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Techniques for RNA DetectionWhere Are We Headed?August 27, 2014

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Participating Experts

Martin Stoddart, Ph.D. AO Research Institute DavosDavos, Switzerland

Don WeldonEMD MilliporeTemecula, CA

Webinar Series

Sponsored by:

Techniques for RNA DetectionWhere Are We Headed?August 27, 2014

Dr. Martin Stoddart, Principal InvestigatorAO Research Institute Davos, Switzerland

Techniques for RNA Detection:Where Are We Headed?

Overview

• Description of most common RNA detection

methods

• Considerations regarding their use

• Data from bone tissue engineering

• Refinement of the data obtained

• New avenues and future direction

Northern Blot

Sample

RNA Isolation

2D Gel Electrophoresis

RNA sizeseparation

TransferGel Membrane

Fix RNA

Incubate membrane with probe

Develop

End Point PCR

http://en.wikipedia.org/wiki/Polymerase_chain_reaction

Real Time PCR

Cell Lysis

Reverse Transcription

Polymerase Reaction

2∆∆CtNormalised Gene Expression

Global v CellG

radi

ent

Bin

ary

Increasing Expression Increasing Expression

Incr

easi

ng S

igna

l

In Situ hybridization

Bone tissue engineering

Early osteogenic markers

Alkaline phosphatase (ALP)Runx2

Late osteogenic markers

Alizarin red stainingCalcium45 incorporation

Runx2 - mRNA

human MSCs

early screening method

Day 28

Transcription Factors

• Include Master Genes such as Runx2 (osteoblast) and Sox9 (chondrocyte)

• Bind short, specific sequences in DNA promoters activating gene transcription

• Results in global regulation of many genes

• Provide an indication of cell fate - Early

AAAAA

Runx2P

Osteogenic differentiation

Stimulate with osteogenic mediumContaining dexamethasone

Human mRNA Analysis- Real time PCR

Day 0 Day 1 Day 3 Day 7 Day 14

Other factors?

Where Are We Headed? - 1) mRNA Ratios

Runx2/Sox9 mRNA ratio is correlated toCalcium45 incorporation

n = 12

Loebel C, et al. Tissue Engineering Part A, 2014, Aug 4

Sox9 mRNA silencing can enhanceosteogenesis in vitro

DEX

DEX + Sox9 siRNA

Calcium45 incorporation (day 28) Alizarin red staining (day 28)

n = 6

Loebel C, et al. Tissue Engineering Part A, 2014, Aug 4

Runx2

Runx2

Runx2 Runx2 Runx2

Hypertrophic chondrocyte

Resting Articular chondrocyte

ImmatureChondroblast

Mesenchymal Stem Cell

Preosteoblast ImmatureOsteoblast

MatureOsteoblast Osteocyte

Sox 5, 6, 9

Col X

Col IBSPOPNOCN

OSX

Mesenchymal stromal cell potential

Global v CellG

radi

ent

Bin

ary

Increasing Expression Increasing Expression

Where Are We Headed? 2) Non-destructive – individual cell

20

gold-quencheddye

capture strandTarget mRNA

Cell sortingbased on single probe

Cell sorting based on SMART-FLARE (Sox 9)

Sox9 P3FL= P9 Sox9 mfi low mfi 2,414 purity 95%

Sox9 p4FR= P10 Sox9 mfihigh mfi 7,102 purity 76%

1

4.771698659

0

1

2

3

4

5

6

Sox9 P3 Sox9 P4

RT-PCR

SmartFlare probes can be used for live cell sorting

Experimental Control ProbesAll Good Experiments Include the Proper Controls

Uptake Scramble Housekeeping

CONFIDENTIAL

0 Seed Primary hMSCs

UPTAKE-control 4µl per 200µl

FACS (UPTAKE)Add osteogenic and control medium, respectively

1

2

3

UPTAKE, Sox9 and scrambled Sox9 4µl per 200µl

4

5

6 FACS (UPTAKE, Sox9, scrambled Sox9)

Day

Methodology – Down-regulated gene

FACS (UPTAKE)

UPTAKE-control 4µl per 200µl

Growth medium Differentiation medium

mfi valuecontrol neg -uptake Cy5 1883 *scramble Cy5 967Sox9 Cy5 1588

mfi valuecontrol neg -uptake Cy5 1883 *scramble Cy5 892Sox9 Cy5 932

Sox9 Down-regulation

Sox 9+RunX2

Cell sorting based on double probes

RT-PCR

1

0.336

0.186

0

0.2

0.4

0.6

0.8

1

1.2

P3 Run X2 P4 RunX2 P5 RunX2

RunX21

0.7370.523

0

0.2

0.4

0.6

0.8

1

1.2

P3 Sox9 P4 Sox9 P5 Sox9

Sox9

Varying Differentiation states

1

0.390.51

0

0.2

0.4

0.6

0.8

1

1.2

P3 ALP P4 ALP P5 ALP

ALP

1

1.45

2.47

0

0.5

1

1.5

2

2.5

3

P3 Collagen I P4 Collagen I P5 Collagen I

Collagen I

10.93 0.88

0

0.2

0.4

0.6

0.8

1

1.2

P3 hOC P4 hOC P5 hOC

hOC (Osteocalcin)

Bone tissue engineering

early osteogenic markers

Alkaline phosphatase (ALP)Runx2

Runx2/Sox9 ratio

human MSCs

early screening method

Day 28

Late osteogenic markers

Alizarin red stainingCalcium45 incorporation

Acknowledgements

Musculoskeletal Regeneration Group AO Research Institute Davos

Bojun Li Claudia Loebel

Ursula MenzelFACS

This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no [NMP.2010.2.3-1]

Servier Medical Art- Creative Commons Licence (Slide Images)

Prof. Mauro Alini

Innovationsstiftung

Brought to you by the Science/AAAS Custom Publishing Office

Participating Experts

Webinar Series

Sponsored by:

Techniques for RNA DetectionWhere Are We Headed?August 27, 2014

Martin Stoddart, Ph.D. AO Research Institute DavosDavos, Switzerland

Don WeldonEMD MilliporeTemecula, CA

Techniques for RNA Detection: Where Are We Headed?

