利用TRIzol 试剂和液氮提取大鼠胰腺高质量总RNA

A method with TRIzol  reagent and liquid nitrogen to extract high-quality RNA from rat pancreas

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。

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 260/280 nm, 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, a-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 expres- sion.