使用miScript PCR System对miRNA进行基于SYBR® Green法的real-time PCR分析

  • 灵敏和特异性的进行miRNA谱分析
  • 分析信号通路和疾病相关的miRNAs或全miRNA组
  • 免费提供在线工具,方便进行数据分析
miScript miRNA PCR Arrays是成熟miRNA的特异性正向引物(miScript Primer Assays),可对生物相关的信号通路和全miRNA组平台进行分析。作为miScript PCR System的一部分,miScript miRNA PCR Arrays用于miRNA定量检测。miScript II RT Kit配合miScript HiSpec Buffer得到的cDNA作为real-time PCR的模板,配合miScript miRNA PCR Array和miScript SYBR Green PCR Kit使用。

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miScript miRNA PCR Arrays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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按照信号通路或miRNome排列的miScript miRNA PCR Array布局。|miRNome miScript miRNA PCR Array和miScript miRNA HC PCR Array的孔板E、G布局|Pathway-Focused miScript miRNA PCR Array的E、G规格孔板布局。|miScript miRNA PCR Array的Rotor-Disc转子布局。|miScript miRNA QC PCR Array的A、C、D、F规格孔板布局。|miScript miRNA QC PCR Array的E和G规格孔板布局。|miScript miRNA QC PCR Array的Rotor-Disc转子布局。|全miRNA组谱分析。|对血清或血浆中的游离miRNA表达进行分析,发现潜在的新生物标记物。|
A1至G12孔(1–84)分别含有一种针对通路/疾病/功能相关的成熟miRNA的miScript Primer Assay。H1和H2孔含有重复的C. elegans miR-39 miScript Primer Assay,也可用作芯片数据的标准化(Ce)。H3至H8孔分别含有一种针对不同snoRNA/snRNA的分析,可用作芯片数据的标准化对照(SN1=SNORD61 assay,SN2=SNORD68 assay,SN3=SNORD72 assay,SN4=SNORD95 assay,SN5=SNORD96A assay,SN6=RNU6B/RNU6-2 assay)。H9和H10孔含有重复的逆转录对照(miRTC)。H11和H12孔含有重复的阳性PCR对照(PPC)。|A1至P12孔(1–372)分别含有一种针对通路/疾病/功能相关的成熟miRNA的miScript Primer Assay。P13和P14孔含有重复的C. elegans miR-39 miScript Primer Assay,也可用作芯片数据的标准化(Ce)。P15至P20孔分别含有一种针对不同snoRNA/snRNA的分析,可用作芯片数据的标准化对照(SN1=SNORD61 assay,SN2=SNORD68 assay,SN3=SNORD72 assay,SN4=SNORD95 assay,SN5=SNORD96A assay,SN6=RNU6B/RNU6-2 assay)。P21和P22孔含有重复的miRTC miScript Primer Assay(miRTC)。P23和P24孔含有重复的阳性PCR对照(PPC)。|E和G形式的Pathway-Focused miScript miRNA PCR Array中包括与96孔A、C、D和F形式中相同的试剂,每种试剂重复4个。|1至84孔分别含有一种针对通路/疾病/功能相关基因的miScript Primer Assay。85和86孔含有重复的C. elegans miR-39 miScript Primer Assay,也可用作芯片数据的标准化(Ce)。87至92孔分别含有一种针对不同snoRNA/snRNA的分析,可用作芯片数据的标准化对照(SN1=SNORD61 assay,SN2=SNORD68 assay,SN3=SNORD72 assay,SN4=SNORD95 assay,SN5=SNORD96A assay,SN6=RNU6B/RNU6-2 assay)。93和94孔含有重复的miRTC miScript Primer Assay(miRTC)。95和96孔含有重复的阳性PCR对照(PPC)。97–100孔为空。|每排的1和2孔含有重复的C. elegans miR-39 miScript Primer Assay(39)。每排的3孔含有miR-16 miScript Primer Assay(16)。每排的4孔含有miR-21 miScript Primer Assay(21)。每排的5孔含有miR-191 miScript Primer Assay(191)。每排的6至8孔分别含有一种针对不同snoRNA的分析(61 = SNORD61 assay,95 = SNORD95 assay,96A = SNORD96A assay)。每排的9和10孔含有重复的miRTC miScript Primer Assay(miRTC)。每排的11和12孔含有重复的阳性PCR对照(PPC)。上述形式可实现至多8个cDNA样本的质量评估。|每排的1至4孔含有重复的C. elegans miR-39 miScript Primer Assay(39)。每排的5和6孔含有重复的miR-16 miScript Primer Assay(16)。每排的7和8孔含有重复的miR-21 miScript Primer Assay(21)。每排的9和10孔含有重复的miR-191 miScript Primer Assay(191)。每排的11至16孔分别含有一种针对不同snoRNA的分析(61 = SNORD61 assay,95 = SNORD95 assay,96A = SNORD96A assay)。每排的17至20孔含有重复的miRTC miScript Primer Assay(miRTC)。每排的21至24孔含有重复的阳性PCR对照(PPC)。上述形式可实现至多32个cDNA样本的质量评估。|1和2孔含有重复的C. elegans miR-39 miScript Primer Assay。3孔含有miR-16 miScript Primer Assay。4孔含有miR-21 miScript Primer Assay。5孔含有miR-191 miScript Primer Assay。6至8孔分别含有一种针对不同snoRNA的分析(6孔 = SNORD61 assay,7孔 = SNORD95 assay,8孔 = SNORD96A assay)。9和10孔含有重复的miRTC miScript Primer Assay(miRTC)。11和12孔含有重复的阳性PCR对照(PPC)。13至96孔重复上述排列7次。97至100孔为空。上述形式可实现至多8个cDNA样本的质量评估。|从采用± 5-氮杂-2'-脱氧胞苷(5-aza-2'-dC)去甲基试剂处理的HCT 116结直肠癌细胞中提取总RNA。该试剂通过掺入DNA并与DNA甲基转移酶的活性位点共价结合,不可逆地抑制DNA甲基转移酶介导的甲基化反应。使用miScript II RT Kit及miScript HiSpec Buffer制备cDNA。Human miRNome miScript miRNA PCR Array与miScript SYBR® Green PCR Kit结合使用,通过real-time PCR进行成熟miRNA的表达谱分析。使用在线miScript miRNA PCR Array Data Analysis Tool进行数据分析。[A] 2-ΔCT值散点图显示,2种样本中成熟miRNA的表达水平存在明显差异。[B] 火山图显示了调控miRNA(以±4倍为阈值,并以红线表示)。在5-aza-2'-dC处理的HCT 116细胞中,104种miRNA出现明显上调,30种明显下调。当p值为0.05时(以蓝线表示),104种miRNA中的89种上调明显,30种miRNA中的21种下调明显。|散点图显示了使用miScript miRNA PCR Array,健康血清样本和结肠癌血清样本的混合RNA中的相对miRNA表达水平。|
性能
Discover biomarkers in serum or plasma samples

