miScript Primer Assays
For quantification of mature miRNA using the miScript PCR System
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.
miScript Primer Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
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.
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 Primer Assay — miScript Primer Assays are miRNA-specific forward primers designed to detect mature miRNAs using the most up-to-date information from miRBase.
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. If a miScript Primer Assay for the small RNA of interest is not already available, one can be custom-designed.
miScript Primer Assays are used as part of the miScript PCR System for:
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