Analysis of Epstein-Barr virus gene expression upon phorbol ester and hydroxyurea treatment by real-time quantitative PCR

Summary. Epstein-Barr virus (EBV) has the potential to undergo latent and lytic pathways during infection. However, expression of many of the viral genes during the lytic-latent transition remains unclear. In this study, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydroxyurea (HU), two commonly used modulators of EBV life cycle, on the expression profiles of the entire genome of EBV persistent infected in B95-8 cells. After treatment with TPA for 48h, the copy number of EBV genome in the cells increased about 2.5 fold, whereas HU treatment resulted in a reduction to approximately two-thirds of the original level. Except a small set of genes, the amounts of EBV mRNA are generally less abundant than that of -actin. The expression of a large fraction of the 80 EBV genes was found to be activated after TPA treatment with a noticeable increase of 19 and 21 fold, respectively in BSLF1 and BBLF4. In contrast, treatment of the B95-8 cells with HU, a nucleotide synthesis inhibitor, dramatically suppressed the expression of EBV lytic genes. In summary, we have demonstrated that real-time quantitative PCR is a reliable method to monitor the influence of drug-treatment in EBV genes regulation. Our results also provide a basis for further investigation on how the virus coordinates its own gene expression during latent-lytic pathway transition.     

Evaluation of reference genes and normalization strategy for quantitative real-time PCR in human pancreatic carcinoma

Histologically verified pairs (n=10) of pancreatic tumors and non-neoplastic tissues were used for quantitative real-time PCR and the stability of 24 reference genes was analyzed with geNorm and NormFinder software. Raw C{q} values correlated with the degree of RNA degradation. This correlation was abolished by normalization to C{q} of 18S endogenous control gene. Both geNorm and NormFinder programs suggested EIF2B1, ELF1, MRPL19, and POP4 as the same most stable genes. We have thus identified suitable reference genes for future expression studies in pancreatic carcinoma. Normalization method reducing the effects of RNA degradation on the quality of results was also developed.     

Automated extraction of viral-pathogen RNA and DNA for high-throughput quantitative real-time PCR

The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples (hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTA-blood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 +/- 0.06 for HCV and 0.97 +/- 0.03 for HBV, indicating a linear extraction from 100 to 1.0 x 10(5) HCV IU/ml and from 100 to 1.0 x 10(6) HBV IU/ml. Intra- and interrun variability was below 0.23 log(10) IU/ml for 2.98 to 5.28 log(10) HCV IU/ml and 2.70 to 5.20 log(10) HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log(10) HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log(10) HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log(10) copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log(10) copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.     

母体循环中胎儿游离DNA的定量分析

目的依据孕妇血浆中存在游离胎儿DNA的理论，从孕妇外周血浆中分离出胎儿DNA并加以鉴定，预防x连锁遗传病患儿的出生。方法从孕早期、中期共78名孕妇外周血浆中分离胎儿DNA，用实时荧光定量聚合酶链反应（fluorescence quantitative polymerase chain reaction，FQ—PCR）的方法检测其中的Y性别决定区（sex—determining region Y，SRY）基因。结果孕早期怀男胎的孕妇28名，25名SRY基因阳性，其平均浓度为（58．82±25．22）拷贝／ml；孕中期怀男胎的孕妇20名，SRY基因均为阳性，平均为（152．08±62．61）拷贝／ml；怀女胎的孕妇均为阴性。结论用实时荧光定量PCR的方法最早在孕62天的孕妇外周血浆中就可以检测到胎儿SRY基因，随孕周的增加，母血中胎儿DNA的量也在逐渐增加。实时荧光定量PCR技术在进行无创伤性产前性别诊断中有重要的价值。     

