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Total RNA Extraction of Rice Seeds: Quality Discussion
Xie Yuelan, Cui Huan, Wang Changlong, Chen Zhiqiang, Xiao Wuming
PDF(4235 KB)
PDF(4235 KB)
Total RNA Extraction of Rice Seeds: Quality Discussion
Total RNA isolation from rice seeds plays an important role in the molecular genetic basic experiments of rice. However, it is difficult to obtain high-quality rice seed RNA, which hinders the construction of cDNA library, RNA Seq and gene expression analysis. In order to explore the relevant impact factors of total RNA isolation from rice seeds and find the appropriate isolation methods, this study compared three different isolation methods, including Trizol method, GeneMark Kit Method and Adlaid kit method, different consumable treatment methods, grinding methods and other different factors to improve the effect of total RNA isolation from rice seeds. Different tests results showed that the Aidlad Polysaccharide Extraction Kit isolated total RNA from rice seeds with complete 28S, 18S bands, low degradation and high purity. Among the different consumables treatment methods, the total RNA isolated by the proteinase K-treated consumables degraded less than the other two methods. Besides, the 28S and 18S bands were clearer and more complete, and the total RNA had the highest quality and the best effect. In the grinding methods, the liquid nitrogen freeze-grinding method could inhibit the degradation of RNA by RNase effectively. In this study, the analysis and improvement of the relative factors of rice seed RNA isolation process lays a certain technical foundation for promoting the molecular experiment of rice seed as the research object.
rice seeds / RNA isolation / integrity / isolation methods / lab consumables processing {{custom_keyword}} /
表1 水稻不同组织样品中提取的总RNA的浓度与纯度 |
| 水稻不同组织样品 | 重复 | 浓度/(ng/μL) | OD260/OD280 | OD260/OD230 | RIN值 |
|---|---|---|---|---|---|
| 水稻幼嫩叶片 | 1 | 1864 | 2.19 | 1.91 | 7.3 |
| 2 | 1786 | 2.2 | 1.99 | 7.5 | |
| 水稻萌发后种子 | 1 | 542 | 1.92 | 1.52 | 4.6 |
| 2 | 512 | 1.93 | 1.56 | 4.8 |
表2 不同方式提取水稻种子总RNA的检测结果 |
| 试剂盒 | 重复 | 浓度/(ng/μL) | OD260/OD280 | OD260/OD230 | RIN值 |
|---|---|---|---|---|---|
| GeneMark试剂盒 | 1 | 1046 | 1.95 | 1.90 | 6.4 |
| 2 | 1010 | 1.90 | 1.93 | 6.4 | |
| 艾德莱试剂盒 | 1 | 613 | 2.05 | 2.11 | 7.1 |
| 2 | 586.5 | 2.1 | 2.15 | 7.2 |
图6 基于Agilent 2100 Bioanalyzer对不同耗材处理方法提取水稻种子总RNA的完整性检测结果A：高温灭菌1次,B：高温灭菌2次,C：蛋白酶K处理后高温灭菌2次 |
表3 不同耗材处理方法提取水稻种子总RNA的检测结果 |
| 耗材处理 | 重复 | OD260/OD280 | OD260/OD230 | RIN值 |
|---|---|---|---|---|
| 高温灭菌1次 | 1 | 2.0 | 2.1 | 5.6 |
| 2 | 2.05 | 2.12 | 5.5 | |
| 高温灭菌2次 | 1 | 2.08 | 2.09 | 6.1 |
| 2 | 2.0 | 2.11 | 6.3 | |
| 蛋白酶K处理后高温 灭菌2次 | 1 | 2.15 | 2.13 | 7.3 |
| 2 | 2.1 | 2.2 | 7.4 |
表4 不同研磨方式提取水稻种子总RNA的检测结果 |
| 研磨方式 | 重复 | OD260/OD280 | OD260/OD230 | RIN值 |
|---|---|---|---|---|
| 研磨仪研磨 | - | - | - | - |
| 液氮研磨 | 1 | 2.1 | 2.15 | 7.3 |
| 2 | 2.0 | 2.10 | 7.2 |
| 注：“-”：表示RNA已经全部降解,无检测意义。 |
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PDF(4235 KB)
Collection(s)
图1 不同组织样品提取的总RNA电泳检测结果图2 基于Agilent 2100 Bioanalyzer对不同样品中提取总RNA的完整性检测结果
表1 水稻不同组织样品中提取的总RNA的浓度与纯度图3 不同试剂盒提取水稻种子总RNA的电泳图表2 不同方式提取水稻种子总RNA的检测结果图4 基于Agilent 2100 Bioanalyzer对GeneMar试剂盒和艾德莱试剂盒提取水稻种子总RNA的完整性检测结果图5 不同耗材处理方法提取水稻种子总RNA电泳结果图6 基于Agilent 2100 Bioanalyzer对不同耗材处理方法提取水稻种子总RNA的完整性检测结果表3 不同耗材处理方法提取水稻种子总RNA的检测结果表4 不同研磨方式提取水稻种子总RNA的检测结果图7 不同研磨方式提取水稻种子总RNA电泳结果图8 基于Agilent 2100 Bioanalyzer对不同研磨方式提取水稻种子总RNA的完整性检测结果/
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