Efficient Isolation of High-quality Total RNA from Strawberry
Efficient Isolation of High-quality Total RNA from Strawberry
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DOI：
10.21273/hortsci13683-18
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发表时间：
2019-02-01
期刊：
影响因子：
1.9
通讯作者：
Uno, Yuichi
中科院分区：
文献类型：
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作者：
Ishibashi, Misaki;Nabe, Takeshi;Uno, Yuichi
下一代测序和高通量实时聚合酶链反应分析需要足够产量的高质量RNA。在草莓（Fragaria x ananassa）果实的情况下，成功的RNA分离需要去除大量的抑制物质（多糖和多酚），这些物质大大降低了质量和产量。在这项研究中，我们应用了针对生殖器官的RNA分离方案的各种组合。最好的手工分离方法涉及非离子聚合物和改性的酸性硫氰酸胍-苯酚-氯仿处理，然后苯酚/氯仿/异戊醇萃取。与其他方法相比，该方法得到了显著更高的产量[84.0 μ g/g鲜重（FW）]的更高纯度的RNA（A260/A280 = 1.99; A260/230 = 1.51）。使用自动化方法获得了质量更好的RNA（A260/230 = 2.11），但产量（18.1 μ g/g FW）低于手动获得的产量。该自动化方法包括用非离子聚合物预处理，然后进行基于二氧化硅的系统提取。尽管使用自动和手动方法从容器中获得了用于RNA测序的足够质量的RNA [RNA完整性编号（RIN）>= 6.5和28 S/18 S>= 1.0]，但是手动方法从瘦果和花药中产生高质量的RNA。自动方法具有6倍的高通量能力，而手动方法对不同组织具有更广泛的适用性。
Sufficient yields of high-quality RNA are needed for next-generation sequencing and high-throughput real-time polymerase chain reaction analyses. In the case of strawberry (Fragaria x ananassa) fruits, successful RNA isolation requires removal of abundant inhibitory substances (polysaccharides and polyphenols) that greatly reduce quality and yield. In this study, we applied various combinations of RNA isolation protocols directed at reproductive organs. The best manual isolation method involved nonionic polymer and modified acid guanidinium thiocyanate-phenol-chloroform treatments followed by phenol/chloroform/isoamyl alcohol extraction. Compared with other methods, this approach gave significantly higher yields [84.0 mu g/g fresh weight (FW)] of RNA of greater purity (A260/A280 = 1.99; A260/230 = 1.51). Better-quality RNA (A260/230 = 2.11) was obtained using an automated method, but the yield was lower (18.1 mu g/g FW) than that obtained manually. This automated method consisted of pretreatment with nonionic polymer followed by a silica-based system extraction. Although RNA of sufficient quality [RNA Integrity Number (RIN) >= 6.5 and 28S/18S >= 1.0] for RNA sequencing was obtained from receptacles using both automated and manual methods, the manual method yielded high-quality RNA from achenes and anthers. The automatic method features 6-fold faster high-throughput capacity, whereas the manual method has wider applicability to different tissues.