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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Pathway-Focused or miRNome miScript miRNA PCR Array layout for plate formats A, C, D, F, M.|miRNome miScript miRNA PCR Array and miScript miRNA HC PCR Array layout for plate formats E, G.|Pathway-Focused miScript miRNA PCR Array layout for plate formats E, G.|miScript miRNA PCR Array layout for Rotor-Disc format R.|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.|miScript miRNA QC PCR Array layout for Rotor-Disc format R.|Whole miRNome profiling.|Analysis of expression of circulating miRNAs in serum or plasma holds huge potential as a source of new biomarkers.|
Wells A1 to G12 (1–84) each contain a miScript Primer Assay for a pathway/disease/functionally related mature miRNA. Wells H1 and H2 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells H3 to H8 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells H9 and H10 contain replicate reverse transcription controls (miRTC). Wells H11 and H12 contain replicate positive PCR controls (PPC).|Wells A1 to P12 (1–372) each contain a miScript Primer Assay for a pathway/disease/ functionally related mature miRNA. Wells P13 and P14 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells P15 to P20 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells P21 and P22 contain replicate miRTC miScript Primer Assays (miRTC). Wells P23 and P24 contain replicate positive PCR controls (PPC).|Pathway-Focused miScript miRNA PCR Arrays in formats E and G include 4 replicates of the same assays as provided in the 96-well formats A, C, D, and F.|Wells 1 to 84 each contain a miScript Primer Assay for a pathway/disease/functionally related gene. Wells 85 and 86 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells 87 to 92 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells 93 and 94 contain replicate miRTC miScript Primer Assays (miRTC). Wells 95 and 96 contain replicate positive PCR controls (PPC). Wells 97–100 are empty.|Wells 1 and 2 of each row contain replicate C. elegans miR-39 miScript Primer Assays (39). Well 3 of each row contains a miR-16 miScript Primer Assay (16). Well 4 of each row contains a miR-21 miScript Primer Assay (21). Well 5 of each row contains a miR-191 miScript Primer Assay (191). Wells 6 to 8 of each row each contain an assay for a different snoRNA (61 = SNORD61 assay, 95 = SNORD95 assay, 96A = SNORD96A assay). Wells 9 and 10 of each row contain replicate miRTC miScript Primer Assays (miRTC). Wells 11 and 12 of each row contain replicate positive PCR controls (PPC). These formats enable the quality assessment of up to 8 cDNA samples.|Wells 1 to 4 of each row contain replicate C. elegans miR-39 miScript Primer Assays (39). Wells 5 and 6 of each row contain replicate miR-16 miScript Primer Assays (16). Wells 7 and 8 of each row contain replicate miR-21 miScript Primer Assays (21). Wells 9 and 10 of each row contain replicate miR-191 miScript Primer Assays (191). Wells 11 to 16 of each row contain assays for different snoRNAs (61 = SNORD61 assay, 95 = SNORD95 assay, 96A = SNORD96A assay). Wells 17 to 20 of each row contain replicate miRTC miScript Primer Assays (miRTC). Wells 21 to 24 of each row contain replicate positive PCR controls (PPC). These formats enable the quality assessment of up to 32 cDNA samples.|Wells 1 and 2 contain replicate C. elegans miR-39 miScript Primer Assays. Well 3 contains a miR-16 miScript Primer Assay. Well 4 contains a miR-21 miScript Primer Assay. Well 5 contains a miR-191 miScript Primer Assay. Wells 6 to 8 each contain an assay for a different snoRNA (Well 6 = SNORD61 assay, Well 7 = SNORD95 assay, Well 8 = SNORD96A assay). Wells 9 and 10 contain replicate miRTC miScript Primer Assays (miRTC). Wells 11 and 12 contain replicate positive PCR controls (PPC). This pattern is repeated 7 additional times from wells 13 to 96. Wells 97 to 100 are empty. This format enables the quality assessment of up to 8 cDNA samples.|Total RNA was isolated from HCT 116 colorectal cancer cells that had been treated with ± 5-aza-2'-deoxycytidine (5-aza-2'-dC) demethylation reagent. This reagent irreversibly inhibits DNA methyltransferase driven methylation reactions by incorporating into DNA and covalently binding to the active site of the DNA methyltransferase. cDNA was prepared with miScript HiSpec Buffer using the miScript II RT Kit. The Human miRNome miScript miRNA PCR Array, in combination with the miScript SYBR® Green PCR Kit, was used to profile mature miRNA expression by real-time PCR. Data analysis was performed using the online miScript miRNA PCR Array Data Analysis Tool. [A] A scatter plot of 2-ΔCT values demonstrates that significant differences exist in the mature miRNA expression levels of the 2 samples. [B] A volcano plot shows regulated miRNAs (±4-fold is used as a cutoff and indicated by red lines). 104 miRNAs were strongly upregulated and 30 were strongly downregulated in 5-aza-2'-dC treated HCT 116 cells. When a p value of 0.05 is applied (indicated by blue line), the upregulation of 89 of the 104 miRNAs is significant, and the downregulation of 21 of the 30 miRNAs is significant.|Scatter plots show the relative miRNA expression levels from pooled RNA from healthy and colon cancer serum samples, characterized using miScript miRNA PCR Arrays.|
Performance
发现血清或血浆样本中的生物标记

