【摘要】 目的建立胰腺高质量总RNA快速、经济、稳定的提取方法。方法应用TRIzol试剂和液氮提取大鼠胰腺总RNA,通过总RNA含量和A260/280比值的测定及10 g/L非变性琼脂糖凝胶电泳评估总RNA的质量,并用RT-PCR检测大鼠胰岛素1、胰高血糖素、胰α-淀粉酶和β-actin的表达。结果利用TRIzol和液氮提取的大鼠胰腺总RNA量可达到3～6μg/mg胰腺组织,A260/280比值在1.75～1.89之间。在10 g/L非变性琼脂糖凝胶电泳时,均可见28S及18S条带,并且在28S、18S条带间可见明显的云雾状阴影。大鼠胰岛素1、胰高血糖素、胰α-淀粉酶、β-actin RT-PCR产物电泳结果显示均有清晰的条带。结论应用TRIzol试剂和液氮成功地提取了大鼠胰腺高质量的总RNA,后者可用于RT-PCR研究。更多还原

【Abstract】 Objective To establish a quick,economical and reproducible method for high-quality RNA extraction from pancreas.Methods We utilized TRIzol Reagent and liquid nitrogen to isolate total RNA from the rat pancreas.The RNA quality was determined by detection of its content and optic density(A) at 260/280nm,and electrophoresis in 1% non-denatured agarose gel.Then reverse transcription-polymerase chain reaction(RT-PCR) was performed to detect expression of the pancreas-specific genes.Results The content of the total RNA extracted from the rat pancreas reached 3-6μg/mg pancreatic tissues,and A260/280 ratio was 1.75-1.89.Electrophoresis of the total RNA showed 28S and 18S rRNA bands with clear smear between them.The RT-PCR products of pancreas-specific genes including insulin 1,glucagon,α-amylase and housekeeping gene β-actin all exhibited clear bands on 1% agarose gel,which were located in the expected positions,respectively.Conclusion These results suggest that we have successfully isolated the high-quality and intact RNA from the rat pancreas with TRIzol Reagent and liquid nitrogen.The extracted total RNA can be used in RT-PCR for pancreatic gene expression.更多还原