Real-time PCR分析miRNA之前,预扩增从含少量RNA的样本中获得的cDNA

  • 有限的RNA进行miRNA表达分析的突破技术
  • 无偏差、可重复性结果
  • 可靠的工作流程
  • 完全整合入miScript PCR System工作流程

miScript PreAMP PCR Kit能够预扩增含少量RNA的起始样本,用于miScript PCR System miRNA的定量。miScript PreAMP PCR Kit使用多功能的、基于PCR的预扩增技术,在一个反应中预扩增400个miRNA特异性cDNA靶点。miScript PreAMP PCR Kit配合miScript PreAMP Primer Mixes,能够准确而全面对10 ng总RNA进行表达分析。10 ng的合成cDNA可以作为10个预扩增反应的模板。这样就提供了足够的模板用于多种Pathway-Focused miScript miRNA PCR Arrays或多种重复的miRNome miScript miRNA PCR Array,取决于使用的miScript PreAMP Primer Mix。

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miScript PreAMP PCR Kit (12)
For 12 preamplification reactions: 5x miScript PreAMP Buffer, HotStarTaq DNA Polymerase (2 U/µl), miScript PreAMP Universal Primer, miR-16 miScript Primer Assay, SNORD95 miScript Primer Assay, miRNA reverse transcription control (miRTC), C. elegans miR-39 miScript Primer Assay, RNase-Free Water
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miScript PreAMP PCR Kit (60)
For 60 preamplification reactions: 5x miScript PreAMP Buffer, HotStarTaq DNA Polymerase (2 U/µl), miScript PreAMP Universal Primer, miR-16 miScript Primer Assay, SNORD95 miScript Primer Assay, miRNA reverse transcription control (miRTC), C. elegans miR-39 miScript Primer Assay, RNase-Free Water
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The miScript PreAMP PCR 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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100% increase in miRNAs detected from 10-fold less cDNA after preamplification from serum.|High technical reproducibility.|Preamplification preserves expression patterns in FFPE samples using 1000-fold less input cDNA.|Extremely high correlation of expression between preamplified and nonpreamplified FFPE samples.|
Total RNA was purified from 5 µl human serum using the miRNeasy Serum/Plasma Kit. cDNA was then prepared from 0.7 µl serum equivalence (SE) using the miScript II RT Kit with miScript HiSpec Buffer. cDNA (0.7 µl SE) was used directly for miRNA profiling or one-tenth of the cDNA preparation (0.07 µl SE) was preamplified using the miScript PreAMP PCR Kit with Serum & Plasma miScript PreAMP Pathway Primer Mix prior to profiling. miRNA profiling was performed with the with the Plasma & Serum miScript miRNA PCR Array. Plots of [A] mean CT values achieved and [B] number of miRNAs detected demonstrate highly superior results from 10 fold less starting cDNA due to preamplification.|cDNA was prepared from a human universal reference RNA sample using the miScript II RT Kit with miScript HiSpec Buffer. Three different users performed preamplification using the miScript PreAMP PCR Kit and miFinder miScript PreAMP Pathway Primer Mix. Preamplified cDNA was used for miRNA profiling in duplicate with the miFinder miScript miRNA PCR Array. High levels of correlation were observed between users when CT values were [A] compared on a scatter plot or [B] plotted for all 3 users.|Preamplified cDNA and nonpreamplified cDNA from the same prep were used for miRNA profiling. Scatter plots of x-fold expression change calculations (2-ΔΔCT) between normal and tumor sections demonstrate high correlation between nonpreamplified and preamplified samples. [A] 96-plex miFinder miScript PreAMP Pathway Primer Mix and Array or [B] 384-plex miScript PreAMP miRNome Primer Mix and Array were used. The miRNeasy FFPE Kit was used to purify RNA from normal and tumor lung tissue 5 µM FFPE sections. cDNA was prepared from 10 ng total RNA using the miScript II RT Kit with miScript HiSpec Buffer and preamplified using the miScript PreAMP PCR Kit.|Preamplified cDNA and nonpreamplified cDNA from the same prep were used for miRNA profiling. Scatter plots of ΔCT values (normalized against housekeeping controls) between normal and tumor sections demonstrate high correlation between nonpreamplified and preamplified samples. [A] 96-plex miFinder miScript PreAMP Pathway Primer Mix and Array or [B] 384-plex miScript PreAMP miRNome Primer Mix and Array were used. The miRNeasy FFPE Kit was used to purify RNA from normal and tumor lung tissue 5 µM FFPE sections. cDNA was prepared from 10 ng total RNA using the miScript II RT Kit with miScript HiSpec Buffer and preamplified using the miScript PreAMP PCR Kit.|
Performance
从非常有限量的样本中获得有意义的数据

miScript PreAMP PCR Kit提供了从小样本和RNA含量非常低的样本中获得大量可靠的miRNA谱分析数据的机会。先前不可能的样本现在也可获得关于miRNA表达和功能的宝贵信息。预扩增后,使用Serum & Plasma miScript miRNA PCR Array,少于1 µl血清中的RNA就可获得可靠的miRNA表达谱。