以信号通路为主的miScript miRNA PCR Arrays的检测范围不断扩大,可发现生物过程中miRNAs的作用。使用QIAGEN专有的方法,可选择miScript miRNA PCR Arrays的量,确保芯片时时更新且与生物研究息息相关。目前miRNA研究领域突出的是血清或血浆样本中的miRNAs检测。近期发现血清和血浆中的胞外miRNAs相对稳定,研究集中在miRNA水平的变化,可作为一系列疾病研究的潜在非侵入性生物标记。设计Serum & Plasma miScript miRNA PCR Array,可快速分析84个与血清和血浆最具相关性的miRNAs(参见"Analysis of expression of circulating miRNAs in serum or plasma")。

单个cDNA样本完成所有miRNomes分析

除了用于信号通路研究的miScript miRNA PCR Arrays,还有及时更新的miRNome miScript miRNA PCR Arrays用于包括人类、小鼠、大鼠和狗等多个物种。使用不到1 µg RNA即可快速分析人类的所有miRNome(参见"Whole miRNome profiling")。miScript miRNA PCR Array中的每个引物都是经过实验验证的,确保使用real-time PCR高灵敏度、高特异性检测成熟miRNA。

Principle

miScript PCR System配合SYBR Green-based real-time PCR,高灵敏度、高特异性进行miRNA定量检测和分析。可以完成从RNA中合成cDNA及后续的real-time PCR检测miRNAs涉及的所有步骤。

使用miScript PCR System分析成熟miRNA,需要以下组件:

  • miScript II RT Kit,可简单、单步合成cDNA。单个cDNA合成反应可用于检测数以百计的miRNAs。双缓冲体系满足使用real-time PCR进行miRNA定量的独特要求。
  • miScript SYBR Green PCR Kit,包含QuantiTect SYBR Green PCR Master Mix和反向引物miScript Universal Primer,配合miScript Primer Assay或miScript miRNA PCR Array进行miRNAs检测。
  • miScript miRNA PCR Array,前沿的miScript miRNA PCR Arrays包含成熟miRNA的特异性正向引物(miScript Primer Assays),用于miRNome平台及生物相关的、以信号通路为主的平台。
Procedure
使用miScript miRNA PCR Arrays进行miRNA表达分析简单、可靠。首先,使用miScript HiSpec Buffer配合miScript II RT Kit制备cDNA。然后,加入cDNA预混物、miScript Universal Primer、QuantiTect SYBR Green PCR Master Mix和不含RNase的纯水到miScript miRNA PCR Array。接着,运行real-time PCR循环仪,进行反应。最后,使用miScript miRNA PCR Array Data Analysis Tool分析数据。
miScript miRNA PCR Array板布局

miScript miRNA PCR Arrays适用于96孔板、384孔板或Rotor-Disc 100孔盘三种规格(参见"Pathway-Focused or miRNome miScript miRNA PCR Array layout for plate formats A, C, D, F"、"Pathway-Focused miScript miRNA PCR Array layout for plate formats E, G"、"384HC and miRNome miScript miRNA PCR Array layout for plate formats E, G"和"miScript miRNA PCR Array layout for Rotor-Disc format R")。 每个芯片都包含几个对照检测,检测目的见表。

miScript miRNA PCR Arrays对照检测
对照 目的
C. elegans miR-39 miScript Primer
Assay
使用外源标准Syn-cel-miR-39 miScript miRNA Mimic,得到可供选择的标准化数据
6 snoRNA/snRNA miScript Primer
Assays (miScript PCR Controls)
使用相对定量的ΔΔCT方法得到标准化数据
miRNA reverse transcription control
(miRTC)
评估逆转录性能
Positive PCR control (PPC) 评估PCR性能

有关上述对照的更多信息,参见miScript miRNA PCR Array Handbook

miScript miRNA QC PCR Array板布局

miScript miRNA QC PCR Arrays使用real-time PCR进行多个cDNA样本的质量评估。提供96孔板、384孔板或Rotor-Disc 100三种规格(如需更多信息,参见miScript miRNA PCR Array Handbook)。每个miScript miRNA QC PCR Array包含在miScript miRNA PCR Array上进行对照检测的副孔。96孔板和Rotor-Disc 100规格为至多8个cDNA样本提供质量对照。384孔板规格为至多32个cDNA样本提供质量对照。

miScript miRNA PCR Array Data Analysis Tool

在线提供关于miScript miRNA PCR Arrays的即用型数据分析软件,操作方便。每个miScript miRNA PCR Array带有一个专有的数据分析工具。一旦输入原始CT值,该分析工具即可自动分析PCR对照,使用∆∆CT方法进行相对定量。通过多种格式显示结果,可快速解读数据。

Applications

miScript miRNA PCR Arrays是miScript PCR System产品线的一部分,适用于:

  • 分析疾病或信号通路相关的miRNA
  • 全miRNA组分析
  • 生物标记物发现

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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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