For SYBR® Green-based, real-time PCR profiling of miRNAs using the miScript PCR System
Sensitive and specific mature miRNA expression profiling
PCR arrays for pathway, disease, or miRNome profiling
Free online data analysis tools
miScript miRNA PCR Arrays are mature miRNA-specific forward primers (miScript Primer Assays) that have been arrayed in biologically relevant pathway-focused, disease-focused, or whole miRNome panels. miScript miRNA PCR Arrays are part of the miScript PCR System for miRNA detection and quantification. cDNA is prepared using the miScript II RT Kit with miScript HiSpec Buffer and is used as a template in real-time PCR with a miScript miRNA PCR Array and the miScript SYBR Green PCR Kit.
Pathway-Focused or miRNome miScript miRNA PCR Array layout for plate formats A, C, D, F, M.
Wells A1 to G12 (1–84) each contain a miScript Primer Assay for a pathway/disease/functionally related mature miRNA. Wells H1 and H2 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells H3 to H8 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells H9 and H10 contain replicate reverse transcription controls (miRTC). Wells H11 and H12 contain replicate positive PCR controls (PPC).
miRNome miScript miRNA PCR Array and miScript miRNA HC PCR Array layout for plate formats E, G.
Wells A1 to P12 (1–372) each contain a miScript Primer Assay for a pathway/disease/ functionally related mature miRNA. Wells P13 and P14 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells P15 to P20 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells P21 and P22 contain replicate miRTC miScript Primer Assays (miRTC). Wells P23 and P24 contain replicate positive PCR controls (PPC).
Pathway-Focused miScript miRNA PCR Array layout for plate formats E, G.
Pathway-Focused miScript miRNA PCR Arrays in formats E and G include 4 replicates of the same assays as provided in the 96-well formats A, C, D, and F.
miScript miRNA PCR Array layout for Rotor-Disc format R.
Wells 1 to 84 each contain a miScript Primer Assay for a pathway/disease/functionally related gene. Wells 85 and 86 contain replicate C. elegans miR-39 miScript Primer Assays that can be used as an alternative normalizer for array data (Ce). Wells 87 to 92 each contain an assay for a different snoRNA/snRNA that can be used as a normalization control for the array data (SN1=SNORD61 assay, SN2=SNORD68 assay, SN3=SNORD72 assay, SN4=SNORD95 assay, SN5=SNORD96A assay, SN6=RNU6B/RNU6-2 assay). Wells 93 and 94 contain replicate miRTC miScript Primer Assays (miRTC). Wells 95 and 96 contain replicate positive PCR controls (PPC). Wells 97–100 are empty.
miScript miRNA QC PCR Array layout for plate formats A, C, D, F.
Wells 1 and 2 of each row contain replicate C. elegans miR-39 miScript Primer Assays (39). Well 3 of each row contains a miR-16 miScript Primer Assay (16). Well 4 of each row contains a miR-21 miScript Primer Assay (21). Well 5 of each row contains a miR-191 miScript Primer Assay (191). Wells 6 to 8 of each row each contain an assay for a different snoRNA (61 = SNORD61 assay, 95 = SNORD95 assay, 96A = SNORD96A assay). Wells 9 and 10 of each row contain replicate miRTC miScript Primer Assays (miRTC). Wells 11 and 12 of each row contain replicate positive PCR controls (PPC). These formats enable the quality assessment of up to 8 cDNA samples.
miScript miRNA QC PCR Array layout for Rotor-Disc format R.
Wells 1 and 2 contain replicate C. elegans miR-39 miScript Primer Assays. Well 3 contains a miR-16 miScript Primer Assay. Well 4 contains a miR-21 miScript Primer Assay. Well 5 contains a miR-191 miScript Primer Assay. Wells 6 to 8 each contain an assay for a different snoRNA (Well 6 = SNORD61 assay, Well 7 = SNORD95 assay, Well 8 = SNORD96A assay). Wells 9 and 10 contain replicate miRTC miScript Primer Assays (miRTC). Wells 11 and 12 contain replicate positive PCR controls (PPC). This pattern is repeated 7 additional times from wells 13 to 96. Wells 97 to 100 are empty. This format enables the quality assessment of up to 8 cDNA samples.
Whole miRNome profiling.
Total RNA was isolated from HCT 116 colorectal cancer cells that had been treated with ± 5-aza-2'-deoxycytidine (5-aza-2'-dC) demethylation reagent. This reagent irreversibly inhibits DNA methyltransferase driven methylation reactions by incorporating into DNA and covalently binding to the active site of the DNA methyltransferase. cDNA was prepared with miScript HiSpec Buffer using the miScript II RT Kit. The Human miRNome miScript miRNA PCR Array, in combination with the miScript SYBR® Green PCR Kit, was used to profile mature miRNA expression by real-time PCR. Data analysis was performed using the online miScript miRNA PCR Array Data Analysis Tool. [A] A scatter plot of 2-ΔCT values demonstrates that significant differences exist in the mature miRNA expression levels of the 2 samples. [B] A volcano plot shows regulated miRNAs (±4-fold is used as a cutoff and indicated by red lines). 104 miRNAs were strongly upregulated and 30 were strongly downregulated in 5-aza-2'-dC treated HCT 116 cells. When a p value of 0.05 is applied (indicated by blue line), the upregulation of 89 of the 104 miRNAs is significant, and the downregulation of 21 of the 30 miRNAs is significant.
Analysis of expression of circulating miRNAs in serum or plasma holds huge potential as a source of new biomarkers.
Scatter plots show the relative miRNA expression levels from pooled RNA from healthy and colon cancer serum samples, characterized using miScript miRNA PCR Arrays.
Performance
Discover biomarkers in serum or plasma samples
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 most exciting areas of current miRNA research involves the assessment of miRNAs present in serum or plasma samples. The discovery of relatively stable, extracellular miRNAs in serum and plasma has generated huge interest in the use of changes in these miRNA levels as potential noninvasive biomarkers for a variety of diseases. As a result, the Serum & Plasma miScript miRNA PCR Array has been developed. This array enables rapid profiling of the 84 most relevant miRNAs associated with serum and plasma (see figure "Analysis of expression of circulating miRNAs in serum or plasma").
Profile entire miRNomes with a single cDNA sample
In addition to pathway-focused arrays, up-to-date miRNome miScript miRNA PCR Arrays are available for a variety of species including human, mouse, rat, dog, and rhesus macaque. With less than 1 µg RNA, the entire human miRNome can be rapidly profiled (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 PCR System enables sensitive, specific miRNA quantification and profiling using SYBR Green 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.
Mature miRNA profiling with the miScript PCR System uses the following components:
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 — 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 PCR Arrays consist of mature miRNA-specific forward primers (miScript Primer Assays) arrayed in biologically relevant pathway- or disease-focused panels or miRNome panels.
Procedure
miRNA expression profiling with miScript miRNA PCR Arrays is straightforward and robust. First, prepare cDNA buffered with miScript HiSpec Buffer using the miScript II RT Kit. Second, add a premix of cDNA, miScript Universal Primer, QuantiTect 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.
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:
