实时荧光定量RT-PCR检测鼻咽癌Survivin mRNA基因表达

Objective:To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeal carcinoma (NPC) tissues.Methods:The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA,a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established,in which chronic nasopharyngitis patients' nasopharynx tissues treated as control group.Results:The expression of Survivin mRNA all could be detected either in CNE-2 cells,NPC tissues or in chronic nasopharyngitis patients' nasopharynx tissues,and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients' nasopharynx tissues,the difference was significant (P＜0.01).The expression of Survivin mRNA could be detected both in stage Ⅰ Ⅱ and stage Ⅲ Ⅳ NPC,and there was no significant difference in relative quantifications of gene expression between these two groups (P＞0.05).There was no relationship between Survivin mRNA expression and age and sex of NPC patients (P＞0.05).Conclusion:Real time fluorescence quantitative RT-PCR is a rapid,effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues.The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.     

实时荧光定量RT-PCR检测鼻咽癌Survivin mRNA基因表达

目的 建立实时荧光定量RT-PCR方法检测鼻咽癌组织中Survivin mRNA的表达.方法 用Trizol方法提取鼻咽癌CNE-2细胞株和鼻咽癌组织总RNA后,将其逆转录为cDNA,建立实时荧光定量RT-PCR方法,检测人鼻咽癌Survivin mRNA的表达,以慢性鼻咽炎患者鼻咽部组织作为对照.结果 鼻咽癌细胞株、鼻咽癌组和慢性鼻咽炎组患者鼻咽部组织均可检测到Survivin基因mRNA的表达,Survivin mRNA在鼻咽癌组织中的表达较慢性鼻咽炎患者鼻咽部组织高,两者差异有显著性意义(P＜0.01).Ⅲ+Ⅳ期和Ⅰ+Ⅱ期鼻咽癌患者均检测到Survivin基因mRNA的表达,但相对表达量无统计学意义.Survivin mRNA表达与鼻咽癌患者的年龄、性别无相关性.结论 实时荧光定量RT-PCR方法是一种快速有效灵敏度高的鼻咽癌Survivin mRNA表达的定量检测方法,Survivin mRNA的过度表达可能在鼻咽癌发生、发展过程中起一定作用.     

实时荧光定量RT-PCR检测白血病细胞WT1 mRNA方法的建立

目的 建立实时荧光定量RT-PCR检测白血病细胞WT1 mRNA的方法。方法 在PCR反应体系中加入了一条与靶基因序列互补的寡核苷酸双荧光标记探针(5′端为荧光报告基团，3′端为荧光淬灭基团)，建立实时荧光定量RT—PCR检测白血病细胞WT1 mRNA的方法，并测试其特异性及敏感性。结果 所建立的实时荧光定量RT—PCR检测WT1 mRNA的方法具有很好的准确性，检测WT1 mRNA的灵敏度达10^-4。方法的变异系数为1．93％，稳定性较好。循环阈值与PCR体系中起始模板量的对数值之问有着良好的线性关系(斜率为-3．5，线性相关系数，-0．997)，可对WT1 mRNA的表达进行准确定量分析。结论 实时荧光定量RT-PCR检测白血病细胞WT1 mRNA的方法具有很好的特异性和敏感性，且操作简便、安全、快速。     

The effect of total-ABL, GUS and B2M control genes on BCR-ABL monitoring by real-time RT-PCR

We compared the effect of control genes (CG): total Abelson (total-ABL), beta-2-microglobulin (B2M) and beta-glucuronidase (GUS), recommended in the Europe Against Cancer (EAC) program, on real-time BCR-ABL monitoring in patients with chronic myeloid leukemia (CML). We focused on the stability of CG expressions during therapy and the effect of the CGs on BCR-ABL ability to characterize the disease status and disease prognosis, issues that have not been addressed yet. The results showed B2M as a very convenient CG for BCR-ABL monitoring. On the contrary, the widely used total-ABL was not confirmed as appropriate for normalization of gene expression in CML.     

Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation.

