NanoString数字式单分子基因…

 NanoString nCounter® Analysis System

    它的优势在于直接测量mRNA的表达水平，无需进行复杂的酶催化反应；高通量，高灵敏度，结果偏差较小；处理样品的容量高；能够直接读出数字结果。 该技术的核心是各种不同的“标签”，这些标签能够结合到复杂生物样品的各种特异分子上，并在不同的位置产生不同的彩色光。用nCounter系统和芯片、TaqMan PCR等mRNA检测方法比较，发现它的灵敏度比芯片高，与实时PCR的相当。适用于数字化基因表达谱分析、数字化小RNA分析、基因拷贝数变异检测。
 

nCounter Workflow

The technical expertise needed to master the nCounter workflow... is the ability to count to three.

Scientists now have the power to look at more genes, across more samples, and do it faster and more cost-effectively.

Simplified, User-friendly and Efficient

1. Hybridization NanoString’s Technology employs two ~50 base probes per mRNA that hybridize in solution. The Reporter Probe carries the signal; the Capture Probe allows the complex to be immobilized for data collection.

2. Purify and ImmobilizeAfter hybridization, the excess probes are removed and the probe/target complexes aligned and immobilized in the nCounter Cartridge.

3. CountSample Cartridges are placed in the Digital Analyzer for data collection. Color codes on the surface of the cartridge are counted and tabulated for each target molecule.

 数字式单分子基因表达谱分析系统 
 

美国NanoString 是继生物芯片技术和新一代测序技术（NGS)后在基因表达谱分析上展示出强大应用前景的新技术公司。nCounter Analysis System是直接对基因表达进行多重计数的全新数字式技术，利用分子条形码和单分子成像来检测及统计每一个反应体系中特定转录本的数量，表现出极高的灵敏度、精确度和重复性。该技术整个系统包括全自动的样品处理工作站、数字化成像分析仪、CodeSet（分子条形码）和相关试剂，可以使研究人员用少量的实验材料进行特定或一组基因的表达特征研究。该技术上无需使用酶，无需反转录，也不需要做PCR 扩增，从而可进一步减少误差的产生，因此nCounter 在表达谱定量分析领域具有无可比拟的优势。
 
Nanostring Website：http://www.nanostring.com/
 

 
 
应用领域：
 
l          基因表达谱研究 Gene Expression
 
 800个基因分析通量
 
 具有弹性的样品需求，适用于血液样本与FFPE样本
 
 只需要15分钟的手动操作时间
 
 
 
l          小RNA分析 miRNA Analysis
 
人类，小鼠 及大鼠的miRNA分析皆适用 

 完整包含miRBase资料库的miRNA(人类、小鼠、大鼠)
 
 
 
l          拷贝数多样性分析 Copy Number Variation
 
 同时检视人类基因组中800个区域
 
 准确的标记感兴趣的区域
 
 
 
l          帮助二代测序进行后期验证 NGS Validation
 
 
 
技术原理：
 
1，荧光编码探针与目标分子杂交
 
荧光编码探针具有两个部份
 
(1)报告探针(Reporter Probe)是一段带有50 mer 的核酸及
 
   不同荧光颜色排列组合而成的探针，为主要的信号来源。
 

(2)捕获探针:是一段带有50 mer 的核酸及Biotin ， 能将
 
   目标分子捕获并停留在由Avidin组成的Cartridge上。
 

 2.2，2.杂交过后, 将样品送进Prep Station移除剩余为杂交的探针
 
并且将形成探针/目标分子的复合物与Cartridge進行接合，
 
通電使其皆朝同一個方向排列。
 

 
 
3，将cartridges移动到 DigitalAnalyzer进行信号数据采集

计数在Cartridge表面上的荧光编码探针并且将其列表标识。
 

 
 
Publication List:
 
·         Ioffe YJ, et al. “Phosphatase and tensin homolog (PTEN) pseudogene expression in endometrial cancer: a conserved regulatory mechanism important in tumorigenesis?” Gynecol Oncol. 2011 Oct 14.
 
