实时定量RT-PCR的原理及方法

实时定量RT-PCR广泛应用于定量检测mRAN表达水平,为基础研究、分子药物学和生物技术研究提供了一种有力的方法。该法具有易操作、高通量、敏感性高和特异性强的特点,随着新酶、新探针和新仪器的发展而得到快速的发展。本文将对实时定量RT-PCR的定量原理、仪器应用、探针种类的研究进展以及细胞因子mRNA表达水平检测上的应用作一综述。     

Molecular epidemiology of norovirus variants detected in children under five years of age in Hyderabad,India

PurposeNoroviruses are common viral agents in acute diarrhea in all age groups worldwide. Norovirus has been classified into 10 genogroups, GI to GX with over 48 genotypes among them the GII.4 genotype has evolved over time with a clear pattern of periodic variant replacement. Immunity is strain or genotype specific with little or no protection conferred across genogroups. The present study was aimed to determine the epidemiology, prevalent genotypes of norovirus in children below five years of age in the Hyderabad region, India.MethodsThe stool samples and clinical data were collected from 458 children below 5 years of age comprising of cases with acute gastroenteritis (n ?= ?366) and a control group (n ?= ?92) admitted to the pediatric ward. All the samples were tested for Norovirus by ELISA and RT-PCR. Sequencing was done for predominant strains.Results10.3% (n ?= ?38) of cases and 3.2% (n ?= ?3) of the control group were found to be Norovirus positive. Predominant genotypes were GII-82.5% followed by GI-12.5%.ConclusionSequencing and Phylogenetic analyses of 20 GII.4 strains was done. All of the isolates are clustered away from published the GII.4 variants thus suggesting the appearance of a new variant.     

Sequence analysis of L RNA of Lassa virus

The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses.     

Plasma Content of Extracellular Nucleic Acids in Donors and Patients with Mammary Tumors

The concentrations of extracellular DNA and RNA were measured in the plasma of donors and patients with fibroadenoma and breast cancer. The content of extracellular DNA surpassed the normal in 80% plasma samples from patients with mammary tumors. Extracellular RNA was detected in 30% plasma samples from donors and patients with breast tumors. No correlations were found between plasma concentration of extracellular DNA and size and stage of tumor growth. Hence, measurement of extracellular DNA in the plasma of patients can be used only as an accessory test for tumor diagnosis.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 4, pp. 462–464, April, 2005     

Effect of tissue fixatives on telomere length determination by quantitative PCR

Telomere length is a well established marker of cellular senescence and thus biological age. Quantitative PCR allows the determination even from very low amounts of tissue by using telomere specific and single copy gene primers. Comparing a directly processed tissue sample to a 4% formaldehyde fixed one showed a significantly reduced efficiency of PCR reactions (mainly in single copy gene experiments) in a storage time-dependent manner resulting in an artificial increase in reported relative telomere length. This effect was not seen when the tissue was stored in RNA later solution. In summary, telomere length determination from formaldehyde fixed material by quantitative PCR is not a reliable method. Unfortunately therefore, many easily accessible tissue samples from pathology laboratories are unsuitable for this technique.     

Congenital infection with human herpesvirus 6 variant B associated with neonatal seizures and poor neurological outcome

Human herpesvirus 6 (HHV 6) has neurotropic and neuroinvasive properties. The virus has been found in the cerebrospinal fluid of many children with aseptic meningoencephalitis. Intrauterine transmission has been documented by HHV 6 DNA detection in cord blood specimens of apparently healthy newborns and in fetuses following spontaneous abortions. A patient is described with early neonatal afebrile seizures resulting from a congenital HHV 6 variant B infection disclosed by repeated detection of viral genome by polymerase chain reaction (PCR) in cerebrospinal fluid in the first days of life. At follow-up, magnetic resonance imaging (MRI) studies disclosed hyperintensities in the periventricular white matter and basal ganglia, associated with cerebral atrophy. Further follow-up at 18 months revealed poor neurological outcome with mild neurodevelopmental retardation, strabismus and hypertonia of legs. This report provides evidence of neurological involvement after HHV 6 vertical transmission, and the association with neurological sequelae.     

