一种用于从冷冻三年的人血浆中提取非病毒mRNA的优化方案。
An optimised protocol for the extraction of non-viral mRNA from human plasma frozen for three years.
作者信息
Wong S C C, Lo E S F, Cheung M T
机构信息
Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region, China.
出版信息
J Clin Pathol. 2004 Jul;57(7):766-8. doi: 10.1136/jcp.2003.007880.
AIMS
To detect non-viral mRNA in human plasma that has been frozen for three years using a new protocol.
METHODS
Plasma from 15 patients with colorectal cancer and 10 normal subjects was separated and frozen with Trizol at -80 degrees C for three years. As a control measure, plasma from 10 of the 15 patients was separated using the same protocol but no Trizol during storage. After three years, all samples were extracted using Trizol and RNeasy before the reverse transcriptase polymerase chain reaction was performed to detect non-viral beta catenin mRNA. In addition, extraction of three plasma samples by Trizol or RNeasy independently was carried out for comparison.
RESULTS
beta Catenin mRNA was detected in all 15 patient plasma samples and only one of the 10 normal subjects. In contrast, no beta catenin mRNA was found in the control and patient samples that were independently extracted by Trizol and RNeasy kit.
CONCLUSIONS
This new protocol is a reliable method for extracting non-viral mRNA from the plasma of patients with cancer after longterm storage for three years. Extractions using Trizol and RNeasy kits independently could not isolate mRNA with sufficient quantity and quality for detection.
目的
使用一种新方法检测已冷冻三年的人血浆中的非病毒mRNA。
方法
将15例结直肠癌患者和10名正常受试者的血浆分离，用Trizol在-80℃下冷冻三年。作为对照措施，15例患者中的10例患者的血浆采用相同方法分离，但储存期间不使用Trizol。三年后，在进行逆转录聚合酶链反应以检测非病毒β-连环蛋白mRNA之前，所有样本均用Trizol和RNeasy提取。此外，独立地用Trizol或RNeasy提取三个血浆样本以作比较。
结果
在所有15例患者血浆样本中均检测到β-连环蛋白mRNA，而10名正常受试者中仅1例检测到。相比之下，在由Trizol和RNeasy试剂盒独立提取的对照样本和患者样本中未发现β-连环蛋白mRNA。
结论
这种新方法是一种可靠的方法，可在长期储存三年后从癌症患者血浆中提取非病毒mRNA。单独使用Trizol和RNeasy试剂盒提取无法分离出足够数量和质量用于检测的mRNA。
Zotero 插件