富含RNA酶大鼠腺体组织总RNA提取方法改良

Method Improvement for Extract Total RNA from Rats’Ribonuclease-rich Gland Tissue

为了改良传统提取富含RNA酶大鼠胰腺和腮腺组织总RNA方法,本研究将组织样本分成对照组,0.5%,1.0%和2.0%β-巯基乙醇处理组,对照组采用Trizol法提取总RNA,β-巯基乙醇处理组分别于Trizol试剂中加入0.5%,1%,2%β-巯基乙醇。测定不同处理组单链核酸含量、完整性,A_(260/280)和A_(260/230);采用实时荧光定量PCR检测管家基因β-actin,GAPDH,胰腺淀粉酶AMY2和腮腺水通道蛋白AQP-5表达情况,并通过计算扩增效率,验证β-巯基乙醇是否对后续PCR实验有影响。结果表明,不同处理组单链核酸含量、A_(260/280),A_(260/230)无显著差异,β-巯基乙醇组RNA完整性显著高于对照组,1.0%,2.0%β-巯基乙醇组RNA完整性显著高于0.5%β-巯基乙醇组,1.0%β-巯基乙醇组RNA完整性与2.0%β-巯基乙醇组无显著差异;添加1.0%β-巯基乙醇对后续PCR试验无影响。综上所述,在传统Trizol法内添加β-巯基乙醇可显著提高胰腺和腮腺组织总RNA提取质量,其中添加1.0%β-巯基乙醇是最佳提取方案。

To improve traditional method for extracting total RNA from RNase-rich pancreas and parotid tissues of rats,the samples were divided into control group,0.5%,1.0%and 2.0%β-mercaptoethanol treatment groups.Total RNA of control group was extracted by Trizol method.0.5%,1.0%and 2.0%β-mercaptoethanol were added to the Trizol in theβ-mercaptoethanol treatment groups,respectively.The concentration of nucleic acid,A_(260/280),A_(260/230) and integrity of each treatment groups were determined;RT-qPCR was used to detect the expression of housekeeping genesβ-actin,GAPDH,pancreatic amylase AMY2 and parotid gland aquaporin AQP-5.The amplification efficiency was calculated to verify whether the addition ofβ-mercaptoethanol in RNA extraction had an impact on subsequent PCR The results showed that there were no significant difference in nucleic acid concentration,A_(260/280) and A_(260/230)between different treatment groups,but the RNA integrity of theβ-mercaptoethanol group was significant higher than that of the control group.The RNA integrities of 1.0%β-mercaptoethanol and 2.0%β-mercaptoethanol groups were significant higher than that of the 0.5%β-mercaptoethanol group.There was no significant difference in RNA integrity between the 1.0%β-mercaptoethanol and the 2.0%β-mercaptoethanol groups.There was no effect to subsequent PCR by adding 1.0%β-mercaptoethanol to Trizol.In summary,total RNA quality extracted from pancreas or parotid gland tissue could be significantly improved by addingβ-mercaptoethanol into Trizol reagent,and when adding 1.0%β-mercaptoethanol,it will reach the best consequent.