用实时荧光定量PCR方法检测母血中的胎儿SRY基因

目的　从孕妇外周血浆中分离出胎儿DNA ,并加以鉴定 ,预防X连锁遗传病患儿的出生。方法　从孕早期、中期共 3 0 0名孕妇外周血浆中分离胎儿DNA ,用实时荧光定量聚合酶链反应 (fluorescencequantitativepolymerasechainreaction ,FQ PCR)的方法检测其中的Y性别决定区 (sex determiningregionY ,SRY)基因。结果　孕早期怀男胎的孕妇 82名 ,70名SRY基因阳性 ,其平均浓度为 ( 5 8.82± 2 0 .90 )拷贝 /ml,中位数为 5 8.5 0拷贝 /ml。孕中期怀男胎的孕妇 90名 ,SRY基因均为阳性 ,平均为 ( 15 2 .0 8± 62 61)拷贝 /ml,中位数为 14 9.3 5拷贝 /ml。怀女胎的孕妇均为阴性。结论　用实时荧光定量PCR的方法最早在孕42天的孕妇外周血浆中就可以检测到胎儿SRY基因 ,随孕周的增加 ,母血中胎儿DNA的量也在逐渐增加。实时荧光定量PCR技术在进行无创伤性产前性别诊断中有重要的价值。     

慢性粒细胞白血病病人TCRζ链表达特点

目的： 建立实时定量PCR方法检测TCRζ链表达水平的方法，了解慢性粒细胞白血病（CML）外周血TCRζ链表达水平。方法: 采用SYBR GreenⅠ荧光定量PCR，相对定量检测30例CML患者和30例正常人外周血的单个核细胞的TCRζ链表达情况，以β2微球蛋白基因（β2M）作为内参，根据相对定量公式：2-△△Ct计算CML病人与正常人TCRζ链表达差异倍数。结果: 成功建立SYBR GreenⅠ荧光实时定量PCR检测TCRζ链表达检测技术。18例CML患者TCRζ链出现表达低于正常人，而12例CML患者TCRζ链出现表达高于正常人。结论: CML病人中TCRζ链表达水平可分为表达下调（60%）和表达上调(40%)2组，提示部分CML病人的细胞免疫缺陷可能与其TCRζ链表达下调有关。     

RNA isolation from micro-quantity of articular cartilage for quantitative gene expression by microarray analysis

Isolation of quality RNA from articular cartilage has been challenging due to low cellularity and the high abundance of extracellular matrix and proteoglycan proteins. Recently developed methods for isolation of high quality RNA from cartilage are more applicable to larger cartilage specimens typically weighing at least 25 mg. While these methods generate RNA suitable for analysis, they are less successful with smaller tissue inputs. For the study of small focal defect cartilage specimens an improved RNA extraction method is needed. Here we report a protocol for direct RNA isolation from less than 3 mg of wet weight rabbit articular cartilage for quantitative microarray gene profiling. This protocol is useful for identifying differentially expressed genes in chondrocytes following focal cartilage repair and can potentially be adopted for gene expression analysis of cartilage biopsy specimens from human joints.     

慢性粒细胞白血病病人TCRζ链表达特点

目的:建立实时定量PCR方法检测TCRζ链表达水平的方法,了解慢性粒细胞白血病(CML)外周血TCRζ链表达水平.方法:采用SYBR Green Ⅰ荧光定量PCR,相对定量检测30例CML患者和30例正常人外周血的单个核细胞的TCRζ链表达情况,以β2微球蛋白基因(β2M)作为内参,根据相对定量公式:2-ΔΔCt计算CML病人与正常人TCRζ链表达差异倍数.结果:成功建立SYBR Green荧光实时定量PCR检测TCRζ链表达检测技术.18例CML患者TCRζ链出现表达低于正常人,而12例CML患者TCRζ链出现表达高于正常人.结论:CML病人中TCRζ链表达水平可分为表达下调(60%)和表达上调(40%)2组,提示部分CML病人的细胞免疫缺陷可能与其TCRζ链表达下调有关.     

