An improved Trizol method for extracting total RNA from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves.

IF 3 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS Phytochemical Analysis Pub Date : 2024-10-01 Epub Date: 2024-07-01 DOI:10.1002/pca.3404

Siqiu Xiao, Ying Zhang, Xiaoqing Tang, Jing Yang, Weixue Zhong, Ye Zhang, Ying Liu, Dewen Li

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Abstract

Introduction: High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim.

Objective: To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol.

Methodology: Based on the conventional Trizol method, the modified Trizol method 1 and modified Trizol method 2 were used as the control for extraction of RNA from E. senticosus Maxim leaves. The modified Trizol method 1 added β-mercaptoethanol on the conventional Trizol method. After RNA was dissolved, a mixed solution of phenol, chloroform, and isoamyl alcohol was added to denature protein and inhibit the degradation of RNA. The modified Trizol method 2 adds PVPP to grind on the basis of modified Trizol method 1, so as to better remove phenols from leaves, and eliminates the step of incubation at -20°C to reduce extraction time and RNA degradation. Chloroform, CTAB, and CH₃COONa were used instead of a phenol, chloroform, and isoamyl alcohol mixed solution to ensure complete separation of nucleic acid from plant tissues and to obtain high-purity RNA.

Results: The research results showed that the quality of RNA extracted by conventional Trizol method, modified Trizol method 1, was incomplete, accompanied with different degrees of contamination of polysaccharides, polyphenols, and DNA. The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. The ratio of A260/A280 was in the range of 1.8-2.0, and the yield of RNA was the highest, which was 1.68 and 1.15 times compared with that by conventional Trizol method and modified Trizol method 1 extraction, respectively. The reverse transcription cDNA was further tested through PCR with the specific primers. The amplified fragments are displayed in clear and bright bands in accordance with the expected size.

Conclusion: The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. High-quality RNA has more advantages in molecular biology study of E. senticosus Maxim.

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一种改进的 Trizol 方法，用于从 Eleutherococcus senticosus (Rupr. & Maxim.) Maxim 叶片中提取总 RNA。

简介高质量的核酸是分子生物学实验的基础。传统的 RNA 提取方法并不适合榄香附 Maxim：为了找到一种合适的方法来提高 RNA 提取的质量，我们对 Trizol 的 RNA 提取方法进行了改进：方法：在传统 Trizol 方法的基础上，以改良 Trizol 方法 1 和改良 Trizol 方法 2 作为对照，从仙客来叶片中提取 RNA。改良 Trizol 法 1 在传统 Trizol 法的基础上添加了 β-巯基乙醇。RNA 溶解后，加入苯酚、氯仿和异戊醇的混合溶液，使蛋白质变性，抑制 RNA 降解。改良 Trizol 法 2 在改良 Trizol 法 1 的基础上，在研磨过程中加入了 PVPP，以更好地去除叶片中的酚类物质，并取消了-20℃孵育的步骤，以缩短提取时间，减少 RNA 降解。用氯仿、CTAB和CH3COONa代替苯酚、氯仿和异戊醇混合溶液，以确保核酸从植物组织中完全分离，获得高纯度的RNA：研究结果表明，传统 Trizol 法、改良 Trizol 法 1 提取的 RNA 质量不完全，且伴有不同程度的多糖、多酚和 DNA 污染。改良 Trizol 法 2 能更好地提取洋二仙草马克西姆叶片中的 RNA。A260/A280 的比值在 1.8-2.0 之间，RNA 的产量最高，分别是传统 Trizol 法和改良 Trizol 法 1 提取的 1.68 倍和 1.15 倍。用特定引物对反转录 cDNA 进一步进行 PCR 检测。扩增片段的条带清晰明亮，符合预期大小：结论：改良的 Trizol 方法 2 能更好地从仙客来叶片中提取 RNA。结论：改良的 Trizol 方法 2 能更好地提取马克西姆猴面包树叶片的 RNA，高质量的 RNA 在马克西姆猴面包树的分子生物学研究中更具优势。

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来源期刊

Phytochemical Analysis 生物-分析化学

CiteScore

6.00

自引率

6.10%

发文量

审稿时长

1.7 months

期刊介绍： Phytochemical Analysis is devoted to the publication of original articles concerning the development, improvement, validation and/or extension of application of analytical methodology in the plant sciences. The spectrum of coverage is broad, encompassing methods and techniques relevant to the detection (including bio-screening), extraction, separation, purification, identification and quantification of compounds in plant biochemistry, plant cellular and molecular biology, plant biotechnology, the food sciences, agriculture and horticulture. The Journal publishes papers describing significant novelty in the analysis of whole plants (including algae), plant cells, tissues and organs, plant-derived extracts and plant products (including those which have been partially or completely refined for use in the food, agrochemical, pharmaceutical and related industries). All forms of physical, chemical, biochemical, spectroscopic, radiometric, electrometric, chromatographic, metabolomic and chemometric investigations of plant products (monomeric species as well as polymeric molecules such as nucleic acids, proteins, lipids and carbohydrates) are included within the remit of the Journal. Papers dealing with novel methods relating to areas such as data handling/ data mining in plant sciences will also be welcomed.