Zhao Zhilong, Zong Zhihong, Li Jianxin, Liu Hongxu, Zhao Huiru
Department of Thoracic Surgery, First Hospital, China Medical University,Shenyang, Liaoning 110001, China;Department of Thoracic Surgery, Zhongshan Hospital, Dalian University, Dalian, Liaoning 116001, China.
Zhongguo Fei Ai Za Zhi. 2008 Aug 20;11(4):563-6. doi: 10.3779/j.issn.1009-3419.2008.04.010.
Formalin-fixed and paraffin-embedded tissue (FPE) with long term follow-up and clinicopathological data are extensively available and easily accessible. They represent an extensive source of genetic materials and proteins to be investigated for clinical usage. We conducted the study to explore the possibility of extracting RNA from FPE of lung cancer, and checking up the transcription levels of the promyelocytic leukemia gene (PML) by RT-PCR.
five fresh frozen lung adenocarcinoma tissues and 40 FPEs of lung cancer were chosen; FPEs had been conserved for 60-85 months. All samples had been identified lacking expression of PML protein by immunohistochemical staining. The total RNA was extracted by using Trizol one-step method, and verified by testing OD value. RT-PCR was used to detect the transcription level of PML gene.
The RNAs from both tissues had the OD value ranging from 1.7 to 2.1. RT-PCR results displayed that mRNA of PML (295 bps) and beta-actin (318 bps) was not absent in all cancer tissues.
Trizol one-step method is easily accessible, and reliable for extracting RNA in fragments around 300 bases from FPEs. Loss of PML protein in lung cancer is not caused by downward adjustment of mRNA.
长期随访及临床病理数据的福尔马林固定石蜡包埋组织(FPE)广泛可得且易于获取。它们是用于临床研究的大量遗传物质和蛋白质的来源。我们开展本研究以探索从肺癌FPE中提取RNA,并通过逆转录聚合酶链反应(RT-PCR)检测早幼粒细胞白血病基因(PML)转录水平的可能性。
选取5例新鲜冷冻肺腺癌组织及40例肺癌FPE;FPE已保存60 - 85个月。所有样本经免疫组织化学染色鉴定均缺乏PML蛋白表达。采用Trizol一步法提取总RNA,并通过检测OD值进行验证。RT-PCR用于检测PML基因的转录水平。
两种组织的RNA的OD值均在1.7至2.1之间。RT-PCR结果显示,所有癌组织中均存在PML(295 bp)和β-肌动蛋白(318 bp)的mRNA。
Trizol一步法易于操作,对于从FPE中提取约300个碱基片段的RNA是可靠的。肺癌中PML蛋白的缺失并非由mRNA下调所致。