To analyze the value of real time RT-PCR for monitoring of bcr-abl expression in CML patients after allogeneic or autologous stem cell transplantation (SCT), we generated pairs of PCR-primers and TaqMan probes specific for either the b2a2- or the b3a2-variant of bcr-abl. Either variant could be detected specifically from cDNA from a single K562 (b3a2) and BV173 (b2a2) cell with the respective TaqMan probe. Bcr-abl expression was normalized by comparison with GAPDH expression, and samples were quantitated using standard cDNA dilutions from K562 or BV173 cells. In a retrospective analysis 13 patients with CML after allogeneic (n = 10) or autologous (n = 3) SCT including patients with relapsed or persistent CML were analyzed by both real-time and conventional nested RT-PCR. In addition chimerism was monitored by FISH analysis of sex chromosomes in three patients with relapsed disease. The bcr-abl/GAPDH ratio dropped at least 1000-fold in all seven patients evaluable prior to and after allogeneic SCT as estimated by real-time RT-PCR, and conventional RT-PCR became negative in 6/7 patients. In five patients with relapsed or persistent disease after allogeneic SCT the bcr-abl/GAPDH ratio eventually increased again, and real-time RT-PCR was as sensitive as conventional RT-PCR for detection of bcr-abl. Donor lymphocyte infusions (DLI) were given to all five patients, and the bcr-abl/GAPDH ratio dropped to undetectable levels in two patients both remaining in continuing molecular remission. In contrast, in three other patients the bcr-abl/GAPDH ratio decreased only or did not change significantly after DLI. In three patients undergoing autologous SCT the bcr-abl/GAPDH ratio dropped only 1.1 to 30-fold, and the patients were tested positive with real-time RT-PCR at all time points. These data demonstrate that real-time RT-PCR is valuable to quantitate bcr-abl expression in CML patients after transplantation.     

Disturbance of circadian gene expression in endometrial cancer: detection by real-time quantitative RT-PCR

Circadian genes control the daily changes of the circadian rhythms in a variety of physiological processes, which in turn regulate many functions in the human body. Disruption of circadian rhythms can have a profound influence on our well-being. We established a set of PCR primers and fluorescent probes to analyze the mRNA levels of nine different circadian genes, and used immunohistochemical methods to study four important circadian proteins in 35 endometrial cancers and their paired non-cancerous tissues. Of these, 13 cases showed reduced expression in all nine circadian genes in the cancerous tissues relative to the paired non-cancerous tissues; the remaining cases showed similar reduced expression in 4-8 of the genes analyzed. Conversely, 3 non-cancerous tissues showed reduced expression in all nine circadian genes in comparison with their respective adjacent cancerous tissues, whereas 6 other non-cancerous tissues showed reduced expression in 6-8 of the circadian genes. These results were also confirmed by immunohistochemical study. Expression of the circadian genes is perturbed in endometrial cancer. Based on these results, we suggest that different circadian rhythms occur in endometrial cancer and non-cancerous tissues. Our results may provide the molecular basis for chronotherapy of endometrial cancer.     

bcr-abl P210和abl基因实时荧光定量PCR检测共用质粒标准品的制备及应用

 目的 建立一个荧光实时定量PCR（RQ-PCR）共用质粒标准品,用于同时检测bcr-abl P210白血病融合基因和abl内参基因。方法 分离K562细胞RNA,反转录为cDNA,以此为模板,设计引物进行PCR,PCR产物经琼脂糖电泳、胶回收,T-A载体克隆后,转化JM109工程菌,抽提质粒、测序、测定拷贝／μl,冻存备用。以RQ-PCR和定性巢式PCR进行验证,检测23例CML患者的骨髓标本,并同bcr-abl基因荧光原位杂交（FISH）结果比较。结果 获得了预期的模板质粒,以欧洲抗癌协会推荐的引物探针RQ-PCR检测bcr-abl P210和abl基因,浓度相同时,Ct值相同;反复检测日内差和日间差均<5 %。23例标本按FISH结果≥10%（n=8）、0.5 %～10 %（n=6）和阴性（n=9）分组,RQ-PCR结果分为0.492±0.263、0.023±0.033和（4.33±3.84）×10-4;FISH阴性组和其余两组间P值均＜0.001,三组间P值均＜0.05。结论 该研究建立了bcr-abl P210基因和abl基因共用的质粒标准品,定量准确,性质稳定,能简化操作,满足临床检测和科研的要求。     

