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定量测定黑鲷生长激素受体mRNA的液相杂交/RNase保护法 总被引:4,自引:0,他引:4
目的 建立液相杂交 核糖核酸酶保护测定 (RibonucleaseProtectionAssay,RPA)技术定量检测黑鲷(Sparusmacrocephalus)生长激素受体mRNA水平。方法 将黑鲷生长激素受体cDNA片断亚克隆至pGEM T载体 ,制备特异性的放射性反义RNA探针及正义RNA ,将反义RNA探针与正义RNA及样品总RNA进行液相杂交 ,用RNaseA和RNaseT1 降解杂交产物的单链RNA ,双链杂交体得到保护 ,然后检测杂交体的分子大小及放射强度。结果 检测出了黑鲷生长激素受体反义RNA探针与正义RNA及样品总RNA的特异性杂交片断 ,由此建立了定量测定黑鲷生长激素受体mRNA的液相杂交 RNase保护法 ,并采用该方法在黑鲷的多种组织中检测出了生长激素受体基因的表达 ,表达水平在肝脏中最高。该结果与我们曾采用生长激素放射受体分析法对黑鲷生长激素受体所研究的结果相吻合。结论 为进一步深入研究鱼类生长激素受体分子内分泌的调控理论提供了有力手段。 相似文献
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基因芯片分析技术(二) 总被引:2,自引:0,他引:2
(续 2 0 0 0年第 7期第 7页 )3 基因芯片的使用基因芯片块制作好后 ,关键就是用它来完成特殊的实验。其基本原理就是将研究的细胞或组织 RNA加以标记 ,与基因芯片块上所点印的基因片段进行杂交 ,从而探测特定基因的表达。3 .1 准备探针 与传统的 Northern Blot正好相反 ,这里我们将所有表达的 RNA作为探针来源。所以探针的准备工作要包括 RNA的提取和标记。3 .1 .1 RNA的提取 提取 RNA的方法很多。现在市场上有很多提取 RNA的试剂盒 ,用起来十分方便。不管用什么方法 ,提取 RNA的质量和产量是问题的关键。对于研究基因表达… 相似文献
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目的:制备用于检测小鼠胚胎早期Ucp2基因表达的地高辛标记的特异性RNA探针。方法:提取小鼠胚胎脑组织总RNA,设计引物,通过RT-PCR方法获取Ucp2基因片段,将其克隆到pGEM-T载体。分别利用Sp6、T7和Ucp2特异性引物,PCR扩增获得转录模板,通过Sp6及T7 RNA聚合酶,获得地高辛标记的正义、反义Ucp2 RNA原位杂交探针。检测标记探针的效价后,通过全胚胎原位杂交分析制备探针的特异性和杂交效果。结果:成功获得Ucp2基因正义、反义探针,反义探针能高效灵敏检测到Ucp2基因在小鼠胚胎Ed9.5、Ed10.5神经系统呈现高表达,而正义探针未能检测到表达信号。结论:成功制备了特异高效的地高辛标记Ucp2 RNA原位杂交探针,为进一步研究Ucp2基因在小鼠胚胎组织中的表达,尤其在神经组织的定位奠定基础。 相似文献
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检测Mdrl基因表达水平可预测白血病化疗效果,用原位杂交的方法可检测Mdrl在单个细胞的表达水平。本文用Rt-PCR的方法获得了一段特异的cDNA片段,将其克隆到PGEM4Z载体中,经DNA序列分析证明与文献报道一致,采用地高辛素(DIG)RNA标记试剂盒制备反义RNA探针,已初步用于临床骨髓涂片标本的原位杂交检测。 相似文献
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放射性标记的核酸探针(DNA和RNA探针)目前已被广泛地应用于分子生物学的各个领域,例如克隆的筛选,Southern杂交分析,基因表达水平的测定等等。放射性核酸分子探针是指特定的已知核酸分子片段,内含放射性核素(例:‘千、‘H和”S),并能与被检测的核酸分子退火杂交(核酸序列互补),因此可用于待测核酸样品中特定基因序列片段的探测。1探针的种类和选择根据核酸分子探针的来源及其性质可将其分成3大类,即:DNA探针、RNA探针和人工合成的寡聚核青酸探针。而DNA探针又可分为基因组DNA探针和。DNA探针。探针的选择是根据不… 相似文献
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基因芯片技术及其在植物上的应用 总被引:7,自引:0,他引:7
基因芯片技术(gene chip technology)是采用光导原位合成或缩微印刷等方法,将大量特定的DNA探针片段有序地固定于固相载体的表面,形成DNA微阵列,然后与待测的标记样品靶DNA或RNA分子杂交,对杂交信号进行扫描及计算机检测分析,从而获取所需的生物信息。该技术在植物研究中广泛应用于寻找特异性相关基因和新基因,基因表达分析,基因突变和多态性检测,DNA测序等。 相似文献
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Arianne C Richard Paul A Lyons James E Peters Daniele Biasci Shaun M Flint James C Lee Eoin F McKinney Richard M Siegel Kenneth GC Smith 《BMC genomics》2014,15(1)
Background
Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings. The recent development of a multiplexed, digital technology for nucleic acid measurement enables counting of individual RNA molecules without amplification and, for the first time, permits such a study.Results
Using a set of human leukocyte subset RNA samples, we compared previously acquired microarray expression values with RNA molecule counts determined by the nCounter Analysis System (NanoString Technologies) in selected genes. We found that gene measurements across samples correlated well between the two platforms, particularly for high-variance genes, while genes deemed unexpressed by the nCounter generally had both low expression and low variance on the microarray. Confirming previous findings from spike-in and dilution datasets, this “gold-standard” comparison demonstrated signal compression that varied dramatically by expression level and, to a lesser extent, by dataset. Most importantly, examination of three different cell types revealed that noise levels differed across tissues.Conclusions
Microarray measurements generally correlate with relative RNA molecule counts within optimal ranges but suffer from expression-dependent accuracy bias and precision that varies across datasets. We urge microarray users to consider expression-level effects in signal interpretation and to evaluate noise properties in each dataset independently.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-649) contains supplementary material, which is available to authorized users. 相似文献13.
