For SYBR® Green-based real-time PCR profiling of plant miRNAs using the miScript PCR System
Cat. No. / ID: B44B2FEC-BCB6-40B0-9251-70B5CAE4B62D
Plant miRNAs play a necessary role in processes such as development, disease resistance and stress response. These small non-coding RNAs are thought to regulate gene expression in plants at the post-transcriptional level by targeting mRNAs for cleavage. These targeted alterations in gene expression are mediated by differential expression of hundreds of mature miRNAs that each bind to specific sequences in the target mRNAs. Therefore, profiling to coordinate regulation and expression of mature plant miRNAs provides invaluable insight into mechanisms that an organism is using to develop tissue, protect itself from drought or fend off disease. Until now, universal profiling of mature plant miRNAs by qRT-PCR was virtually nonexistent because polyadenylation-based reverse transcription reactions occur with very low efficiency due to the biochemical nature of plant miRNAs.
Important note: This product line will be discontinued on May 1, 2021 and will be replaced by the miRCURY LNA miRNA PCR system. For more information about this transition, please visit our dedicated knowledge hub or contact us.
Typical RT kits cannot be used to quantify plant miRNAs. Small RNAs possessing a 2’-O-methyl (2’-O-Me) modification on their 3’ terminal base, such as plant mature miRNAs and piwi-interacting RNAs (piRNAs), are refractory to polyadenylation. As a result, they cannot be efficiently reverse transcribed using a polyadenylation-based cDNA synthesis approach, which is the underlying mechanism of various miRNA reverse transcription kits including the miScript II RT Kit. The miScript Plant RT Kit has been designed and optimized for real-time PCR-based small RNA expression analysis together with miScript SYBR Green PCR Kit and miScript miRNA PCR Arrays or miScript Primer Assays. These functionally verified assays and arrays are available for rice, corn, soybean, tobacco and other significant species in agriculture and plant research. The specialized miScript Plant RT Kit overcomes this by first ligating an adaptor to the 3′ terminus of plant miRNAs. Unlike polyadenylation, this ligation reaction is not negatively inhibited by the presence of a 2′-O-Me modification on the 3′ terminal base of plant miRNAs. Reverse transcription can be performed following ligation.
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.
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.
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.
The exclusive, scalable PCR array sets enable you to profile the relative expression of corn, rice or soybean miRNAs:
miScript Corn miRBase Profiler – Profile up to 168 corn miRNAs; the 2-plate set contains 84 miScript Primer Assays on each plate
miScript Rice miRBase Profiler – Profile up to 504 rice miRNAs; the 6-plate set contains 84 miScript Primer Assays on each plate
miScript Soybean miRBase Profiler – Profile up to 336 soybean miRNAs; the 4-plate set contains 84 miScript Primer Assays on each plate
Each array set enables the accurate relative quantification of the most current corn, rice or soybean miRNA genome (miRNome or microRNome) as annotated in miRBase (www.miRBase.org). Individual plate(s) containing miRNAs of interest may be selected, or plates may be used in succession to profile miRNAs from each miRBase release up through the most recent version (currently V20). The flexibility and completeness of the panel ensures that scientists can identify all of the miRNAs that potentially impact a specific biological process. A set of controls present on this array enables data analysis using the ΔΔCT method of relative quantification, assessment of ligation-mediated reverse-transcription performance, assessment of reverse-transcription performance, and assessment of PCR performance. This unique series of PCR arrays delivers the specificity, sensitivity, and scalability to support any miRNA expression research.
miScript miRNA PCR Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease. miRBase Profiler is not associated with miRBase or miRBase.org or the University of Manchester, the BBSRC, the Wellcome Trust, or the Sanger Institute.
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.
| Control | Purpose |
|---|---|
| C. elegans miR-39 miScript Primer Assay | Alternative data normalization using exogenously spiked Syn-cel-miR-39 miScript miRNA Mimic |
| 6 snoRNA/snRNA miScript Primer Assays (miScript PCR Controls) | Data normalization using the ΔΔCT method of relative quantification |
| miRNA reverse transcription control (miRTC) miScript Primer Assay | Assessment of reverse transcription performance |
| Positive PCR control (PPC) | Assessment of PCR performance |
For more information on these controls, consult the miScript miRNA PCR Array Handbook.
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.
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 CT values are entered, the analysis tool automatically interprets the PCR array controls, performs relative quantification using the ∆∆CT method, and presents the results in a variety of visual formats, allowing you to rapidly interpret your data.
miScript miRNA PCR Arrays are used as part of the miScript PCR System for:
