一种适合蚊虫总RNA提取及样本保存的方法
A suitable method for isolating total RNA and preserving samples from mosquitoes
黄文雨；卢 莹；尹灿灿；马高风；刘 啸；邵黄芳；李梦楠；孙恩涛
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DOI：10.7679/j.issn.2095-1353.2019.156
作者单位：皖南医学院检验学院，芜湖 241002；皖南医学院检验学院，芜湖 241002；皖南医学院检验学院，芜湖 241002；皖南医学院检验学院，芜湖 241002；皖南医学院检验学院，芜湖 241002；皖南医学院检验学院，芜湖 241002；皖南医学院检验学院，芜湖 241002；皖南医学院检验学院，芜湖 241002
中文关键词：蚊虫；SDS法；改良SDS法；RNA提取；样本保存
英文关键词：mosquitoes; SDS method; modified SDS method; RNA isolation; samples preservation
中文摘要：
【目的】 建立适合蚊虫总RNA提取的方法，探讨4种不同保存样本方法对蚊虫总RNA提取的影响。【方法】 以室温、4 ℃冰箱、﹣20 ℃冰箱、﹣80 ℃冰箱4种不同方法保存蚊虫为研究对象，运用改良SDS法、传统SDS法和TRIzol法分别提取蚊虫总RNA，用紫外分光光度计、琼脂糖凝胶电泳和RT-PCR检测提取RNA的质量，并比较提取效果。【结果】 改良SDS法、传统SDS法和TRIzol法提取蚊虫总RNA的浓度值分别为1.223 3 ± 0.374 3、0.675 0 ± 0.319 8、0.310 0 ± 0.123 6，3种方法的浓度值差异具有统计学意义（F＝14.767，P < 0.001）。经LSD检验，改良SDS法提取蚊虫总RNA的浓度高于传统SDS法、TRIzol法（P＝0.005、P < 0.001）；改良SDS法提取的蚊虫总RNA纯度较高，A260/A280、A260/A230分别在1.8-2.0及2.0-2.2之间；电泳检测结果表明28 s、18 s及5.8 s条带清晰完整，条带之间无弥散，且无苯酚、蛋白质和gDNA污染；β⁃actin扩增显示符合RT-PCR的实验要求。在一个月内，﹣20 ℃、﹣80 ℃冰箱保存的蚊虫样本，其总RNA的提取未见明显降解。【结论】 改良SDS法是一种经济便利、可提取高质量蚊虫RNA的方法。﹣20 ℃、﹣80 ℃冰箱在一个月内对蚊虫样本具有较好的保存效果。
英文摘要：[Objectives] To determine the best methods for isolating and preserving the total RNA of mosquitoes. [Methods] The ability of three methods to isolate total RNA from mosquitoes; the modified SDS, traditional SDS and TRIzol reagent methods, were compared. The quality of total RNA obtained was evaluated with ultraviolet spectrophotometry, agarose gel electrophoresis and RT-PCR assay. In addition, mosquito RNA samples isolated by the modified SDS method were preserved under four different conditions; room-temperature, 4 ℃, -20 ℃ and -80 ℃, and the quality of the RNA preserved under each condition compared. [Results] The average concentration values of total RNA isolated by the modified SDS method, traditional SDS method and TRIzol reagent method were 1.223 3±0.374 3, 0.675 0±0.319 8 and 0.310 0±0.123 6, respectively. Analysis of variance indicates that there was a significant difference among these three methods (F=14.767, P<0.001). LSD test results suggest that the modified SDS method achieved higher concentration values of total RNA than the other two methods (P=0.005, P<0.001). The average spectrophotometric values of total RNA isolated by the modified SDS method were in the ranges 1.8-2.0 (A260/A280) and 2.0-2.2(A260/A230). The agarose gel bands of the 28S rRNA, 18S rRNA and 5S rRNA were clear, indicating a high quality of RNA devoid of polyphenols, protein and gDNA contamination. The RNA β⁃actin gene could be amplified by RT⁃PCR assay. There was no significant degradation of total RNA samples that had been frozen at -20 ℃ and -80 ℃ after one month. [Conclusion] Our data provide strong evidence that the modified SDS method is a cost-effective and convenient way to isolate high quality mosquito RNA. Freezing mosquito RNA samples at -20 ℃ and –80 ℃ can preserve their quality for up to a month.