qiagen microrna seminar
DESCRIPTION
This presentation gives an overview on how new tools from QIAGEN facilitate miRNA research, from purification through to functional analysis.TRANSCRIPT
Sample & Assay Technologies
Facilitating miRNA research –
from purification to functional
analysis
The applications presented here are for research purposes. Not for use in diagnostic procedures.
Sample & Assay Technologies- 2 -
microRNAs - micromanagers of gene expression
Characteristic of miRNAs Naturally occurring, endogenous small RNA Regulate at least 1/3 of the protein encoding genes by binding to the mRNA > 1200 miRNAs in human (miRBase V15.0) A mature miRNA is approximately 20 - 22 nt long miRNA seed sequences (2-7 nt) are crucial in target selection Imperfectly base pair to the target and result in translational repression A given miRNA may have multiple target transcripts
Target mRNAs miRNA binding sites are typically at the 3’-UTR of the target transcripts Typically multiple miRNA binding sites are present for a given transcript
Ω
Seed region
5’ 3’
miRNA
Target mRNA
5’3’
Ω
miRNA
5’3’
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Biogenesis of microRNA
Pri-miRNA= Primary microRNAPre-miRNA = Precursor microRNA
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QIAGEN solutions for microRNA research
miRNeasy Kits1
miScript PCR System2
miScript RT2 miRNA PCR Arrays 3
miScript miRNA Mimics and Inhibitors4
miScript Target Protector5
Sample & Assay Technologies
Efficient purification of miRNA from
different starting materials
Tissue
Cells
Blood
Serum/Plasma
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miRNeasy - copurification or enrichment of miRNA
Flexible protocols: Copurification of miRNA & total
RNA miRNA enriched fraction
Formats:Single spin 96-well plate Manual or automated on
QIAcube
Principle: Proven silica membrane
technology
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miRNA isolation from different starting material
Human
Animal
Blood
Serum/plasma
Species Sample specification Kit
Cells
Tissue unfixed FFPE fixed sections fixed in PAXgene containers
miRNA miRNA + protein
Blood
Serum/plasma
Cells
Tissue unfixed FFPE fixed sections fixed in PAXgene containers
miRNA miRNA + protein
miRNeasy Protect Animal Blood Kit miRNeasy Mini Kit (UDP)
PAXgene Blood miRNA Kit
QIAamp Circulating Nucleic Acid Kit (SP)
miRNeasy Mini/96 Kit AllPrep® DNA/RNA/Protein Mini Kit*
miRNeasy Mini/96Kit miRNeasy FFPE Kit PAXgene Tissue miRNA Kit
miRNeasy Kit AllPrep® DNA/RNA/Protein Mini Kit*
Plant RNeasy Lipid Tissue Mini Kit* (UDP) RNeasy Mini Kit* (UDP)
miRNeasy Mini/96Kit miRNeasy FFPE Kit PAXgene Tissue miRNA Kit
*additionally needed RNeasy MinElute® Cleanup Kit; UDP = User Developped Protocol; SP = Supplementary Protocol
Virus depending on virus
Sample & Assay Technologies
Detection - The miScript PCR System
miRNA quantification by real-time PCR
mature microRNA
precursor microRNA
other small non-coding RNAs
mRNAs
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miScript PCR SystemAll from the same cDNA
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miScript PCR SystemReverse transcription - principle
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miScript RT Protocol: One-step reaction
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miScript PCR SystemReal-time PCR detection of mature miRNAs
mRNA miRNA
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miScript RT SystemLinear cDNA synthesis from 10 pg – 1µg RNA
1 µg to 10 pg of input RNA from HeLa S3 cells, used for miScript RT reaction in 20 µl volume. 2µl of this cDNA (100 ng to 1 pg) was used in PCR
2 cells
2x105 cells
Detection to <20 copies
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Specificity of miScript Primer Assays: Let-7 Isoforms
Human Let-7 Isoforms
Cross-reactivity
Sample & Assay Technologies
The miScript PCR System
miScript Control Assays
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miScript Control Assays
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miScript Control Assays: Constant level of expression across cell types
Constant level of expression across cell types
Similar small size as miRNA
A wide range of expression level to choose the most suitable control
Cross species
Sample & Assay Technologies
microRNA expression profiling
RT2 miRNA PCR Arrays
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RT2 miRNA PCR Array SystemmicroRNA profiling
RT2 miRNA First Strand Kit Preferentially reverse transcribe mature miRNA Built-in external RNA control One-step reaction
PCR Arrays: pathways and genomes Cancer Cell Differentiation & Development Immunopathology Inflammation miFinder Whole Genome (miRNome) – Sanger miRBase V14.0
Optimized RT2 SYBR® Green Mastermix
RT2 miRNA Data Analysis Excel Template & Web Portal
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cDNA synthesis 2 hours
Load plates 2 minutes
Run 40 cycle qPCR program 2 hours
Upload and analyze data 15 minutes
How RT2 miRNA PCR Arrays work
Compatible with many cyclers
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Anatomy of a miRNA PCR Array
Each plate contains:4 housekeeping snoRNA genes4 quality controls (2 reverse transcription & 2 PCR controls)Available for human, mouse, and rat
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Data Analysis
Web-based software No installation needed
From raw Ct values to fold change results Using ∆∆C(t) Method
Multiple analysis formats Scatter plot Volcano plot Multi-group plot Clustergram
Also available as downloadable Excel templates
Clustergram
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Volcano Plot
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Scatter Plot
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Cancer-specific miRNA upregulated in colon tumor.