·         Fresneau B., et al. “Vinblastine in the treatment of children and adolescents with refractory immune thrombocytopenia.” Am J Hematol. 2011 Sep;86(9):785-7.
 
·         Guttman M., et al. “lincRNAs act in the circuitry controlling pluripotency and differentiation” Nature. 2011 Aug 28.
 
·         Zhang W, et al. "Type-A response regulators are required for proper root apical meristem function through post-transcriptional regulation of PIN auxin efflux carriers" The Plant Journal. 2011 Jun 6
 
·         Novikov, Aleksey, et al. "Mycobacterium tuberculosis Triggers Host Type I IFN Signaling To Regulate IL-1b Production in Human Macrophages". J Immunol.  (2011)
 
·         Finlay W. McNab et al. “Programmed death ligand 1 is over-expressed by neutrophils in the blood of patients with active Tuberculosis” Eur. J. Immunol. 2011. 41: 1941–1947.
 
·         Haynes K and Silver P. “Synthetic Reversal Of Epigenetic Silencing” J. Biol. Chem. 2011  jbc.C111.229567.
 
·         Orlovsky K, et al. “Down-regulation of homeobox genes MEIS1 and HOXA in MLL-rearranged acute leukemia impairs engraftment and reduces proliferation.” PNAS May 10, 2011 vol. 108 no. 19 7956-7961.
 
·         Reis PP, et al. “mRNA transcript quantification in archival samples using multiplexed, color-coded probes.” BMC Biotechnol. 2011 May 9;11(1):46.
 
·         Gamer LW, et al. “BMPR-II is dispensable for formation of the limb skeleton” Genesis. 2011 May 2.
 
·         Rabini M, et al. “Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells” Nature Biotechnology (2011).
 
·         Cools T, et al. “The Arabidopsis thaliana Checkpoint Kinase WEE1 Protects against Premature Vascular Differentiation during Replication Stress.” Plant Cell. 2011 Apr 15.
 
·         Kulkarni MM.  “Digital Multiplexed Gene Expression Analysis Using the NanoString nCounter System” Current Protocols in Molecular Biology, April 2011.

·         Beaume M, et al. “Orientation and expression of methicillin-resistant Staphylococcus aureus small RNAs by direct multiplexed measurements using the nCounter of NanoString technology.” J Microbiol Methods; 2011 Feb; 84(2):327-34
 
·         Baugh LR, et al. “Sensitive and Precise Quantification of Insulin-Like mRNA Expression in Caenorhabditis elegans.” PLoS ONE (2011) 6(3).
 
·         Yun L, et al. “Effective posttransplant antitumor immunity is associated with TLR-stimulating nucleic acid–immunoglobulin complexes in humans” J Clin Invest doi:10.1172/JCI44581.
 
·         Chaussabel S, et al. “Blood Transcriptional Fingerprints to Assess the Immune Status of Human Subjects" Immunologic Signatures of Rejection (2011), Part 3, 105-125.
 
·         Wurbel MA, et al. “CCL25/CCR9 Interactions Regulate Large Intestinal Inflammation in a Murine Model of Acute Colitis” PLoS One. 2011 Jan 25;6(1):e16442.
 
·         Andersen JB, et al. “An integrated genomic and epigenomic approach predicts therapeutic response to zebularine in human liver cancer”.  Sci. Transl. Med. 2, 54ra77 (2010).
 
·         Kim C, et al. “Gene-expression-based prognostic assays for breast cancer” Nature Reviews Clinical Oncology 7, 340-347 (June 2010).
 
·         Guiducci C., et al. “TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus” Nature.  465, 937–941 (17 June 2010).
 
·         Sun Z, et al. “Integrated Analysis of Gene Expression, CpG Island Methylation, and Gene Copy Number in Breast Cancer Cells by Deep Sequencing”  PloS ONE Vol 6-2, e17490 (2011).
 