The range of pathway-focused miScript miRNA PCR Arrays is continually expanding to enable new discoveries about the roles of miRNAs in biological processes. The content of miScript miRNA PCR Arrays is selected using our proprietary methodology, which ensures that each array is up-to-date and biologically relevant. One of the most exciting areas of current miRNA research involves the assessment of miRNAs present in serum or plasma samples. The discovery of relatively stable, extracellular miRNAs in serum and plasma has generated huge interest in the use of changes in these miRNA levels as potential noninvasive biomarkers for a variety of diseases. As a result, the Serum & Plasma miScript miRNA PCR Array has been developed. This array enables rapid profiling of the 84 most relevant miRNAs associated with serum and plasma (see figure "Analysis of expression of circulating miRNAs in serum or plasma").

Profile entire miRNomes with a single cDNA sample

In addition to pathway-focused arrays, up-to-date miRNome miScript miRNA PCR Arrays are available for a variety of species including human, mouse, rat, dog, and rhesus macaque. With less than 1 µg RNA, the entire human miRNome can be rapidly profiled (see figure "Whole miRNome profiling"). Each assay in a miScript miRNA PCR Array has been bench verified to ensure sensitive and specific detection of mature miRNA by real-time PCR.

原理

The miScript PCR System enables sensitive, specific miRNA quantification and profiling using SYBR Green real-time PCR. The miScript PCR System covers all the steps involved in conversion of RNA to cDNA and subsequent real-time PCR detection of miRNAs.