Construction of a risk model of steroid-induced necrosis of the femoral head and prediction of potential Chinese medicine treatments北大核心

背景:随着疾病治疗模式的改变,人们已经意识到中医药在激素性股骨头坏死治疗过程中的重要性,因此利用生物信息学从分子水平分析激素性股骨头坏死的发病机制,构建疾病风险模型,并预测具有潜在治疗作用的中药,为后期中医药治疗激素性股骨头坏死提供一定的理论依据。目的:基于生物信息学挖掘激素性股骨头坏死的竞争性内源RNA(ceRNA)调控网络,分析其在激素性股骨头坏死中的分子调控机制,预测相关疾病靶点并构建疾病风险模型,同时预测具有潜在治疗作用的中药。方法:检索GEO数据库,下载激素性股骨头坏死的矩阵文件GSE123568和基因注释文件GPL15207。借助R语言等软件分析得到差异表达的长链非编码RNA与mRNA,并通过公共数据库预测与差异表达长链非编码RNA关联的miRNA-mRNA,再将预测到的mRNA与差异表达mRNA取交集,整合得到ceRNA网络。随后采用STRING数据库和Cytoscape软件筛选关键基因,利用R语言分析关键基因的功能与相关通路,并挖掘关键ceRNA网络。最后根据关键基因构建激素性股骨头坏死的风险模型,并进行中药预测。结果与结论:(1)与健康对照相比,激素性股骨头坏死患者共有7个长链非编码RNA和1763个mRNAs存在差异表达;(2)筛选出STAT3、KAT2B、AGO4、JAK2、JAK1、PTGS2共6个关键基因;(3)关键基因所富集的功能包括对肽激素的反应、白细胞介素6介导的信号通路、细胞对白细胞介素6的反应等生物学过程,涉及JAK-STAT、脂肪细胞因子、催乳素等信号通路;(4)4种mi RNAs(mi R-135a-5p、mi R-137、mi R-17-5p、miR-20b-5p)和2种长链非编码RNA(SNHG11、C20orf197)可能在导致激素性股骨头坏死发生发展过程中发挥关键作用;(5)KAT2B最有可能是激素性股骨头坏死发生发展的风险因子;(6)郁金、淫羊藿、黄芪具备治疗激素性股骨头坏死疾病靶点的可能。通过对激素性股骨头坏死相关长链非编码RNA介导的ceRNA网络进行分析,识别出潜在的疾病靶点、信号通路及潜在治疗中药,为进一步阐明其发病机制,并为后续的实验研究提供参考依据。     

Possible Relation of p53 and mdm-2 Oncoprotein Expression in Thyroid Carcinoma: A Molecular-Pathological and Immunohistochemical Study on Paraffin-Embedded Tissue

Routinely processed tissues from a series of benign and malignant thyroid lesions were immunohistochemically investigated with antibodies against p53 and mdm-2. p53 was immunolocalized in <10% of nuclei in 2/80 nodular goiters, 2/60 follicular adenomas, 26/68 follicular carcinomas, 7/40 papillary carcinomas, 3/10 “insular” carcinomas, and 10/31 anaplastic carcinomas. More than 10% positively stained nuclei were found in 2 widely invasive follicular, 2 insular, and 15 anaplastic carcinomas. All p53-positive cases showed a concomitant immunohistochemical mdm-2 expression; an immunohistochemical colocalization on serial section was demonstrated in 12 anaplastic carcinomas. Screening by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of these 12 cases revealed no relevant mutations in the coding regions of exons 2–11 of the p53 gene. Additionally, 1 follicular adenoma, 6 follicular carcinomas (4 minimally and 2 widely invasive), 1 papillary, and 2 poorly differentiated insular carcinomas were mdm-2 positive without immunohistochemically detectable p53 expression. These results provide evidence that wild-type p53 expression in thyroid carcinomas may be associated with mdm-2 induced formation of stable complexes. However, the role of p53 mutations and p53 protein inactivation owing to other factors (e.g., mdm-2) in the progression of thyroid carcinomas is still poorly understood.