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Paula Fernandez Max Solenthaler Olivier Spertini Stephane Quarroz Alicia Rovo Pierre-Yves Lovey Leda Leoncini Sylvie Ruault-Jungblut Mathilde D’Asaro Olivier Schaad Mylène Docquier Patrick Descombes Thomas Matthes Swiss Cytometry Society 《PloS one》2012,7(11)
Background
The diagnosis of malignant hematologic diseases has become increasingly complex during the last decade. It is based on the interpretation of results from different laboratory analyses, which range from microscopy to gene expression profiling. Recently, a method for the analysis of RNA phenotypes has been developed, the nCounter technology (Nanostring® Technologies), which allows for simultaneous quantification of hundreds of RNA molecules in biological samples. We evaluated this technique in a Swiss multi-center study on eighty-six samples from acute leukemia patients.Methods
mRNA and protein profiles were established for normal peripheral blood and bone marrow samples. Signal intensities of the various tested antigens with surface expression were similar to those found in previously performed Affymetrix microarray analyses. Acute leukemia samples were analyzed for a set of twenty-two validated antigens and the Pearson Correlation Coefficient for nCounter and flow cytometry results was calculated.Results
Highly significant values between 0.40 and 0.97 were found for the twenty-two antigens tested. A second correlation analysis performed on a per sample basis resulted in concordant results between flow cytometry and nCounter in 44–100% of the antigens tested (mean = 76%), depending on the number of blasts present in a sample, the homogeneity of the blast population, and the type of leukemia (AML or ALL).Conclusions
The nCounter technology allows for fast and easy depiction of a mRNA profile from hematologic samples. This technology has the potential to become a valuable tool for the diagnosis of acute leukemias, in addition to multi-color flow cytometry. 相似文献20.
Alison P Sanders Heather H Burris Allan C Just Valeria Motta Katherine Svensson Adriana Mercado-Garcia Ivan Pantic Joel Schwartz Martha M Tellez-Rojo Robert O Wright Andrea A Baccarelli 《Epigenetics》2015,10(3):221-228
Preterm birth is a leading cause of infant mortality and can lead to poor life-long health and adverse neurodevelopmental outcomes. The pathophysiologic mechanisms that precede preterm labor remain elusive, and the role that epigenetic phenomena play is largely unstudied. The objective of this study was to assess the association between microRNA (miRNA) expression levels in cervical cells obtained from swabs collected during pregnancy and the length of gestation. We analyzed cervical samples obtained between 16 and 19 weeks of gestation from 53 women in a prospective cohort from Mexico City, and followed them until delivery. Cervical miRNA was extracted and expression was quantified using the NanoString nCounter Analysis System. Linear regression models were used to examine the association between miRNA expression levels and gestational age at delivery, adjusted for maternal age, education, parity, body mass index, smoke exposure, and inflammation assessed on a Papanicolaou smear. We identified 6 miRNAs that were significantly associated with gestational age at the time of delivery, including miR-21, 30e, 142, 148b, 29b, and 223. Notably, per each doubling in miR-21 expression, gestations were 0.9 (95% CI: 0.2–1.5) days shorter on average (P = 0.009). Per each doubling in miR-30e, 142, 148b, 29b, and 223 expression, gestations were shorter by 1.0 to 1.6 days. The predicted targets of the miRNAs were enriched for molecules involved in DNA replication and inflammatory processes. The levels of specific miRNAs in the human cervix during pregnancy are predictive of gestational age at delivery, and should be validated in future studies as potential biomarkers of preterm birth risk.Key words: Cervix, delivery, gestational age, labor, microRNA, Pap smear, preterm 相似文献