实时定量RT—PCR芯片技术筛选乳腺癌术后复发相关基因

目的筛选并建立与乳腺癌术后复发转移相关的基因表达谱,初步探讨其与疾病发展的关系。方法81例乳腺癌患者入选,其中41例术后5年随访期内出现复发者为研究组,40例未复发仍存活者为对照组。提取所有病例石蜡包埋组织RNA,质量鉴定后,应用实时定量RT—PCR芯片技术平行检测84个已知的与乳腺癌转移特性相关的基因以及5个管家基因和7个质控对照,分析两组问差异表达基因。结果研究组与对照组相比较,12个基因表达有差异,其中9个基因表达上调,3个基因表达下调（P〈0．05）。表达异常的基因与细胞周期调控,细胞内激素信号传导,细胞增殖、分化和凋亡以及细胞迁移和粘附等功能相关。结论RT—PCR芯片技术能快速、准确地提供肿瘤相关特性基因表达谱的信息;乳腺癌术后复发相关基因差异表达分析可为预防和控制该病的复发提供新线索。     

Differential expression of 37 selected genes in hormone-refractory prostate cancer using quantitative taqman real-time RT-PCR

Progression of prostate cancer to androgen independence remains the primary obstacle to improved survival. The development of more effective treatments depends on our understanding of the molecular events associated with the hormone-refractory stage. We quantified, among 90 screened genes, the expression of 37 target genes, using real-time quantitative RT-PCR. Gene expression was studied in 13 samples of HPRC compared to 33 clinically localised cancers and normal prostate tissue. We identify 19 genes with significant differential expression in HRPC compared to localised prostate cancer. Genes with decreased expression included receptors for growth factors, MMR genes and the serine protease hepsin. Analysis of increased gene expression confirmed the importance of AR upregulation and highlighted genes not previously linked to HRPC, including enzymes involved in steroid synthesis and the antiapoptotic factor survivin. Progression of prostate cancer to the hormone-refractory state is associated with differential gene expression, which may prove useful for both understanding disease progression and the development of new therapeutic approaches.     

A new molecular breast cancer subclass defined from a large scale real-time quantitative RT-PCR study

Background  

Current histo-pathological prognostic factors are not very helpful in predicting the clinical outcome of breast cancer due to the disease's heterogeneity. Molecular profiling using a large panel of genes could help to classify breast tumours and to define signatures which are predictive of their clinical behaviour.     

Biomarker selection for detection of occult tumour cells in lymph nodes of colorectal cancer patients using real-time quantitative RT-PCR

Accurate identification of lymph node involvement is critical for successful treatment of patients with colorectal carcinoma (CRC). Real-time quantitative RT–PCR with a specific probe and RNA copy standard for biomarker mRNA has proven very powerful for detection of disseminated tumour cells. Which properties of biomarker mRNAs are important for identification of disseminated CRC cells? Seven biomarker candidates, CEA, CEACAM1-S/L, CEACAM6, CEACAM7-1/2, MUC2, MMP7 and CK20, were compared in a test-set of lymph nodes from 51 CRC patients (Dukes'' A–D) and 10 controls. Normal colon epithelial cells, primary tumours, and different immune cells were also analysed. The biomarkers were ranked according to: (1) detection of haematoxylin/eosin positive nodes, (2) detection of Dukes'' A and B patients, who developed metastases during a 54 months follow-up period and (3) identification of patients with Dukes'' C and D tumours using the highest value of control nodes as cutoff. The following properties appear to be of importance; (a) no expression in immune cells, (b) relatively high and constant expression in tumour tissue irrespective of Dukes'' stage and (c) no or weak downregulation in tumours compared to normal tissue. CEA fulfilled these criteria best, followed by CK20 and MUC2.