For miRNA detection by real-time PCR using the miScript PCR System
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
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).
For 2000 reactions: QuantiTect SYBR Green PCR Master Mix, miScript Universal Primer
miRNA expression in colorectal cancer.
cDNA was prepared from human colon adenocarcinoma total RNA and normal adjacent colon total RNA. miRNome profiling was performed using the Human miRNome miScript miRNA PCR Array. Fifty-six miRNAs were strongly downregulated and 50 were strongly upregulated in the adenocarcinoma tissue (red lines indicate ±4-fold change).
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.
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 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.
miScript Primer Assay or miScript Precursor Assay — miScript Primer Assays are miRNA-specific forward primers designed to detect mature miRNAs. miScript Precursor Assays consist of forward and reverse primers targeting precursor miRNA stem-loop sequences. Assays are designed using the most up-to-date sequence information from miRBase.
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
Mature miRNA and precursor miRNA detection in parallel
For 50 total RNA preps: 50 RNeasy MinElute Spin Columns, Collection Tubes (1.5 ml and 2 ml), QIAzol Lysis Reagent, Ce_miR-39_1 miScript Primer Assay, RNase-free Reagents and Buffers
