使用miScript PCR System,对包含miRNA的总RNA进行逆转录

  • 灵敏和特异性的检测miRNA
  • 利用单一cDNA定量检测多种miRNA
  • 利用同一cDNA定量检测miRNA和mRNA
  • 利用同一cDNA定量检测成熟miRNA和miRNA前体
  • 专有技术合成RNA,评估逆转录效率

miScript II RT Kit是miScript PCR System的重要组成部分,用于miRNA定量检测。配合miScript SYBR Green PCR Kit和miScript PCRarrays(miRNome或Pathway-Focused miScript miRNA PCR Arrays)或所需的检测(用于miRNA或其他非编码RNA的miScript Primer Assays、用于miRNA前体的miScript Precursor Assays或用于mRNA的QuantiTect Primer Assays),miScript II RT Kit合成的cDNA可作为real-time PCR 的模板。

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miScript II RT Kit (12)
For 12 cDNA synthesis reactions: miScript Reverse Transcriptase Mix, 10x miScript Nucleics Mix, 5x miScript HiSpec Buffer, 5x miScript HiFlex Buffer, RNase-Free Water
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miScript II RT Kit (50)
For 50 cDNA synthesis reactions: miScript Reverse Transcriptase Mix, 10x miScript Nucleics Mix, 5x miScript HiSpec Buffer, 5x miScript HiFlex Buffer, RNase-Free Water
218161
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The miScript II RT Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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miScript II RT Kit buffers.|Selective conversion of mature miRNAs into cDNA in miScript HiSpec Buffer.|Simultaneous conversion of all RNA species into cDNA in miScript HiFlex Buffer.|Reproducible results in technical and biological replicates.|
Two buffers are supplied with the miScript II RT Kit. Use miScript HiSpec Buffer for cDNA synthesis to enable either mature miRNA profiling (using miScript miRNA PCR Arrays) or mature miRNA quantification using individual miScript Primer Assays. Use miScript HiFlex Buffer for cDNA synthesis to enable quantification of mature miRNA, precursor miRNA, noncoding RNA (ncRNA), and/or mRNA from the same cDNA. cDNA prepared using either miScript HiSpec Buffer or miScript HiFlex Buffer can be used with miScript PCR Controls.|In a reverse transcription reaction with miScript HiSpec Buffer, mature miRNAs are polyadenylated by poly(A) polymerase and converted into cDNA by reverse transcriptase with oligo-dT priming. The cDNA is then used for real-time PCR quantification of mature miRNA expression.|In a reverse transcription reaction with miScript HiFlex Buffer, miRNAs and other noncoding RNAs (ncRNAs) are polyadenylated by poly(A) polymerase and converted into cDNA by reverse transcriptase with oligo-dT priming. mRNAs are converted into cDNA by reverse transcriptase using both oligo-dT and random priming. Detection of mature miRNA, precursor miRNA, other ncRNA, and mRNA can be performed using the appropriate assays.|Total RNA was purified using the miRNeasy Mini Kit from [A] 2 identical HeLa S3 cell pellets with identical passage numbers and [B] 2 HeLa S3 cell pellets collected from different parental stocks at different times. Using the miScript PCR System, cDNA was prepared and used as a template in real-time PCR with the miFinder miScript miRNA PCR Array. Plotting CT values of the replicates against each other demonstrated the high technical and biological reproducibility of the results. These experiments were performed by a first-time user of the miScript PCR System.|
Performance

miScript II RT Kit配合miScript PCR System的其他组分,灵敏和特异性地检测并定量miRNA。由于许多miRNA表达水平低,灵敏度特别重要,以确保数据的可靠。miScript PCR System灵敏度高、动态范围广和持续高扩增率。其卓越的性能使得初次尝试时即可获得高度重复性结果(参见"Reproducible results in technical and biological replicates")。

高特异性

miRNAs存在许多miRNA亚型家族,有些只是单个碱基的不同。这使得miRNA定量面临很大挑战。miScript PCR System可有效识别紧密相关的miRNA亚型,即使是只有单个碱基不同的miRNA也可(见表)。

miScript PCR System的特异性
miRNA序列
Let-7b UGAGGUAGUAGGUUGUGUGGUU
Let-7c UGAGGUAGUAGGUUGUAUGGUU
miR-98 UGAGGUAGUAAGUUGUAUUGUU
Let-7d AGAGGUAGUAGGUUGCAUAGUU
Let-7e UGAGGUAGGAGGUUGUAUAGUU
Let-7a UGAGGUAGUAGGUUGUAUAGUU
Let-7f UGAGGUAGUAGAUUGUAUAGUU
Let-7g UGAGGUAGUAGUUUGUACAGUU
Let-7i UGAGGUAGUAGUUUGUGCUGUU
用于PCR的cDNA Let-7b Let-7c miR-98 Let-7d Let-7e Let-7a Let-7f Let-7g Let-7i
Let-7b 100.00 1.78 0.00 0.00 0.00 0.01 0.00 0.00 0.01
Let-7c 0.54 100.00 0.00 0.00 1.01 0.12 0.00 0.00 0.00
miR-98 0.00 0.21 100.00 0.06 0.01 0.05 0.00 0.01 0.13
Let-7d 0.05 0.02 0.00 100.00 0.00 0.38 0.00 0.00 0.01
Let-7e 0.05 0.01 0.00 0.02 100.00 0.23 0.00 0.00 0.01
Let-7a 0.07 0.64 0.00 0.54 3.86 100.00 0.06 0.04 0.02
Let-7f 0.55 0.12 0.01 0.11 0.03 1.05 100.00 0.14 0.13
Let-7g 0.58 0.17 0.01 0.08 0.00 0.04 0.01 100.00 0.17
Let-7i 0.13 0.02 0.01 0.02 0.00 0.01 0.00 0.10 100.00
Let-7亚型的RNA序列已列出。粗体有下划线部分为碱基变化。使用miScript II RT Kit和miScript HiFlex Buffer,每个Let-7亚型(Let-7a-Let-7i和miR-98)的合成miRNAs用于cDNA合成反应。相同的实验也可使用miScript HiSpec Buffer,获得相同的结果(数据未列出)。不同cDNA的等分试样作为real-time PCR反应的模板,配合适合不同亚型miScript Primer Assay和miScript SYBR® Green PCR Kit使用。%相对检测值为所有与miScript Primer Assays不匹配和匹配的CT值的差值(%相对检测值= 2-ΔCT x 100)。其值表明miScript PCR System有效识别紧密相关的亚型家族的能力。
Principle