Mature miRNA profiling with the miScript PCR System uses the following components:

miScript II RT Kit — this kit enables simple, single-step cDNA synthesis. A single cDNA synthesis reaction can be used for detection of hundreds of miRNAs. The dual buffer system meets the distinctive needs of miRNA quantification using real-time PCR.
miScript PreAMP PCR Kit and Primer Mixes — this kit enables an optional preamplification step when working with limited RNA amounts.
miScript SYBR Green PCR Kit — this kit includes QuantiTect SYBR Green PCR Master Mix and the miScript Universal Primer, a reverse primer that allows detection of miRNAs in combination with a miScript Primer Assay or miScript miRNA PCR Array.
miScript miRNA PCR Array — Cutting-edge miScript miRNA PCR Arrays consist of mature miRNA-specific forward primers (miScript Primer Assays) arrayed in biologically relevant pathway- or disease-focused panels or miRNome panels. 
操作流程
miRNA expression profiling with miScript miRNA PCR Arrays is straightforward and robust. First, prepare cDNA buffered with miScript HiSpec Buffer using the miScript II RT Kit. Second, add a premix of cDNA, miScript Universal Primer, QuantiTect SYBR Green PCR Master Mix, and RNase-free water to a miScript miRNA PCR Array. Third, run the reaction in a real-time PCR cycler. Finally, analyze the data using the miScript miRNA PCR Array Data Analysis Tool.
 
miScript miRNA PCR Array plate layout

miScript miRNA PCR Arrays are available in 96-well, 384-well, and Rotor-Disc 100 formats (see figures "Pathway-Focused or miRNome miScript miRNA PCR Array layout for plate formats A, C, D, F, M", "Pathway-Focused miScript miRNA PCR Array layout for plate formats E, G", "miRNome miScript miRNA PCR Array and miScript miRNA HC PCR Array layout for plate formats E, G", and "miScript miRNA PCR Array layout for Rotor-Disc format R"). Each array contains several control assays. The purpose of these assays is described in the table below.

Controls in miScript miRNA PCR Arrays
Control Purpose
C. elegans miR-39 miScript Primer
Assay
Alternative data normalization using
exogenously spiked Syn-cel-miR-39
miScript miRNA Mimic
6 snoRNA/snRNA miScript Primer
Assays (miScript PCR Controls)
Data normalization using the ΔΔCT
method of relative quantification
miRNA reverse transcription control
(miRTC) miScript Primer Assay
Assessment of reverse transcription
performance
Positive PCR control (PPC) Assessment of PCR performance

For more information on these controls, consult the miScript miRNA PCR Array Handbook.

miScript miRNA QC PCR Array plate layout

miScript miRNA QC PCR Arrays enable assessment of the quality of multiple cDNA samples using real-time PCR. miScript miRNA QC PCR Arrays are available in 96-well, 384-well, and Rotor-Disc 100 formats (see figures miScript miRNA QC PCR Array layout for plate formats A, C, D, F, miScript miRNA QC PCR Array layout for plate formats E and G, and "miScript miRNA QC PCR Array layout for Rotor-Disc format R"). Each miScript miRNA QC PCR Array includes replicates of C. elegans miR-39 miScript Primer Assay, 3 mature miRNA miScript Primer Assays (miR-16, miR-21, miR-191), 3 snoRNA miScript Primer Assays (SNORD61, SNORD95, SNORD96A), miRTC miScript Primer Assay, and a positive PCR control. For more information, consult the miScript miRNA PCR Array Handbook. The 96-well plate and Rotor-Disc 100 formats allow quality control of up to 8 cDNA samples. The 384-well format allows quality control of up to 32 cDNA samples.

miScript miRNA PCR Array Data Analysis Tool

Free, easy-to-use data analysis software is available online for miScript miRNA PCR Arrays. Each miScript miRNA PCR Array comes with a dedicated data analysis tool. Once raw CT values are entered, the analysis tool automatically interprets the PCR array controls, performs relative quantification using the ∆∆CT method, and presents the results in a variety of visual formats, allowing you to rapidly interpret your data.

应用

miScript miRNA PCR Arrays are used as part of the miScript PCR System for:

Pathway- and disease-focused miRNA profiling
miRNome profiling
Biomarker discovery

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Kit Handbooks
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For SYBR Green-based, real-time PCR profiling of microRNAs using pathway-focused arrays, HC arrays, and miRNome arrays
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Instrument Technical Documents
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For pathway-focused miRNA or miRNome analysis
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For gene expression and genomic analysis
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