miScript Primer Assay Plate
For quantification of mature miRNA using the miScript PCR System with custom-formatted assay plates
Customer-configurable miScript Primer Assay Plates enable flexible design of miRNA experiments to suit individual requirements. miRNA-specific primers for any miRNAs listed in the current version of the miRBase can be selected and placed in any of the wells of a 96-well plate or a 384-well plate with the easy-to-use drag and drop tool at the GeneGlobe web portal. Each miRNA-specific assay is supplied in the well as a lyophilized primer that can be easily reconstituted to obtain the concentrations required for miRNA detection by real-time PCR using either a standard real-time cycler or the Fluidigm BioMark System. The 96-well plate option allows two different scales for ordering. A minimum of 24 primers must be ordered for a 96-well plate and 96 primers for a 384-well plate.
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.
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:
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. If a miScript Primer Assay for the small RNA of interest is not already available, one can be custom-designed.
Applications
miScript Primer Assay Plates are used as part of the miScript PCR System for:
|
