Absolute quantification of microRNAs by using a universal reference

MicroRNAs (miRNAs) are a species of small RNAs ∼21–23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/CD133(−) hematopoietic progenitor cells.     

RT—PCR法同时测定APP与tau mRNA的含量

β－淀粉样蛋白前体（ＡＰＰ）与ｔａｕ蛋白是两种与Ａｌｚｈｅｉｍｅｒ病的病理改变相关的蛋白质,近年来也发现它们或其修饰产物在包含体肌炎及慢性氯喹中毒的肌细胞中沉积。为更好地研究这两种相关蛋白质在肌细胞中的表达,我们建立了用逆转录－多聚酶链反应同时定量这两种相关蛋白质在肌细胞中的表达,我们建立了用逆转录－多聚酶链反应同时定量这两种基因转录产物的方法。本法选用甘油醛－３－磷酸脱氢酶（Ｇ３ＰＤ）作为内标,     

基于DNA扣除法的Real-time RT-PCR对工程乳酸菌外源基因表达的绝对定量分析

本研究旨在利用Real-time RT-PCR对外源基因在工程乳酸菌中的表达进行定量分析,建立一种新的Real-time RT-PCR分析方法。采用玻璃珠热酚法提取工程乳酸菌总RNA,对外源目的基因的反转录(含有cDNA和DNA)样品和非反转录(仅含DNA)样品进行Real-time PCR检测,根据经典绝对定量方法并结合DNA扣除法进行分析,将得到的Ct值通过标准曲线换算为样品拷贝数,通过从反转录样品中扣除DNA样品的拷贝数的量,去除了DNA对实验结果的影响,得出最终的定量结果。采用以上方法分析工程乳酸乳球菌NZ9000中外源纤维素酶基因CBHⅡ的表达情况,对表达量较低的目的基因进行转录水平的分析,避免了RNA的损失,得到了外源基因表达的量为(1.28±0.02)×10-1 copies/cfu。这种基于DNA扣除法的Real-time RT-PCR绝对定量方法可以有效地对外源基因在工程乳酸菌中的表达进行分析。     

拟南芥CHS基因表达的实时荧光定量PCR检测

在简要介绍实时荧光定量PCR反应和定量原理的基础上,采用TaqMan荧光定量PCR技术,研究了UV-B辐射对拟南芥(Arabidopsis thaliana)CHS(查耳酮合成酶基因)表达的诱导,获得了与传统Northern杂交一致的结果.实时荧光定量PCR用于基因表达的定量检测,具有特异性强、自动化程度高、高效快捷,避免使用放射性同位素,能同时对多个样品中的起始模板进行准确定量等特点,因此该方法已逐渐被广泛用于基因表达的定量分析.     

拟南芥CHS 基因表达的实时荧光定量PCR 检测

在简要介绍实时荧光定量PCR反应和定量原理的基础上, 采用TaqMan荧光定量PCR技术, 研究了UV-B辐射对拟南芥(Arabidopsis thaliana)CHS(查耳酮合成酶基因)表达的诱导, 获得了与传统Northern杂交一致的结果。实时荧光定量PCR用于基因表达的定量检测, 具有特异性强、自动化程度高、高效快捷, 避免使用放射性同位素, 能同时对多个样品中的起始模板进行准确定量等特点, 因此该方法已逐渐被广泛用于基因表达的定量分析。     

Influence of reagent formulation on mRNA quantification by RT-PCR using imported external standard curves

Use of an imported external standard curve is common in real-time quantitative RT-PCR. Two practical strategies for long-term experiments include importing a grand mean standard curve to all accumulated runs or using daily imported standard curves, fixing the slope at the beginning of the experiment and calibrating successive runs with curves generated from this imported slope, adding a single standard that registers the variation in the y-intercept. This study determines the influence that a change in reagent lots has on these two calibration approaches when determining mRNA copy numbers of the ornithine decarboxylase and porphobilinogen deaminase genes. Two sets of determinations were run with the use of lot A and lot B. A marked decrease in the crossing points (Cp) in the standards for both genes at all concentration levels was observed with the change in lots. A grand mean standard curve was generated for each gene and each set and comparisons between the sets were performed. Statistically significant differences were found with respect to the y-intercept but not the slope, suggesting that the change of reagent lot affected the detection sensitivity but not the efficiency of the reaction. The excellent correlation coefficients obtained for these curves for each gene were not achieved when overall data from both sets were combined to generate an overall grand mean standard curve. We conclude that when faced with a change of RT-PCR reagent lot that will affect the detection sensitivity of the method, samples should be calculated with either the daily imported standard curves or with the respective grand mean standard curve for each lot.     

Real—time TaqMan RT—PCR快速检测犬瘟热病毒方法的研究

按照犬瘟热（CDV）N基因序列,设计合成了特异性引物和探针,经各反应条件的优化,建立了Real—time荧光定量RT—PCR技术,对细胞培养物、肝脏、肺脏、脑、脾脏、淋巴结以及鼻腔拭子等组织病料中的CDV进行了特异性检测和敏感性试验。同时,利用建立的Real—time荧光定量RT—PCR方法与常规RT—PCR以及韩国BIOINDIST生产的BITRAPIDCDV检测试剂盒对57份临床样品进行了检测。结果：用20pmol／mL的引物浓度各luL和20pmol／mL的探针浓度0.3uL,获得的荧光信号最强,曲线平滑。敏感性高,可检测到1.24×3ng／uL的病毒RNA;特异性强,与NDV、AIV、NiPV等RNA病毒不发生交叉反应。试验重复性的变异系数（CV）分别为2．3％、2．5％和4.2％;与常规RT—PCR和BIOINDIST生产的BITRAPIDCDV检测试剂盒相比较,该方法具有快速、特异、敏感、可定量,并可同时检测大量样品等优点。