Evaluation of the Abelson gene as a control gene for real-time quantitative PCR in multiple myeloma

The Abelson (ABL) gene was the best control gene for quantitative PCR (qPCR)-based diagnosis and minimal residual disease detection in leukemic patients. However, there is still no concerted effort focused on the optimization of control genes in multiple myeloma (MM). The results of this study will provide a basis for the use of ABL as a control gene for the quantification of aberrantly expressed genes in MM. We analyzed ABL, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase, and β₂-microglobulin genes expression in bone marrow samples of 62 MM patients and 31 healthy donors. MAGE-C1, the most commonly expressed cancer–testis antigen gene in MM, which is to be a potential clinical indicator for auxiliary diagnosis and prognostic evaluation in MM, was also detected. Our experimental results show that the copy numbers of the four control genes were well correlated in all samples detected. The quantitative data for MAGE-C1 using the four control genes also had high correlations. ABL and GAPDH genes were stably expressed in healthy donors and MM patients during therapy and did not vary with disease state. ABL is stable in the MM bone marrow mononuclear fraction, and it could be used as a reliable control gene for the normalization of qPCR studies in MM.     

妊娠糖尿病孕妇外周血中胎儿游离DNA的定量检测

目的依据孕妇血浆中存在游离胎儿DNA的理论,探讨其在诊断及预防妊娠期糖尿病（GDM）中的应用价值。方法用血浆DNA抽提法提取62例孕中晚期孕妇外周血中胎儿游离DNA（其中病例组32例,对照组30例）,通过实时荧光定量PCR（RQ-PCR）法检测两组孕妇外周血胎儿DNA浓度。结果 39例孕男胎均出现SRY阳性信号,妊娠期糖尿病组孕妇外周血中胎儿游离DNA-SRY水平明显高于对照组（P〈0.05）。结论妊娠期糖尿病孕妇外周血中游离胎儿DNA水平的异常升高有望成为预测及诊断GDM发病与疾病程度的一项有效指标。     

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.     

Rapid and quantitative detection of human adenovirus DNA by real-time PCR

Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was 相似文献   

实时荧光定量PCR方法检测非霍奇金淋巴瘤患者B淋巴细胞刺激因子表达水平

目的:建立实时荧光定量PCR方法(RFQ-PCR)检测非霍奇金淋巴瘤(NHL)患者B淋巴细胞刺激因子(BAFF)表达水平。方法:47例NHL患者及20例健康对照,采用实时荧光定量PCR方法检测其外周血单个核细胞中的B淋巴细胞刺激因子表达水平。结果:RFQ-PCR检测BAFF含量的示灵敏度为10 pg/ml,低浓度样本批内和批间变异系数(CV)分别为8.56%和11.32%,高浓度样本批内和批间CV分别为0.76%和4.58%。NHL患者和健康对照者BAFF mRNA表达量分别为(0.48±0.023,0.25±0.023),两者具有显著性差异(P=0.0001)。结论:RFQ-PCR检测BAFF mRNA含量的方法,具有较好的检测灵敏度和重复性。NHL患者BAFF mRNA高表达,提示BAFF可能在NHL发生发展中发挥重要作用。     

Detection of gene amplification in intraductal and infiltrating breast cancer by laser-assisted microdissection and quantitative real-time PCR.

Gene amplification is one essential mechanism leading to oncogene activation which is supposed to play a major role in the pathogenesis of invasive breast cancer. However, using standard methodologies the detection of gene amplifications has been limited especially in small-sized lesions, like pre-invasive precursor lesions. The combination of two novel technologies, laser-based microdissection and quantitative real-time PCR, facilitates the detection of low-level amplifications in morphologically defined lesions. As a model system we investigated in situ breast cancer (ductal carcinoma in situ, DCIS) classified according to the morphology-based Van Nuys grading system for amplification of growth-regulatory genes. In this study 83 formalin-fixed, paraffin-embedded archival DCIS specimens were examined after laser-based microdissection by quantitative real-time PCR using the TaqMan detection system for amplification of the c-erbB2, topoisomerase IIalpha, c-myc and cyclinD1 gene. In a subset of 17 DCIS with adjacent infiltrating tumour components we compared intraductal and invasive tumour components in parallel for differences in amplification status. The combination of these new techniques represents an excellent tool to gain new insights into carcinogenesis by analyzing genetic alterations in morphologically identified heterogeneous lesions in breast cancer progression within the very same specimen or even tissue slide.