快速高效从石蜡包埋肿瘤组织中提取micro-RNA方法的研究

AN EFFICIENT AND RAPID METHOD FOR MICRORNA FROM PARAFFIN-EMBEDDED TUMOR TISSUE

目的探索一种从石蜡包埋组织标本中快速、便捷、低成本的提取miRNAs的方法。方法收集2年内石蜡包埋的人乳腺癌组织和保存6个月的MMTV基因工程小鼠乳腺癌组织石蜡样本，同时选用进口A公司生产的提取石蜡组织miRNAs试剂盒作为对照；通过2种方法提取人和鼠的石蜡切片中miRNAs做对比分析，包括采用凝胶电泳验证，检测miRNA纯度、浓度分析。为了进一步验证其提取效果，采用定量PCR对所提取的总miRNAs进行miRNA-375和miR-218检测。结果①采用本实验方法从石蜡包埋乳腺癌组织中提取的miRNAs其纯度在1．86—2．210D之间，其浓度可以满足常规实验要求（最低38．50mg／L）；②荧光定量PCR能够检测到miRNA-375和miRNA-218，其拷贝数与对照组比较，2种方法差异无统计学意义（P〉0．05）。③该方法提取时间短、成本低，成本是试剂盒法的1／10，提取时间至少节省1／3。结论从石蜡包埋组织标本中提取miRNAs方法切实可行，为从石蜡组织样本提取miRNA进行分子诊断和肿瘤相关的研究提供了便利，对采用回顾性石蜡标本研究miRNAs提供了有力的帮助。

Objective To look for better and low cost method for extracting microRNAs （miRNAs） from paraffin-embedded tissue for miRNA diagnosis or other miRNAs assay. Methods The samples were collected from paraffin-embedded human breast cancer tissue stored over two years and MMTV transgenic mouse paraffin-embedded breast tissue over six months. The miRNAs extract paraffin tissue kit served as control. To detect the quality of miRNAs obtained by these two methods,used 2% gel electrophoresis and the OD value to confirm the quality and concentration. Furthermore, verified miRNAs by testing miRNA-375 and miRNA-218 with quantitative PCR. Results （1）miRNAs extracted by our method met the experimental requirements （A260/280 = 1.86 -2.21OD）, and the lowest concentration was 38.50mg/L. （2）The quantitative PCR can detect miRNAs and its copies number compared with the control, the difference was no significant （P 〉 0.05 ）. （3）This method has relatively lower cost, about 1/10 cost of kit method,while the extraction process decreased about 1/3 time. Conclusion The method we optimized for extracted miRNAs from paraffin-embedded tissue is feasible, it provides a convenient experimental method, and a better approach to detect more retrospective specimens of paraffin-embedded tissue samples.