Don WeldonLead Application Scientist EMD Millipore

Scientists realized early on the best way to understand a biological organism is to observe it in its natural environment.

vs..

Observe with minimal /no disturbance

Identify individualsof interest

Leave with minimal /no disturbance

Ultimately we want to:

When Studying RNA:Let’s take the approach of biologists…not archeologists

Validation of Probes

150 (kDa)

250 (kDa)

blot: EGFR(~180kDa)

Normalized signal intensity = Blot/CBB

Unf

lare

d

Scra

mbl

e SF EG

FR

SF

No Changein Cell Viability

No Impacton Gene Expression

No Changein Protein Expression

150 (kDa)

250 (kDa)

2.07 2.07 2.12

Genome Wide Array ScreenViaCount™ Viability Assay

WorkflowWorkflow is a simple addition to Cell Culture

1. Reconstitute & Dilute

2. Add to cells in culture Allow to incubate overnight

3. Detect RNA levels via microscopy or Flow

Time Lapse MicroscopyDetection of ERBB2 in low and high expressing cells

Time Lapse MicroscopyDetection of ERBB2 in low and high expressing cells

Example of Live Cell RNA Detection

Live cell sorting of immune cells based on RNA

c-Myc Levels Decrease During MonocyteDifferentiation

J. Auwerx, Experientia 47 (1991)

Background:• c-Myc is a transcription factor • Downstream target genes play a role in cellular proliferation• Many cancers are known to show constitutive c-Myc expression

THP-1 Monocytes(high MYC) Undifferentiated THP-1

(high MYC)

Sorted cells returned to culture for

4 days to complete differentiation and further

functional characterization

THP-1 Monocytes(high MYC)

Enrich withFACS

after 1 day

Differentiate to Macrophages

with PMA

Add MYCSmartFlare™

Probe

Pre-Macrophages(decreased MYC)

Detecting RNA Levels During DifferentiationEnrichment Based on Decreased Expression

Enter Without Disturbing the Cell

Identify the IndividualCells of Interest

Leave Without Disturbing the Cell

Cell-based Phenotype: Bacterial killing assay

High Low

Characterization of Protein ProductsMilliplex cytokine assay post- stimulation (LPS)

Undifferentiated THP-1(high MYC)

Pre-Macrophages(decreased MYC)

FunctionalAssays

Detecting Differences in RNA ExpressionMultiplex protein and cell-based phenotype assays confirm function

0.

150.

300.

450.

600.

750.

Day 0 Day 5

MYC

mR

NA

Sm

artF

lare

MFI

MYC mRNA detection by SmartFlare™ Probe

0.

0.25

0.5

0.75

1.

1.25

Day 0 Day 5

Rel

ativ

e M

YC m

RN

A

MYC mRNA detection by qRT-PCR

Correlation of MYC RNA detection by SmartFlare™ Probe and qRT-PCR

MYC levels decrease during macrophage differentiation

RT-PCR and Live Cell RNA DetectionMYC mRNA expression decreases during monocyte differentiation

Undifferentiated THP-1 MonocytesTHP-1 Monocytes PMA 2 daysMonocytes PMA 5 days (macrophages)

MYC levels decrease during macrophage differentiation

Monitoring mRNA Expression ChangesMorphology and Expression

00.5

11.5

22.5

3

0 1 2 3 4 5 6 7 8

TNFa

(ng/

ml)

Stimulation (hours)

TNFalpha secretion over time in LPS-stimulated sort products

MYC highMYC low

Con

cent

ratio

n (p

g/m

l)

MYC mRNA sort products show distinct cytokine profiles

Cytokines secretion kinetics in response to LPS stimulation in MYC sort products

Stimulation (hours)

0

1

2

3

4

5

6

7

Con

cent

ratio

n (n

g/m

l) MYC high

MYC low

Detection of Distinct Cytokine ProfilesProfiles and Response to Stimulation are Unique to Cell Type

Multiplex

Live Cell Time-Lapse Imaging MovieRNA detection during a functional assay

Live Cell Time-Lapse Imaging MovieRNA detection during a functional assay

Summary

3

Native RNA was detected in live cells during differentiation

Protocol was simplified with direct addition to cell culture

Cells were enriched based on their RNA content through FACS

Additional functional assays were performed on the sort products

Starter Kit

Uptake

• Use as an initial test to determine the cell’s ability to endocytoseSmartFlare™ probes.

Scramble

• Use to determine the level of background fluorescence inside the cell and compare to target fluorescence.

Housekeeping

• Housekeeping genes can be used as positive controls to confirm the system is working in your culture conditions.

All good experiments utilize the proper controls

Visit www.emdmillipore.com/smartflare to see more technical information and application notes and to browse our list of SmartFlare™ probes available.

AcknowlegementsThe Application Development Team who Performed the Experiments:Yuko WilliamsAlex KoHaizhen Liu

EMD Millipore

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Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office

Webinar Series

Sponsored by:

Techniques for RNA DetectionWhere Are We Headed?August 27, 2014

Martin Stoddart, Ph.D. AO Research Institute DavosDavos, Switzerland

Don WeldonEMD MilliporeTemecula, CA

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Techniques for RNA DetectionWhere Are We Headed?August 27, 2014