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miScript Primer Assays

For quantification of mature miRNA using the miScript PCR System

Features

  • Sensitive and specific detection and quantification of miRNA
  • Quantification of miRNA and mRNA from the same cDNA
  • Convenient ordering via GeneGlobe

Product Details

miScript Primer Assays are miRNA-specific primers available for all human, mouse, rat, and dog miRNAs listed in the current version of miRBase. miScript Primer Assays are part of the miScript PCR System for miRNA detection and quantification. cDNA is prepared using the miScript II RT Kit with miScript HiSpec Buffer (for mature miRNA detection only) or miScript HiFlex Buffer (for parallel quantification of all RNA species using appropriate assays). Either cDNA is used as a template in real-time PCR with a miScript Primer Assay and the miScript SYBR Green PCR Kit.

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 miRNA PCR Assays for better miRNA quantification and profiling.

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

Performance

Reproducible, reliable results are ensured using the miScript Primer Assays with the miScript PCR System for mature miRNA detection. As many miRNAs are expressed at low levels, sensitivity is critical to ensure reliable data. The miScript PCR System provides exceptional sensitivity, detecting as low as 10 miRNA copies (see figure  Accurate detection of as low as 10 miRNA copies). Accurate, reproducible miRNA data is provided from as little as 10 pg total RNA (see figure  Broad dynamic range and high amplification efficiencies). Amplification efficiencies are consistently high, enabling accurate expression analysis of multiple miRNAs using the ∆∆CT method.
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.

For mature miRNA detection and quantification using the miScript PCR System, the following components are used:

  • 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 — the kit and primer mixes enable unbiased preamplification of limited miRNA amounts, allowing subsequent miRNA profiling.
  • 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.

Procedure

For detection of mature miRNA, cDNA prepared in a reverse transcription reaction using miScript HiSpec Buffer or miScript HiFlex Buffer from the miScript II RT Kit serves as the template for real-time PCR. Real-time PCR is performed using an miRNA-specific miScript Primer Assay (forward primer) and the miScript SYBR Green PCR Kit, which contains the miScript Universal Primer (reverse primer) and QuantiTect SYBR Green PCR Master Mix.

For detection of other noncoding RNA, cDNA prepared in a reverse transcription reaction using miScript HiFlex Buffer from the miScript II RT Kit serves as the template for real-time PCR. Real-time PCR is performed using a miScript Primer Assay (forward primer) and the miScript SYBR Green PCR Kit, which contains the miScript Universal Primer (reverse primer) and QuantiTect SYBR Green PCR Master Mix. 

Applications

miScript Primer Assays are used as part of the miScript PCR System for:

  • Mature miRNA quantification
  • snoRNA detection

Supporting data and figures

Resources

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

FAQ

3322 - What is the concentration of the reconstituted miScript Primer Assay (100) and miScript Precursor Assay (100) tubes?

After reconstitution of the lyophilized primers in 550ul nuclease-free water or TE, the miScript Primer Assay and miScript Precursor Assay 100 reaction tubes will be at 10X stock concentration.  They should be used at 1X concentration (0.5 uM) in the PCR/RT-PCR reaction (eg. for a 25ul reaction, 1/10th of the total volume, or 2.5ul, should be primer). For Fluidigm® BioMarkTM System related applications, please refer to the miScript Microfluidics Handbook for primer concentrations and reconstitution recommendations.

Are miScript Primer Assay included in miScript miRNA PCR Arrays the same primers as the miScript Primer Assays available in individual tubes?

Yes, miScript Primer Assays included in miScript miRNA PCR Arrays are the same primers as the miScript Primer Assays available in individual tubes.

FAQ ID - 3441

 

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
Are miScript Primer Assays available for plant mature miRNAs?
Yes, miScript Primer Assays are available for plant miRNAs at www.qiagen.com/GeneGlobe.
FAQ ID - 3439
How reliable are the results from the miScript Primer Assays?
Assuming the use of good, consistent experimental technique, real-time PCR methods such as the miScript Primer Assays provide very reproducible results. To insure the reliability of your results and to reliably detect smaller fold changes in miRNA expression from the PCR Assays, the performance of replicate determinations (such as biological triplicates) is highly recommended.
FAQ ID -2730
Are plant miScript Primer Assay designs wet-bench validated?

Any plant miScript Primer Assay included on a plant miScript miRNA PCR Array has been bench validated.

FAQ ID - 3440