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