·         Bock C, et al. “Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines”.  Cell. 44, 439–452, (2011).
 
·         Peter IS,  et al. “The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage” Developmental Biology, Special Section: Gene Regulatory Networks for Development (340)(2), 188-199., 15 April 2010.
 
·         Anakk S, et al. “Combined deletion of Fxr and Shp in mice induces Cyp17a1 and results in juvenile onset cholestasis”. The Journal of Clinical Investigation. 10.1172 (2010).
 
·         Kabacik S, et al. “Gene expression following ionising radiation: Identification of biomarkers for dose estimation and prediction of individual response”. Int. J. Radiat. Biol., Vol. 87; 1-15 (2010).
 
·         Strobl-Mazzulla HP, et al.  “Histone Demethylase JmjD2A Regulates Neural Crest Specification”. Developmental Cell. Volume 19, Issue 3, 14 September 2010, Pages 460-468.
 
·         Yassour M, et al. “Strand-specific RNA Sequencing Reveals Extensive Regulated Long Antisense Transcripts that are Conserved Across Yeast Species”.  Genome Biology, August 26, 2010; 11:R87.
 
·         Garay J, et al. “Crosstalk Between PKA and Epac Regulates the Phenotypic Maturation and Function of Human Dendritic Cells”.  The Journal of Immunology 2010; 185:3227-3238.
 
·         D’Angelo JA, et al. “The Cystine/Glutamate Antiporter Regulates Dendritic Cell Differentiation and Antigen Presentation”. The Journal of Immunology 2010; 185:3217-3226.
 
·         Materna SC, et al. “High accuracy, high-resolution prevalence measurement for the majority of locally expressed regulatory genes in early sea urchin development”.  Gene Expression Patterns doi:10.1016/j.gep.2010.04.002 (2010).
 
·         Dixit E, et al.  “Peroxisomes Are Signaling Platforms for Antiviral Innate Immunity”.  Cell(2010 May) Vol. 141, Issue 4, 668-681.
 
·         Nam J, et al. “Functional cis-regulatory genomics for systems biology”.  Proc Natl Acad Sci USA 2010 Feb 23;107(8):3930-5.
 
·         Smith ER, et al. “Nuclear entry of activated MAPK is restricted in primary ovarian and mammary epithelial cells”.  PLoS One 2010 Feb 18;5(2):e9295.
 
·         Ouellet M, et al.  “A rapid and inexpensive labeling method for microarray gene expression analysis”.  BMC Biotechnology 2009 Nov 25;9(1):97.
 
·         Amit I, et al.  “Unbiased Reconstruction of a Mammalian Transcriptional Network Mediating Pathogen Responses”.  Science2009 Oct 9; Vol. 326. no. 5950, 257-263.
 
·         Palamanda JR, et al. “Evaluation of CYP1A1 and CYP2B1/2 m-RNA Induction in Rat Liver Slices Using the NanoString Technology: A Novel Tool for Drug Discovery Lead Optimization”.  Drug Metabolism Letters 2009 Aug 3;(3):171-5.
 
·         Payton JE, et al.  “High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples”.  The Journal of Clinical Investigation 119(6): 1714-1726 (2009).
 
·         Vladislav MA, et al.  “Multiplexed measurements of gene signatures in different analytes using the NanoString nCounter Assay System”.  BMC Research Notes 2: 80 (2009).
 
·         Yi-Hsien S, et al.  “A Perturbation Model of the Gene Regulatory Network for Oral and Aboral Ectoderm Specification in the Sea Urchin Embryo”.  Developmental Biology 329: 410-421 (2009).
 
·         Birtwell S, et al.  Baugh LR, et al. “Sensitive and Precise Quantification of Insulin-Like mRNA Expression in Caenorhabditis elegans.”PLoS ONE (2011) 6(3).
 
·         Geiss GK, et al.  “Direct multiplexed measurement of gene expression with color-coded probe pairs”.  Nature Biotechnology 26: 317-25 (2008).