单细胞绿藻佐夫色绿藻中的RNA纯化

Sean D. Gallaher; Melissa S. Roth

doi:10.21769/BioProtoc.2793

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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 email

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: 7345

评审: Adam IdoineClaudia CatalanottiAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Proceedings of the National Academy of Sciences of the United States of America, featuring study using the protocol.

May 2017

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

从标记的细胞类型中分离细胞核、提取RNA并去除核糖体RNA以用于RNA-Seq 分析

Mauricio A. Reynoso [...] Kaisa Kajala

2018年04月05日 15030 阅读

从顽拗型树种泡桐中提取RNA

Niveditha Ramadoss and Chhandak Basu

2018年07月20日 6550 阅读

一种简便高效的拟南芥胚胎RNA提取方法

Fernanda Marchetti [...] Eduardo Zabaleta

2025年02月20日 856 阅读

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)

Algae (藻类)

RNA purification (RNA纯化)

RNA-Seq (RNA-Seq)

Cell wall (细胞壁)

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

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

Equipment

Refrigerated centrifuge (Eppendorf, model: 5810 R )
Refrigerated microfuge (Eppendorf, model: 5424 R )
Clean bench
Ice buckets (Corning, catalog number: 432122 )
PIPETMAN Classic pipet (Gilson, model: P20, catalog number: F123600 )
PIPETMAN Classic pipet (Gilson, model: P200, catalog number: F123601 )
PIPETMAN Classic pipet (Gilson, model: P1000, catalog number: F123602 )
Homogenizer (MP Biomedicals, model: FastPrep-24^TM 5G )
Homogenizer adaptor (MP Biomedicals, model: CoolPrep^TM 24x2mL )
Vacuum manifold (QIAGEN, model: QIAvac 24 Plus )
Heat block (VWR, catalog number: 75838-286 )
Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: NanoDrop^TM 2000 )
Bioanalyzer (Agilent Technologies, model: 2100 Bioanalyzer )

Procedure

English

中文翻译

文章信息

版权信息

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.