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RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis

单细胞绿藻佐夫色绿藻中的RNA纯化

Sean D. Gallaher Sean D. Gallaher
MR Melissa S. Roth

发布: 2018年04月05日第8卷第7期 DOI: 10.21769/BioProtoc.2793 浏览次数: 8060

评审: Adam IdoineClaudia CatalanottiAnonymous reviewer(s)

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Abstract

Chromochloris zofingiensis is a unicellular green alga that is an emerging model species for studies in fields such as biofuel production, ketocarotenoid biosynthesis and metabolism. The recent availability of a high-quality genome assembly facilitates systems-level analysis, such as RNA-Seq. However, cells of this alga have a tough cell wall, which is a challenge for RNA purification. This protocol was designed specifically to breach the cell wall and isolate high-quality RNA suitable for RNA-Seq studies.

Keywords: Chromochloris zofingiensis (佐夫色绿藻) search Algae (藻类) search RNA purification (RNA纯化) search RNA-Seq (RNA-Seq) search Cell wall (细胞壁) search

Background

Chromochloris zofingiensis is a small unicellular green alga from the chlorophyte lineage (Dönz, 1934). Previously, this species has been described in the literature with the genera Muriella, Chlorella, and Mychonastes (Fučíková and Lewis, 2012). There are strong economic interests in C. zofingiensis because it is capable of producing large quantities of lipids for biofuels and shows promise as a source of the commercially valuable nutraceutical astaxanthin (Breuer et al., 2012; Mulders et al., 2014; Liu et al., 2016). Recently, a high-quality, chromosome-level genome assembly and accompanying annotations were published, which facilitates systems level analyses like RNA-Seq (Roth et al., 2017). The following protocol was designed to produce highly purified total RNA, including miRNA, from liquid cultures of C. zofingiensis suitable for RNA-Seq. C. zofingiensis cells are protected by a robust cell wall, which this protocol was designed to penetrate. Starting material for the protocol can be up to 2.5 x 108 total cells. This protocol should yield at least 20 µg of highly purified RNA suitable for the preparation of RNA-Seq libraries by standard kits, such as the Illumina TruSeq Stranded Total RNA kit.

Materials and Reagents

  1. 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
  2. Serological pipettes, 10 ml (Fisher Scientific, catalog number: 13-676-10J )
  3. Lysing Matrix D Tubes (2 ml) (MP Biomedicals, catalog number: 116913050 )
  4. Tapered end, metal spatula (Fisher Scientific, catalog number: 14-374 )
  5. VacConnectors (QIAGEN, catalog number: 19407 )
  6. C. zofingiensis (SAG, catalog number: 211-14 )
  7. Proteose medium (UTEX Culture Collection of Algae, Proteose medium)
  8. Chu’s medium (UTEX Culture Collection of Algae, Chu’s medium)
  9. Liquid nitrogen (LN2) (various)
  10. Wet ice (various)
  11. Dry ice (various)
  12. RNaseZap (250 ml spray bottle) (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 )
  13. RNase-free ethanol (EtOH) (100% and 70%) (various)
  14. TRIzol (100 ml) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15596026 )
  15. Chloroform:isoamyl alcohol (24:1) (Sigma-Aldrich, catalog number: C0549-1PT )
  16. MaXtract High Density (100 x 15 ml) (QIAGEN, catalog number: 129065 )
  17. miRNeasy Mini Kit (50) (QIAGEN, catalog number: 217004 ), which includes:
    1. QIAGEN miRNeasy mini columnsa.QIAGEN miRNeasy mini columns
    2. 1.5 and 2 ml collection tubes
    3. QIAGEN buffer RWT
    4. QIAGEN buffer RPE
    5. RNase-free water
  18. RNase-Free DNase Set (50 samples) (QIAGEN, catalog number: 79254 ), which includes:
    1. RNase-free DNase I
    2. QIAGEN buffer RDD
  19. 3 M sodium acetate (NaOAc), pH = 8, RNase-free (various)
  20. 1 M Tris hydrochloride (Tris-HCl), pH = 8, RNase-free (various)
  21. 5 M sodium chloride (NaCl), RNase-free (various)
  22. 0.5 M ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), pH = 8, RNase-free (various)
  23. 10% sodium dodecyl sulfate (SDS) (various)
  24. 20 mg/ml Proteinase K (Fisher Scientific, catalog number: BP1700-100 )
  25. Agilent RNA 6000 Nano kit (Agilent Technologies, catalog number: 5067-1511 )
  26. Lysis buffer (see Recipes)

Equipment

  1. Refrigerated centrifuge (Eppendorf, model: 5810 R )
  2. Refrigerated microfuge (Eppendorf, model: 5424 R )
  3. Clean bench
  4. Ice buckets (Corning, catalog number: 432122 )
  5. PIPETMAN Classic pipet (Gilson, model: P20, catalog number: F123600 )
  6. PIPETMAN Classic pipet (Gilson, model: P200, catalog number: F123601 )
  7. PIPETMAN Classic pipet (Gilson, model: P1000, catalog number: F123602 )
  8. Homogenizer (MP Biomedicals, model: FastPrep-24TM 5G )
  9. Homogenizer adaptor (MP Biomedicals, model: CoolPrepTM 24x2mL )
  10. Vacuum manifold (QIAGEN, model: QIAvac 24 Plus )
  11. Heat block (VWR, catalog number: 75838-286 )
  12. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  13. Bioanalyzer (Agilent Technologies, model: 2100 Bioanalyzer )

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Gallaher, S. D. and Roth, M. S. (2018). RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis. Bio-protocol 8(7): e2793. DOI: 10.21769/BioProtoc.2793.

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分类

植物科学 > 植物分子生物学 > RNA > RNA 提取

分子生物学 > RNA > RNA 提取

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