miScript PCR System应用SYBR Green-based real-time PCR,能够灵敏和特异性地进行miRNA的定量和谱分析。miScript PCR System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。

miScript PCR System包含以下组分:

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 Primer Assay或miScript Precursor Assay,前者为miRNA特异性正向引物,用于检测成熟miRNAs。后者包含正向和反向引物,靶向作用于miRNA前体的颈环序列。该产品使用miRBase中的最新序列信息设计。
miScript miRNA PCR Array,miRNome或Pathway-Focused miScript miRNA PCR Arrays被预先设定,单次使用的PCR孔板/孔盘可快速分析成熟miRNAs。
miScript miRNA PCR Array data analysis tool,免费的基于网络的工具,简化了用于miScript miRNA PCR Arrays相对定量的ΔΔCT方法。
miScript II RT Kit双缓冲体系

miScript II RT Kit包含miScript Reverse Transcriptase Mix、10x miScript Nucleics Mix、5x miScript HiSpec Buffer和5x miScript HiFlex Buffer。miScript Reverse Transcriptase Mix是一种经优化的poly(A)聚合酶和逆转录酶混合物。miScript Nucleics Mix包含dNTPs、rATP、oligo-dT引物和内部合成RNA对照(miRNA逆转录对照[miRTC]),可评估使用miScript miRNA PCR Arrays进行谱分析实验的逆转录性能(获取更多信息,请参考miScript miRNA PCR Array Handbook)。

miScript II RT Kit提供两种缓冲液:miScript HiSpec Buffer和miScript HiFlex Buffer,满足使用real-time PCR进行miRNA定量研究的特别要求(参见"miScript II RT Kit buffers")。miScript HiSpec Buffer配合miScript miRNA PCR Arrays和miScript Primer Assays进行成熟miRNA谱分析和定量实验时,提供理想的结果。miScript HiFlex Buffer适合于低通量miRNA定量实验,从同一样本进行如miRNA、mRNA和前体miRNA等不同种类的RNA的定量实验除外。

miScript HiSpec Buffer进行逆转录实验

使用miScript HiSpec Buffer进行逆转录反应,确保成熟miRNAs的选择性转化,miScript PCR Controls靶向转至cDNA。使用oligo-dT引物,成熟miRNAs通过poly(A)聚合酶加尾后,被逆转录至cDNA(参见"Selective conversion of mature miRNAs into cDNA in miScript HiSpec Buffer")。加尾和逆转录在同一试管内同时进行。oligo-dT引物有一个3'退化锚点和一个在5'端的通用标签序列,可在real-time PCR步骤扩增成熟miRNA。miScript Primer Assays配合miScript SYBR Green PCR Kit,确保通过real-time PCR灵敏、特异性的定量miRNA。加尾和通用标签保证miScript Primer Assays不会检测到基因组DNA。

miScript HiFlex Buffer进行逆转录实验

使用miScript HiFlex Buffer进行逆转录反应,可使所有种类的RNA转化至cDNA(参见"Simultaneous conversion of all RNA species into cDNA in miScript HiFlex Buffer")。使用oligo-dT引物,成熟miRNAs通过poly(A) 聚合酶加尾后,被逆转录至cDNA。加尾和逆转录在同一试管内同时进行。oligo-dT引物有一个3'退化锚点和一个在5'端的通用标签序列,可在real-time PCR步骤扩增成熟miRNA。miScript Primer Assays配合miScript SYBR Green PCR Kit,确保通过real-time PCR定量miRNA。加尾和通用标签确保miScript Primer Assays不会检测到基因组DNA。其它种类RNA(包括miRNA前体、其它非编码RNA和mRNA)使用oligo-dT和随机引物转化至cDNA。配合miScript SYBR Green PCR Kit,使用合适的检测进行real-time PCR的RNAs检测(如用于非编码RNA检测的miScript Primer Assays 、用于miRNA前体检测的miScript Precursor Assays和用于mRNA检测的QuantiTect Primer Assays)。

Procedure
逆转录反应操作简单。包含miRNA的总RNA作为逆转录反应的起始材料。逆转录是一个简单的流程,包括在37oC条件下进行1个小时的孵育反应及在95oC条件下的简单孵育后进行的灭活反应。
Applications

miScript II RT Kit作为miScript PCR System的一部分,用于:

  • 成熟miRNA定量检测和谱分析
  • 同时检测miRNA和mRNA
  • 同时检测成熟miRNA和miRNA前体
  • 检测snoRNA和其他非编码RNA

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Kit Handbooks
3

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For real-time PCR analysis of microRNA using SYBR Green detection
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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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