalog number: 4980-1L )
  2. Lamina flow hood (Chin-Chih H&W ENTERPRISE, catalog number: BSC-4 )
  3. 20 °C incubator (Firstek, catalog number: RI-560 )
  4. 37 °C incubator with shaker (Yihder, catalog number: LM-570RD )
  5. Centrifuge (Beckman Coulter, model: Avanti® J-26XP )
  6. Rotor (Beckman Coulter, model: JA-14 )
  7. Stereomicroscope (Nikon Instruments, model: SMZ745 )
  8. Stainless steel surgical blade (No. 10) and holder (Feather Safety Razor, Japan)
  9. Vorterxer (Scientific Industries, model: Vortex-Genie 2 )
  10. Centrifuge (Eppendorf, model: 5810 R )
  11. High-capacity swing-bucket rotor with four 250 ml buckets (Eppendorf, model: A-4-62 )
  12. -80 °C freezer
  13. 100 ml glass beaker (Corning, PYREX®, catalog number: 1000-100 )
  14. Macropipette (Socorex, catalog number: 835.05 )
  15. Sonicator (Sonics & Materials, model: VCX 400 )
  16. Ultrasonic cleaner (Delta, model: DC200H )
  17. Nitrogen gas spray unit for aluminum block bath evaporation head (TAITEC, model: DTU-2B )
  18. Centrifuge (Eppendorf, model: 5427 R )
  19. Acquity CSH C18 column (particle size of 1.7 μm, 2.1 x 100 mm) (WATERS, catalog number: 186002352 )
  20. Ultra-performance liquid-chromatography (UPLC) system (WATERS, catalog numbers: 176001285 , 700003616 , 700002764 )
  21. SYNAPT G1 HDMS system (WATERS)
  22. Autoclave (Tomin Medical Equipment, catalog number: TM-328 )
  23. 4 °C cooling cabinet (Firstek, catalog number: CC-2 )

Software

  1. MassLynx4.1 (WATERS)
  2. Extended Statistics (EZinfo, WATERS)
  3. MetaboAnalyst (http://www.metaboanalyst.ca)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Yang, H., Hung, C., Pan, Y., Lo, S. J. and Chiu, D. T. (2017). Lipidomic Analysis of Caenorhabditis elegans Embryos. Bio-protocol 7(18): e2554. DOI: 10.21769/BioProtoc.2554.

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分类

生物化学 > 脂质 > 脂质测定

系统生物学 > 代谢组学 > 全生物体

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