Siqiu Xiao, Ying Zhang, Xiaoqing Tang, Jing Yang, Weixue Zhong, Ye Zhang, Ying Liu, Dewen Li
{"title":"一种改进的 Trizol 方法，用于从 Eleutherococcus senticosus (Rupr. & Maxim.) Maxim 叶片中提取总 RNA。","authors":"Siqiu Xiao, Ying Zhang, Xiaoqing Tang, Jing Yang, Weixue Zhong, Ye Zhang, Ying Liu, Dewen Li","doi":"10.1002/pca.3404","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong>High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim.</p><p><strong>Objective: </strong>To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol.</p><p><strong>Methodology: </strong>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 CH<sub>3</sub>COONa 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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>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.</p>","PeriodicalId":20095,"journal":{"name":"Phytochemical Analysis","volume":" ","pages":"1613-1619"},"PeriodicalIF":3.0000,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"An improved Trizol method for extracting total RNA from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves.\",\"authors\":\"Siqiu Xiao, Ying Zhang, Xiaoqing Tang, Jing Yang, Weixue Zhong, Ye Zhang, Ying Liu, Dewen Li\",\"doi\":\"10.1002/pca.3404\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong>High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim.</p><p><strong>Objective: </strong>To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol.</p><p><strong>Methodology: </strong>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 CH<sub>3</sub>COONa 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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>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.</p>\",\"PeriodicalId\":20095,\"journal\":{\"name\":\"Phytochemical Analysis\",\"volume\":\" \",\"pages\":\"1613-1619\"},\"PeriodicalIF\":3.0000,\"publicationDate\":\"2024-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Phytochemical Analysis\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1002/pca.3404\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/7/1 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Phytochemical Analysis","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1002/pca.3404","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/7/1 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
An improved Trizol method for extracting total RNA from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves.
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.
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.
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