摘要
凝胶电泳技术通常被用于总RNA完整性检测,一般认为28S和18S rRNA条带亮度的比值大于等于2表示总RNA完整性良好,该比值越小表明总RNA降解越严重。为了检测这一标准在水产虾蟹类中是否继续适用,分别对凡纳滨对虾rRNA和mRNA的完整性进行了分析。用TRIzol分离纯化的凡纳滨对虾总RNA经凝胶电泳检测,发现其28S∶18S rRNA的比值远小于2;但是以同样的总RNA为模板进行RT-PCR,能顺利扩增出长约1 100 bp的ACT和e EF1A基因序列。进一步的3'∶5'分析显示这2个内参基因mRNA的3'∶5'ratio分别为2.79和1.53,直接表明被测mRNA完整性良好。因此,凝胶电泳低估了水产虾蟹类总RNA的完整性,建议采用3'∶5'分析技术对水产虾蟹类总RNA完整性进行检测。
The denaturing agarose gel electrophoresis and ethidium bromide staining is the most common method used to assess the integrity of total RNA. The 28 S rRNA band is approximately twice as intense as the 18 S rRNA band after running an intact total RNA sample on a denaturing gel. If the 28S∶ 18 S rRNA ratio is less than 2,it is likely that the RNA sample suffered degradation during preparation. The less the ratio is,the more is the degradation. In order to check whether it works for agarose gel analysis to assess the integrity of total RNA extracted from Penaeus vannamei,the integrity of rRNA and mRNA was assessed using two different methods,gel electrophoresis and 3＇∶ 5＇ assay in this paper. Total RNA was extracted from Penaeus vannamei with TRIzol reagent and treated by DNase I. A very low value of 28S∶ 18 S rRNA ratio,far less than 2,was observed using agarose gel analysis,which suggested the degradation of total RNA sample during preparation. However,using the same total RNA as template,two genes of about 1 100 bp were successfully amplified by reverse transcription PCR. Furthermore,a 3＇∶ 5＇ assay was carried out based on the same total RNA. The 3＇∶ 5＇ assay was independent of the rRNA integrity and could assess the mRNA integrity directly. A reverse transcription reaction was primed using oligo（d T）,and a real time quantitative PCR assay was used to quantitate the levels of 3＇ and 5＇ target sequences from ACT and e EF1 A mRNA. The 3＇∶ 5＇ ratio of ACT and e EF1 A mRNA were 2. 79 and 1. 53,respectively,which indicated the high integrity of mRNA. So gel electrophoresis of RNA underestimated the integrity of total RNA. The reliable assessment of the integrity of total RNA would need the 3＇∶ 5＇ assay.
出处
《浙江农业学报》
CSCD
北大核心
2016年第12期2007-2013,共7页
Acta Agriculturae Zhejiangensis
基金
南海水产经济动物增养殖广东普通高校重点实验室开放课题(2011007)
江苏省水产养殖学品牌专业项目(PPZY2015B159)
