miScript Plant miRNA Solutions

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

S_1084_5_GEN_disclaimer
Corn miRBase Profiler

Cat. No. / ID: B44B2FEC-BCB6-40B0-9251-70B5CAE4B62D

miRNome panels of miRNA assays in 96-well, 384-well, or Rotor-Disc formats
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Product
Corn miRBase Profiler
Soybean miRBase Profiler
Rice miRBase Profiler
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miScript Plant miRNA Solutions are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Features

  • Universal reverse transcription of all mature plant miRNAs
  • Functionally validated assays and arrays for corn, rice and soy
  • Free online data analysis tools

Product Details

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.

Performance

miScript plant miRNA quantification: go where other RT reactions cannot

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.


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.

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.

 

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.

Controls in miScript miRNA PCR Arrays
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 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 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.

Applications

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

Pathway- and disease-focused miRNA profiling
miRNome profiling
Biomarker discovery

Resources

Brochures & Guides (1)
QIAGEN provides solutions for the quantification of plant miRNAs with the miScript Plant qPCR System.
Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.

FAQ

Does the miScript Plant RT Kit contain controls?

Yes, the miScript Plant RT Kit contains control elements. The miScript Plant Ligation Control (miPLC) is used to assess ligation-mediated reverse-transcription performance. The miScript Plant Reverse Transcription Control (miPRC) is used to assess reverse-transcription performance.

FAQ ID - 3435
Are any of the miScript II RT Kit components interchangeable with the miScript Plant RT Kit components?

No, the components of the miScript II RT Kit are not interchangeable with the miScript Plant RT Kit components.

FAQ ID - 3436
Can the miScript Plant RT Kit be used to quantify piRNA expression?

Yes, the miScript Plant RT Kit can be used to quantify piRNA expression. The miScript Plant RT Kit represents an unbiased, universal cDNA synthesis kit for small RNAs with 3’-end, 2’-O-Me modifications.

FAQ ID - 3434

Can you describe the adaptor found in the 10x miScript Plant Adaptor?

The miScript Plant Adaptor is a 17 bp, pre-adenylated DNA oligonucleotide that is blocked on the 3’ end to prevent ligation side-products.

FAQ ID - 3442
What is the recommended RNA and RNA input range for the miScript Plant RT Kit?

Total RNA, containing miRNAs, should be used as the starting material for the miScript Plant RT Kit. The recommended RNA input is a maximum of 500 ng to a minimum of 50 pg.

FAQ ID - 3446
What is the amplicon size of PCR products generated with the miScript Plant qPCR System?
The PCR amplicon size is approximately 80 bp.
FAQ ID - 3443
Are miScript Primer Assay included in miScript miRNA PCR Arrays the same primers as the miScript Primer Assays available in individual tubes?

Yes, miScript Primer Assays included in miScript miRNA PCR Arrays are the same primers as the miScript Primer Assays available in individual tubes.

FAQ ID - 3441

 

What should I use to isolate total RNA from plant tissues and what is the maximum recommended amount of tissue input?
A supplementary miRNeasy Mini Kit protocol to isolate total RNA, including small RNAs, from plant tissues is available in Appendix A of the miScript qPCR System Handbook. The maximum recommended amount of plant tissue input is 50 mg.
FAQ ID - 3438
Are miScript Primer Assays available for plant mature miRNAs?
Yes, miScript Primer Assays are available for plant miRNAs at www.qiagen.com/GeneGlobe.
FAQ ID - 3439
Can cDNA synthesized with the miScript Plant RT Kit be used in real-time PCR with TaqMan® MicroRNA Assays?

No, cDNA synthesized with the miScript Plant RT Kit cannot be used in real-time PCR with TaqMan® MicroRNA Assays. Instead, cDNA synthesized with the miScript Plant RT Kit should be used with miScript Primer Assays or miScript miRNA PC Arrays.

See trademarks

FAQ ID - 3445
Can the miScript II RT Kit be used to quantify plant miRNAs?

No, the miScript II RT Kit 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.

FAQ ID - 3431
What is the composition of the 5x miScript Plant RT Buffer?

The composition of the 5x miScript Plant RT Buffer is proprietary.

FAQ ID - 3437
Are plant miScript Primer Assay designs wet-bench validated?

Any plant miScript Primer Assay included on a plant miScript miRNA PCR Array has been bench validated.

FAQ ID - 3440
Can the miScript II RT Kit be used to quantify piRNAs?

No, the miScript II RT Kit cannot be used to quantify piRNAs. 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 reverse transcription kits including the miScript II RT Kit.

FAQ ID - 3432
What is the dissociation curve temperature of PCR products generated with the miScript Plant qPCR system?

The dissociation curve temperature is generally 77-82°C.

FAQ ID - 3444
Can the miScript Plant RT Kit be used to quantify animal miRNAs?

No, the miScript Plant RT Kit should not be used to quantify animal miRNAs (human, mouse, rat, etc.). The miScript PCR System, including the miScript II RT Kit (218160, 218161), should be used for quantification of animal miRNAs.

FAQ ID - 3433