For SYBR^® Green-based real-time PCR profiling of plant miRNAs using the miScript PCR System

Performance

Discover biomarkers in any sample

The range of pathway-focused miScript miRNA PCR Arrays is continually expanding to enable new discoveries about the roles of miRNAs in biological processes. The content of miScript miRNA PCR Arrays is selected using our proprietary methodology, which ensures that each array is up-to-date and biologically relevant.  One of the new exciting areas of research is the effects of miRNA in plant gene expression.  Whether studying stress response, root development or other plant specific pathways, miRNA are present and interacting with cellular expression.  miRNA have been indicated as key drivers of physiological change in animals, but research in plant miRNA is at the earliest stages.

Profile entire miRNomes with a single cDNA sample

Many species of plant have lists of known miRNA, but whole miRNA profiling of species during stress or developmental stages can be informative in identification of specific function during these stages.  Whole miRNome profiling is an important step in biomarker discovery, where quantification of the entire set of miRNA may show important physiological change (see figure "Whole miRNome profiling"). Each assay in a miScript miRNA PCR Array has been bench verified to ensure sensitive and specific detection of mature miRNA by real-time PCR.

Principle

The miScript Plant PCR System enables sensitive, specific miRNA quantification and profiling using SYBR Green real-time PCR. The miScript Plant PCR System covers all the steps involved in conversion of RNA to cDNA and subsequent real-time PCR detection of miRNAs.

Mature miRNA profiling with the miScript PCR System uses the following components:

miScript Plant RT Kit – Built specifically for plant samples. 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 – This kit enables an optional preamplification step when working with limited RNA amounts.

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 miRNA PCR Array – Cutting-edge miScript miRNA Plant PCR Arrays consist of mature miRNA-specific forward primers (miScript Primer Assays) arrayed in biologically relevant panels or miRNome arrays.

Procedure

miRNA expression profiling with miScript miRNA PCR Arrays is straightforward and robust. First, prepare cDNA buffered with miScript HiSpec Buffer using the miScript Plant  RT Kit. Second, add a premix of cDNA, miScript Universal Primer, miScript SYBR Green PCR Master Mix and RNase-free water to a miScript miRNA PCR Array. Third, run the reaction in a real-time PCR cycler. Finally, analyze the data using the miScript miRNA PCR Array Data Analysis Tool.

miScript miRNA PCR Array plate layout

miScript miRNA PCR Arrays are available in 96-well, 384-well and Rotor-Disc 100 formats (see figures "Pathway-Focused or miRNome miScript miRNA PCR Array layout for plate formats A, C, D, F, M", "Pathway-Focused miScript miRNA PCR Array layout for plate formats E, G", "miRNome miScript miRNA PCR Array and miScript miRNA HC PCR Array layout for plate formats E, G", and "miScript miRNA PCR Array layout for Rotor-Disc format R"). Each array contains several control assays. The purpose of these assays is described in the table below.

For more information on these controls, consult the miScript miRNA PCR Array Handbook.

miScript miRNA QC PCR Array plate layout

miScript miRNA QC PCR Arrays enable assessment of the quality of multiple cDNA samples using real-time PCR. miScript miRNA QC PCR Arrays are available in 96-well, 384-well, and Rotor-Disc 100 formats (see figures miScript miRNA QC PCR Array layout for plate formats A, C, D, F, miScript miRNA QC PCR Array layout for plate formats E and G, and "miScript miRNA QC PCR Array layout for Rotor-Disc format R"). Each miScript miRNA QC PCR Array includes replicates of C. elegans miR-39 miScript Primer Assay, 3 mature miRNA miScript Primer Assays (miR-16, miR-21, miR-191), 3 snoRNA miScript Primer Assays (SNORD61, SNORD95, SNORD96A), miRTC miScript Primer Assay, and a positive PCR control. For more information, consult the miScript miRNA PCR Array Handbook. The 96-well plate and Rotor-Disc 100 formats allow quality control of up to 8 cDNA samples. The 384-well format allows quality control of up to 32 cDNA samples.

miScript miRNA PCR Array Data Analysis Tool

Free, easy-to-use data analysis software is available online for miScript miRNA PCR Arrays. Each miScript miRNA PCR Array comes with a dedicated data analysis tool. Once raw C_T values are entered, the analysis tool automatically interprets the PCR array controls, performs relative quantification using the ∆∆C_T method, and presents the results in a variety of visual formats, allowing you to rapidly interpret your data.

Applications

miScript miRNA PCR Arrays are used as part of the miScript PCR System for: