miScript SYBR® Green PCR Kit

For miRNA detection by real-time PCR using the miScript PCR System

Features

  • Sensitive and specific detection and quantification of miRNA
  • Quantification of hundreds of miRNAs from a single cDNA
  • Quantification of miRNA and mRNA from the same cDNA
  • Includes ROX dye for optional normalization of fluorescence

Product Details

The miScript SYBR Green PCR Kit is an integral component of the miScript PCR System for miRNA detection and quantification. The kit is based on the proven technology of QuantiTect SYBR Green Kits. cDNA generated by the miScript II RT Kit is used as the template for real-time PCR with the miScript SYBR Green PCR Kit and either PCR arrays (miRNome or Pathway-Focused miScript miRNA PCR Arrays) or appropriate assays (miScript Primer Assays for miRNA or other noncoding RNA, miScript Precursor Assays for precursor miRNA, or QuantiTect Primer Assays for mRNA).

Important note: This product line will be discontinued on May 1, 2021 and will be replaced by the miRCURY LNA miRNA PCR system. We strongly recommend using the miRCURY LNA SYBR Green PCR Kit for better miRNA quantification and profiling.

For more information about this transition, please visit our dedicated knowledge hub or contact us.

Performance

The miScript SYBR Green PCR Kit, together with the components of the miScript PCR System, ensures sensitive, specific miRNA detection and quantification. The miScript PCR System provides exceptional sensitivity and a broad dynamic range. Amplification efficiencies are consistently high, enabling accurate expression analysis of multiple miRNAs using the ∆∆CT method (see figure  miRNA expression in colorectal cancer). The high performance of the miScript PCR System ensures highly reproducible results between technical and biological replicates at the first attempt.

Important note: This product line will be discontinued on May 1, 2021 and will be replaced by the miRCURY LNA miRNA PCR system. We strongly recommend using miRCURY LNA SYBR Green PCR Kit for better miRNA quantification and profiling.

For more information about this transition, please visit our dedicated knowledge hub or contact us.

See figures

Principle

The miScript PCR System enables sensitive, specific miRNA quantification and profiling using SYBR Green-based 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.

The miScript PCR System comprises:

  • 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 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 Primer Assay — miScript Primer Assays are miRNA-specific forward primers designed to detect mature miRNAs.
  • miScript miRNA PCR Array — miRNome or Pathway-Focused miScript miRNA PCR Arrays are preformatted, single-use PCR arrays for rapid profiling of mature miRNAs.
  • miScript miRNA PCR Array data analysis tool — this complimentary, Web-based tool simplifies the ΔΔCT method of relative quantification for miScript miRNA PCR Arrays.

 

Real-time PCR detection of miRNA

The miScript SYBR Green PCR Kit includes QuantiTect SYBR Green PCR Master Mix and the miScript Universal Primer, which is specific for the universal tag sequence added to the 5' end of the cDNA during reverse transcription. A single cDNA synthesis reaction is sufficient to interrogate hundreds of miRNAs. cDNA prepared using miScript HiSpec Buffer can be used for mature miRNA quantification with miScript miRNA PCR Arrays or miScript Primer Assays. cDNA prepared using miScript HiFlex Buffer can be used for detection of different RNA species with appropriate primers in the real-time PCR step: miScript Primer Assays for miRNA or other noncoding RNA, miScript Precursor Assays for precursor miRNA, or QuantiTect Primer Assays for mRNA. 
 

Procedure

Reverse transcription is performed using the miScript II RT Kit. The resultant cDNA is used as a template for real-time PCR performed with the miScript SYBR Green PCR Kit, together with appropriate assays or arrays. This makes miRNA quantification and profiling a straightforward procedure, accessible to any lab with a real-time PCR cycler.

Applications

The miScript SYBR Green PCR Kit is used as part of the miScript PCR System for:

  • Mature miRNA quantification and profiling
  • miRNA and mRNA detection in parallel
  • snoRNA and other noncoding RNA detection

Supporting data and figures

Resources

Brochures & Guides (3)
Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow
Kit Handbooks (2)
For real-time PCR analysis of microRNA using SYBR Green detection
miRNA Research (1)

FAQ

What is the sequence of the miScript Universal Primer?
The sequence of the miScript Universal Primer in the miScript SYBR Green kit is proprietary.
FAQ ID -2994
Is the expression level of miRNA precursors lower compared to that of mature miRNAs?
In general, precursor miRNA (pre-miRNA) levels are lower than those of mature miRNAs. However this is not always the case. Sometimes the levels of both mature and pre-miRNA may be similar, or pre-miRNA levels may be higher. For this reason, researchers often want to quantify pre-miRNA as well as mature miRNA.  QIAGEN offers miScript precursor miRNA assays as well as mature assays for many miRNAs. 
FAQ ID -2807