与未扩增样本结果的高度相关性

由miScript PreAMP PCR Kit可靠、无偏差的扩增后获得的数据与未扩增样本的结果有高度的相关性,这样确保了结果的可信度。比较肿瘤和正常肺组织FFPE样本miRNA表达谱的结果,也证明了其高度的相关性(参见"Preamplification preserves expression patterns in FFPE samples using 1000 fold less input cDNA"和"Extremely high correlation of expression between preamplified and nonpreamplified FFPE samples")。

可重复的结果和可靠的操作流程

使用miScript PreAMP PCR Kit进行预扩增非常可靠,确保获得一致、可重复的结果。即使是不同的实验者进行预扩增和miRNA表达谱实验,也可获得高度重复的结果(参见"High technical reproducibility")。

Principle

miScript PCR System配合SYBR® Green real-time PCR能够灵敏、特异性的进行miRNA定量和分析。miScript PCR System可以完成从RNA中合成cDNA和后续的real-time PCR检测miRNAs涉及的所有步骤。miScript PreAMP PCR Kit是miScript PCR System的一部分,使得研究者能够使用非常有限量的起始RNA进行miRNA分析实验(参见Janas, M.M. et al. (2012) Reduced Expression of Ribosomal Proteins Relieves MicroRNA-Mediated Repression. Molecular Cell 46, 171.)。这对RNA含量较少的样本是非常必要的,如体液、福尔马林固定石蜡包埋(FFPE)样本以及细胞量较少的样本,如激光捕获显微切割(LCM)样本、流式分选细胞、循环肿瘤细胞和细针穿刺活检样本额。小样本中获得的少量RNA通常不足够进行可靠的miRNA分析实验,即使使用如real-time RT-PCR等灵敏的技术。

miScript PreAMP PCR Kit配合miScript PreAMP Primer Mixes能够对10 ng总RNA进行准确全面地表达分析,是一项重大的技术突破。10 ng的合成cDNA可以作为10个预扩增反应的模板。这样就提供了足够的模板用于多种Pathway-Focused miScript miRNA PCR Arrays或多种重复的miRNome miScript miRNA PCR Array,取决于使用的miScript PreAMP Primer Mix。miScript PreAMP PCR Kit使用多功能的、基于PCR的预扩增技术,在一个反应中预扩增400个miRNA特异性cDNA靶点。一般情况下,不论是96-plex或384-plex反应都能扩增2000–4000倍。

Procedure

少量RNA样本获得miRNA表达谱需要3个步骤:使用miScript II RT Kit进行逆转录,使用miScript PreAMP PCR Kit和miScript PreAMP Primer Mix进行预扩增,以及使用相应的miScript miRNA PCR Array和miScript SYBR Green PCR Kit进行real-time PCR。将miScript PreAMP PCR Kit和miScript PreAMP Primer Mix的预扩增整合入miScript PCR System miRNA表达谱的工作流程,确保了实验方案的可靠性和结果的可靠性和可重复性。

miScript PreAMP Primer Mixes适用于所有miScript miRNA PCR Arrays。也可使用miScript PreAMP PCR Kit配合25个miScript Primer Assays混合液进行预扩增。

预扩增质控

miScript PreAMP PCR Kit包括4个miScript Primer Assays用于预扩增质控实验,用来检验预扩增的cDNA。下表列出了质控和它们的作用。如需获得更多的详细信息,请参考miScript PreAMP Handbook

质控 作用
miR-16 miScript Primer Assay 确定预扩增cDNA的优化稀释因子,如果不知道模板的起始量
SNORD95 miScript Primer Assay 使用相对定量∆∆CT法将组织和细胞样本的数据标准
确定预扩增cDNA的优化稀释因子,如果不知道模板的起始量,且无miR-16
C. elegans miR-39 miScript Primer Assay 测量miRNeasy Serum/Plasma Spike-In Control(如果纯化过程中加入),用于确定血清和血浆样本的回收率
miRTC miScript Primer Assay 评估逆转录效率
Applications

miScript PreAMP PCR Kit作为miScript PCR System的一部分,用于:

  • 成熟miRNA的定量和谱分析
  • 从血清或血浆中发现生物标记物

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Brochures & Guides
1
Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow
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Kit Handbooks
3

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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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For preamplification of cDNA from samples containing low RNA amounts prior to real-time PCR analysis of microRNA
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