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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.

QIAGEN microRNA Seminar

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This presentation gives an overview on how new tools from QIAGEN facilitate miRNA research, from purification through to functional analysis.

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Page 1: QIAGEN microRNA Seminar

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.

Page 2: QIAGEN microRNA Seminar

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’

Page 3: QIAGEN microRNA Seminar

Sample & Assay Technologies- 3 -

Biogenesis of microRNA

Pri-miRNA= Primary microRNAPre-miRNA = Precursor microRNA

Page 4: QIAGEN microRNA Seminar

Sample & Assay Technologies- 4 -

QIAGEN solutions for microRNA research

miRNeasy Kits1

miScript PCR System2

miScript RT2 miRNA PCR Arrays 3

miScript miRNA Mimics and Inhibitors4

miScript Target Protector5

Page 5: QIAGEN microRNA Seminar

Sample & Assay Technologies

Efficient purification of miRNA from

different starting materials

Tissue

Cells

Blood

Serum/Plasma

Page 6: QIAGEN microRNA Seminar

Sample & Assay Technologies- 6 -

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

Page 7: QIAGEN microRNA Seminar

Sample & Assay Technologies- 7 -

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

Page 8: QIAGEN microRNA Seminar

Sample & Assay Technologies

Detection - The miScript PCR System

miRNA quantification by real-time PCR

mature microRNA

precursor microRNA

other small non-coding RNAs

mRNAs

Page 9: QIAGEN microRNA Seminar

Sample & Assay Technologies- 9 -

miScript PCR SystemAll from the same cDNA

Page 10: QIAGEN microRNA Seminar

Sample & Assay Technologies- 10 -

miScript PCR SystemReverse transcription - principle

Page 11: QIAGEN microRNA Seminar

Sample & Assay Technologies- 11 -

miScript RT Protocol: One-step reaction

Page 12: QIAGEN microRNA Seminar

Sample & Assay Technologies- 12 -

miScript PCR SystemReal-time PCR detection of mature miRNAs

mRNA miRNA

Page 13: QIAGEN microRNA Seminar

Sample & Assay Technologies- 13 -

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

Page 14: QIAGEN microRNA Seminar

Sample & Assay Technologies- 14 -

Specificity of miScript Primer Assays: Let-7 Isoforms

Human Let-7 Isoforms

Cross-reactivity

Page 15: QIAGEN microRNA Seminar

Sample & Assay Technologies

The miScript PCR System

miScript Control Assays

Page 16: QIAGEN microRNA Seminar

Sample & Assay Technologies- 16 -

miScript Control Assays

Page 17: QIAGEN microRNA Seminar

Sample & Assay Technologies- 17 -

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

Page 18: QIAGEN microRNA Seminar

Sample & Assay Technologies

microRNA expression profiling

RT2 miRNA PCR Arrays

Page 19: QIAGEN microRNA Seminar

Sample & Assay Technologies- 19 -

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

Page 20: QIAGEN microRNA Seminar

Sample & Assay Technologies- 20 -

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

Page 21: QIAGEN microRNA Seminar

Sample & Assay Technologies- 21 -

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

Page 22: QIAGEN microRNA Seminar

Sample & Assay Technologies- 22 -

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

Page 23: QIAGEN microRNA Seminar

Sample & Assay Technologies- 23 -

Volcano Plot

Page 24: QIAGEN microRNA Seminar

Sample & Assay Technologies- 24 -

Scatter Plot

Page 25: QIAGEN microRNA Seminar

Sample & Assay Technologies- 25 -

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?

Page 26: QIAGEN microRNA Seminar

Sample & Assay Technologies- 26 -

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

Page 27: QIAGEN microRNA Seminar

Sample & Assay Technologies

microRNA functional analysis

miScript miRNA Mimics and Inhibitors

miScript Target Protectors (New)

Page 28: QIAGEN microRNA Seminar

Sample & Assay Technologies- 28 -

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

Page 29: QIAGEN microRNA Seminar

Sample & Assay Technologies- 29 -

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

Page 30: QIAGEN microRNA Seminar

Sample & Assay Technologies- 30 -

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

Page 31: QIAGEN microRNA Seminar

Sample & Assay Technologies- 31 -

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

Page 32: QIAGEN microRNA Seminar

Sample & Assay Technologies- 32 -

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

Page 33: QIAGEN microRNA Seminar

Sample & Assay Technologies- 33 -

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

Page 34: QIAGEN microRNA Seminar

Sample & Assay Technologies- 34 -

Thank you!