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miScript Precursor Assays

For quantification of precursor miRNA using the miScript PCR System

  • Sensitive and specific detection and quantification of precursor miRNA
  • Detection of precursor and mature miRNA in parallel
  • Convenient ordering via GeneGlobe

miScript Precursor Assays are precursor-miRNA–specific forward and reverse primers used in combination with the miScript SYBR Green PCR Kit for real-time PCR of precursor miRNA. miScript Precursor Assays are available for all human, mouse, and rat miRNAs listed in the current version of miRBase, and are continually updated. cDNA is prepared using the miScript II RT Kit with miScript HiFlex Buffer. This cDNA is used as a template in real-time PCR with a miScript Precursor Assay and QuantiTect SYBR Green PCR Master Mix from the miScript SYBR Green PCR Kit.

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Cat No./ID: 218600
miScript Precursor Assay (100)
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10x miScript Precursor Assay (contains one miRNA-precursor–specific forward primer and one miRNA-precursor–specific reverse primer)
miScript Precursor Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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Parallel detection of mature and precursor miRNA.

Total RNA was purified from HeLa S3 cells using the miRNeasy Mini Kit. cDNA was prepared using the miScript II RT Kit with the provided miScript HiFlex Buffer. In real-time PCR, miScript Primer Assays were used for mature miRNA quantification and miScript Precursor Assays were used for precursor miRNA quantification. The miScript PCR Control for RNU6-2 was used for normalization. Real-time PCR was carried out on the Rotor-Gene Q cycler. Using the miScript PCR System, mature miRNAs and their precursor miRNAs can be efficiently quantified in parallel real-time PCR reactions.

Performance

miScript Precursor Assays accurately detect precursor miRNAs and can be used in experiments in which precursor miRNAs and mature miRNAs are detected in parallel (see figure Parallel detection of mature and precursor miRNA).

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 precursor miRNA detection and quantification using the miScript PCR System, the following components are necessary:

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 mature miRNAs in combination with a miScript Primer Assay or miScript miRNA PCR Array. For detection of precursor miRNA, the miScript Universal Primer is not used.
miScript Precursor Assay — the miScript Precursor Assay consists of precursor-miRNA–specific forward and reverse primers designed to detect precursor miRNA using the most up-to-date information from miRBase. Precursor miRNA can be detected in parallel with mature miRNA (detected using a miScript Primer Assay) and mRNA (detected using a QuantiTect Primer Assay) from the same cDNA prep. The miScript Universal Primer is not used for precursor miRNA detection.
Precursor miRNA studies

miScript Precursor Assays consist of a precursor-miRNA–specific forward and reverse primer targeting the stem-loop sequence of the precursor miRNA under study. The stem-loop sequence targeted by the miScript Precursor Assay is present in both primary miRNA (pri-miRNA) and precursor miRNA, therefore miScript Precursor Assays provide quantification of both primary and precursor miRNA simultaneously. Detection of precursor miRNA enables studies of miRNA biogenesis, regulation, and function. Relative levels of mature and precursor miRNA vary for different miRNAs. In addition, mature miRNAs may be expressed from different precursor miRNAs under different conditions or in different tissues.

Procedure

For detection of precursor miRNA, 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 Precursor Assay in combination with QuantiTect SYBR Green PCR Master Mix provided in the miScript SYBR Green PCR Kit.

Applications

miScript Precursor Assays are used as part of the miScript PCR System, for precursor miRNA detection.

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