A Modified Method using TRIzolA® Reagent and Liquid Nitrogen Produces High-Quality RNA from Rat Pancreas
A Modified Method using TRIzolA® Reagent and Liquid Nitrogen Produces High-Quality RNA from Rat Pancreas
复制标题
DOI：
10.1007/s12010-008-8391-0
复制
发表时间：
2009-08-01
影响因子：
3
通讯作者：
Lu, Shemin
中科院分区：
文献类型：
--
作者：
Li, Dongmin;Ren, Wuchao;Lu, Shemin
为了建立一种经济、重复性好的高质量胰腺RNA提取方法，我们用TRIzolA(R)试剂和液氮提取大鼠胰腺总RNA。在实验初期，我们对胰腺匀浆方法、动物胰腺组织采集时间、1mlTRIzolA(R)试剂中胰腺组织重量三个影响因素进行了优化。通过检测总RNA含量及其在260/280 nm波长处的吸光度、未变性琼脂糖凝胶显示RNA和进行胰腺特异基因的RT-PCR来判断RNA质量。在液氮中研磨20-30 mg大鼠胰腺组织，1min后浸泡在1ml TRIzolA(R)试剂中提取的总RNA的A(260)/A(280)比值为1.75-1.89，28S/18S核糖体RNA条带的比值大于1.8。此外，从研磨组而不是常规组提取的RNA扩增了Pdx1开放阅读框的全长。从提取的RNA中成功地获得了胰腺外分泌部和内分泌部的胰腺特异基因的RT-PCR产物。结果表明，用TRIzolA(R)试剂和液氮成功地提供了一种经济、快速、重复性好的从大鼠胰腺中提取高质量、完整的RNA的方法。
To establish an economical and reproducible method for the high-quality RNA extraction from pancreas, we isolated total RNA from rat pancreas with TRIzolA (R) reagent and liquid nitrogen. In the initial stage, we optimized three influential factors, the way to homogenize pancreas, the time to collect the pancreatic tissue from animals, and the weight of the pancreatic tissue in 1 ml of TRIzolA (R) reagent. The RNA quality was determined by detecting total RNA content and its absorbance at 260/280 nm wavelength, visualizing RNA in non-denatured agarose gel and performing RT-PCR of pancreas-specific genes. The A (260)/A (280) ratio of the total RNA extracted by grinding 20-30 mg of rat pancreatic tissue removed from the rats in liquid nitrogen within 1 min and then immersed in 1 ml of the TRIzolA (R) Reagent was 1.75-1.89, and the ratio of 28S/18S ribosomal RNA bands was more than 1.8. Furthermore, full length of Pdx1 open-reading frame was amplified with RNA extracted from the grinding group rather than from the conventional group. The RT-PCR products of pancreas-specific genes from both exocrine and endocrine parts of pancreas were successfully derived from the extracted RNA. The results suggested that we successfully provided an economical, fast, and reproducible method to obtain the high-quality and intact RNA from rat pancreas with TRIzolA (R) Reagent and liquid nitrogen.