Total RNA Extraction from Grape Berry Skin for Quantitative Reverse Transcription PCR and Microarray Analysis
从葡萄果皮上提取总RNA进行定量反转录PCR和基因芯片分析
发布: 2016年04月05日第6卷第7期 DOI: 10.21769/BioProtoc.1777 浏览次数: 8425
评审: Tie LiuAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2015
Abstract
Extraction of high quality RNA is an essential step for quantitative reverse transcription PCR (qRT-PCR) and microarray analysis. However, it is not easy to extract high quality RNA from fruit materials, which contain high amounts of polysaccharides, lipids and secondary metabolites. Wan and Wilkins (1994) had developed ‘Hot Borate Method’ to isolate high quality RNA. Here, we describe a modified protocol of the ‘Hot Borate Method’ to isolate high quality RNA from grape berry skin for qRT-PCR and microarray analysis (Suzuki et al., 2015a; Suzuki et al., 2015b).
Keywords: RNA extraction (RNA的提取)
Grape berry skin (葡萄浆果皮)
Hot Borate Method (热硼酸法)
Materials and Reagents
- 50 ml centrifuge tubes (e.g., the Falcon tubes)
- 1.5 ml microcentrifuge tubes
- 2 ml microcentrifuge tubes
- Stainless steel spoon
- Grape berry skins
Notes:- Skins isolated from grape berries using tweezers (Figure 1A, Video 1) . Skins were put in 15 or 50 ml tubes and frozen in liquid nitrogen immediately (Figure 1B, Video 1). These were stored at -80 °C. We recommend that preparation of about 20 pieces (about 1 g skin) because there are variations among berries.
- This method could be applied to all grape varieties.
- Skins isolated from grape berries using tweezers (Figure 1A, Video 1) . Skins were put in 15 or 50 ml tubes and frozen in liquid nitrogen immediately (Figure 1B, Video 1). These were stored at -80 °C. We recommend that preparation of about 20 pieces (about 1 g skin) because there are variations among berries.
- Liquid nitrogen
- Proteinase K solutions (20 mg/ml) (Siyaku, Wako Pure Chemical Industries, catalog number: 160-22752 )
- Lithium chloride (LiCl) (2 M, 10 M) (Siyaku, Wako Pure Chemical Industries, catalog number: 127-01165 )
Note: DEPC treated and autoclaved. - 2 M potassium acetate (KOAC) (Siyaku, Wako Pure Chemical Industries, catalog number: 160-03175 )
Note: DEPC treated and autoclaved. - 10 mM Tris-HCl (pH 7.5) (Siyaku, Wako Pure Chemical Industries, catalog number: 207-06275 )
Note: DEPC treated and autoclaved. - 1 M potassium chloride (KCl) (Siyaku, Wako Pure Chemical Industries, catalog number: 163-03545 )
Note: DEPC treated and autoclaved. - Ethanol [70%, 99.8% (v/v)] (Siyaku, Wako Pure Chemical Industries, catalog number: 057-00456 )
- RNeasy Plant Mini Kit (QIAGEN, catalog number: 74903 or 74904 )
- RNase free water
- Sodium borate decahydrate (Borax) (Siyaku, Wako Pure Chemical Industries, catalog number: 194-01415 )
- Ethylene glycol tetraacetic acid (EGTA) (Dojindo, catalog number: 346-01312 )
- 1% (w/v) sodium dodecyl sulfate (SDS) (Siyaku, Wako Pure Chemical Industries, catalog number: 191-07145 )
- 1% (w/v) deoxycholate sodium salt (Siyaku, Wako Pure Chemical Industries, catalog number: 192-08312 )
- 10 mM dithiothreitol (DTT) (Siyaku, Wako Pure Chemical Industries, catalog number: 045-08974 )
- 1% (w/v) Triton X-114 (Sigma-Aldrich, catalog number: X114 )
- 2% (w/v) polyvinylpyrrolidone (PVP-40) (Sigma-Aldrich, catalog number: PVP40 )
- Diethyl pyrocarbonate (DEPC) (Sigma-Aldrich, catalog number: D5758 ) treated and autoclaved
- Hot Borate Extraction Buffer (see Recipes)
Equipment
- Crusher (Automill) (Tokken, model: TK-AM5-H )
- Shaking water bath (42 °C) (TAITEC CORPORATION, model: THERMO MINDER SM-05 with PERSONAL-11)
- Centrifuge with angle rotor for 50 ml tube (SAKURA, model: 50A-7 with 50F-8A)
- Centrifuge with angle rotor for 1.5 ml tube (SAKURA, model: SS1500X with 15M-24AM)
- Vacuum equipment (EYELA, model: CVE-2000 )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Suzuki, M. and Shiratake, K. (2016). Total RNA Extraction from Grape Berry Skin for Quantitative Reverse Transcription PCR and Microarray Analysis. Bio-protocol 6(7): e1777. DOI: 10.21769/BioProtoc.1777.
- Suzuki, M., Nakabayashi, R., Ogata, Y., Sakurai, N., Tokimatsu, T., Goto, S., Suzuki, M., Jasinski, M., Martinoia, E., Otagaki, S., Matsumoto, S., Saito, K. and Shiratake, K. (2015b). Multiomics in grape berry skin revealed specific induction of the stilbene synthetic pathway by ultraviolet-C irradiation. Plant Physiol 168(1): 47-59.
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分类
植物科学 > 植物分子生物学 > RNA
分子生物学 > RNA > RNA 提取
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