Human Cancer miRNA RT2 Array: Colon Cancer
Are there specific miRNA expression differences in tumor versus normal adjacent tissue?
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Application Data: Cell Differentiation & Development
Human Cell Differentiation & Development RT2 miRNA PCR Array identifies 26 brain- and 20 muscle-specific miRNA potentially
regulating tissue-specific gene expression.
26 Brain-Specific miRNA
20 Muscle-Specific miRNA
Sample & Assay Technologies
microRNA functional analysis
miScript miRNA Mimics and Inhibitors
miScript Target Protectors (New)
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Tools to study miRNA functions
Synthetic RNA duplexMimics the effect of an endogenous miRNA
Single stranded modified RNATargets of miRNA should increase when miRNA is inhibited
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miScript Target ProtectorPrinciple
Target mRNA A
Target mRNA B
Target mRNA C
one and the same miRNA can bind to several targets no target is expressed ()
Inhibitor binds to miRNA each target is expressed ()
miScript Target Protector binds only to one specific target specific target is expressed while other targets are not expressed
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miScript Target Protector Protection of a luciferase reporter against endogenously expressed miR-16
Reliable, reproducible miRNA inhibition after transfection of miScript Target Protector miR-16
Reliable, reproducible miRNA inhibition after transfection of miScript Target Protector miR-16
Verification of miRNA binding site
RLU
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miScript Target Protector miR-9 causes down regulation of an important gene for cell viability
Determination of miRNA role in a pathway or process
miScript Target Protector miR-9 inhibits the down regulation by miR-9 allowing the cell viability gene to be expressed
miScript Target Protector miR-9 inhibits the down regulation by miR-9 allowing the cell viability gene to be expressed
Untreated cells Cell death caused by miR-9
Cell survival after treatment with TP
TP = Target Protector
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miScript Target ProtectorTarget Specificity of the Target Protector
Transfection of miR-1 mimic leads to down-regulation of different miR-1 targets including ADAR and HDAC4
After cotransfection of a target protector designed against the miR-1 MRE in ADAR, ADAR protein was no longer downregulated by a miR-1 mimic
HDAC4 downregulation was not affected by the presence of the ADAR target protector
55 kD
130 kD
ADAR
TUBB
HDAC4
TUBB
UT 1
Neg ctrl
0.10.5 0.05
ADAR Target Protector [µM]
UT1
Neg ctrl
0.10.5 0.05
ADAR Target Protector [µM]
miR-1 mimic was co-transfected in all samples except UT
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Typical workflowmiRNA research
A complete offering for miRNA research!A complete offering for miRNA research!
miScript Precursor Assay miScript PCR Starter KitRotor-Gene QQIAgility
PAXgene Tissue miRNA Kit
PAXgene Blood miRNA Kit
QIAamp Circulating Nucleic Acid Kit
miRNeasy Protect Animal Blood Kit
miScript PCR Control Set
QIAcube
miScript Target Protector
Mimic and Inhibitor Sets
miScript RT2 miRNA PCR Arrays
miScript PCR Control
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Thank you!