Trizol RNA提取法在雪貂组织的流感病毒H3N2定量PCR检测中优于RNeasy mini kit（Qiagen）柱子法

目的比较RNeasy mini kit（Qiagen）柱子法和Trizol法提取RNA在雪貂肺和肠组织H3N2 SYBRGreen PCR定量中的去干扰作用,从而找到适合该定量体系的RNA提取方法。方法比较Trizol法,RNeasy minikit柱子法,改良RNeasy mini kit柱子法1和改良RNeasy mini kit柱子法2提取肺肠组织RNA的质量,RNA逆转录成cDNA后,利用SYBR Green PCR方法检测样品中H3N2的载量,比较产物的特异性,综合评价RNA提取方法。结果 4种方法提取的肺和肠组织的RNA质量不相同,紫外分光光度仪测得RNeasy mini kit柱子法提取的RNA的A260/280低于1.8,其余3种方法的A 260/280在1.8～2.1之间,A 260/230只有Trizol法能达到2.0左右,其余3种方法均远低于2.0。电泳可见Trizol法提取的RNA有3条带：28 s、18 s和5 s,而RNeasy mini kit柱子法的RNA除了有28 s、18 s外,还有基因组DNA,改良RNeasy mini kit柱子法1和2提取的RNA只有28 s和18 s带。RNA逆转录后进行荧光定量PCR,从溶解曲线看,不论是鼻甲刮取物还是肺肠组织的样品模板,4种方法获得的样品与标准品均为单一峰溶解曲线峰,并且波峰位置重叠。而鼻甲刮取物定量的产物跑琼脂糖凝胶电泳均为单一的特异性条带,而肺肠组织的产物电泳发现：Trizol法获得的模板定量产物与标准品一致,为单一的特异性条带,而其它3种方法获得的模板则均有非特异性条带。结论鼻甲刮取物的病毒定量选用RNeasy mini kit柱子法提取定量结果与Trizol法一样可靠,说明对于简单样品该体系及引物非常适用,结果可信。对于组织样品肺和肠,Trizol法获得的样品定量结果比其它3种方法可靠。

Trizol Method for RNA Isolation from Ferret Tissues is Better Than RNeasy Mini Kit（Qiagen） for Diagnosis of Influenza Virus H3N2 Infection

Objective Comparing the methods for total RNA isolation from ferret tissues with Trizol,RNeasy mini kit,modified RNeasy mini kit1 and modified RNeasy mini kit2 in removing SYBR Green real time PCR inhibitors for diagnosis of influenza H3N2 virus infection.Methods Comparing the total RNA quality isolated by methods Trizol,RNeasy mini kit,modified RNeasy mini kit1 and modified RNeasy mini kit2,respectively,and also the results of assay of SYBR Green real time PCR for the influenza virus H3N2 in tissue total RNA.Results The quality of RNA isolated by the above four methods were measured using a Nano drop 2.0 UV spectrophotometer.A260/280 of the RNA isolated by RNeasy mini kit was below 1.8,and the other three were between 1.8 and 2.1.For A260/230,only the RNA isolated by Trizol was about 2.0,the other three were below 2.0.RNA isolated by Trizol ran in the 1.5% agrose gel electrophoresis showed three bands,28 s,18 s,and 5 s,while from the same tissues isolated by RNeasy mini kit showed three bands and genomic DNA,28 s and 18 s.As for modified RNeasy mini kit1 and modified RNeasy mini kit2,there were only two bands 28 s and 18 s.Judging form the melting curve of SYBR Green real time PCR,the templates obtained from both turbinates and lung or intestine by the four methods were normal with the same wave crest position for samples and standard.Though the melting curve of tissues was specific,the products of SYBR Green real time PCR were different： the product by Trizol method was a single specific band on the 1.5% agrose gel electrophoresis.By RNeasy mini kit,modified RNeasy mini kit1 and modified RNeasy mini kit2,the products were multi-bands.The products of turbinate samples were all a single specific band by the four different methods.Conclusions For turbinate swab samples,the four methods are all right for virus quantification,but for the ferret tissues including lung and intestine,the classical Trizol method is as creditable for as SYBR Green real time PCR.