10/31/2017

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The world leader in serving science

Dee Duangdao, Ph.D.

Real-Time PCR Field Applications Scientist

Overview of the QuantStudioTM 3 & 5 Real-Time PCR System

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Touchscreen (stand-

alone capabilities,

PIN-protected user

accounts, and dye

calibration/RNaseP

functionality)

USB port for

template upload

and data download

Motorized

block drawer

(controlled by

touchscreen)

USB ports

WiFi adapter

port (optional use)

Ethernet port :

RJ45

(10/100Mbps)

RS232 port (Service

only)

Power port: 100/240

VAC

Fuse cover

QuantStudio™ 3 & 5 Real-Time PCR Systems: The Basics

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CMOS

White LED

Excitation

filter wheel

mirror

lens

Emission

filter wheel

384 well plate

excitation emission

QuantStudio™ 5 Real-Time PCR Systems: The Basics

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Technical Specifications

QuantStudio™ 3 (96 well block 0.1ML)

QuantStudio™ 5 (384-well block)

Block configurations 10-30 ml

5-20 ml

Run time <35 minutes (with fast mastermix) <35 minutes

Excitation source Bright white LED Bright white LED

Optical Detection 4 coupled filters 5 coupled filters

21 CFR p11

enablement

No Yes

Detection Sensitivity 10 log dynamic range sensitivity

1 copy

1.5 fold differences in target

quantities

10 log dynamic range sensitivity

1 copy

1.5 fold differences in target

quantities

On-Board Memory 10GB (2,000 - 6,000 run files) 10GB (2,000 - 6,000 run files)

Note: Data file is always saved to the instrument!

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5

A Flexible Optical Technology : OptiFlex™ System

Channel Dye

Examples Excitation

Filter Emission

Filter QuantStudio™ 3

(96-well) QuantStudio™ 5

(384-well)

1 FAM™, SYBR®, SYTO®9, Fluorescein, PTS Dye 470 ± 15nm 520 ± 15nm ✔ ✔

2 VIC®, JOE™, TET™,

HEX™ 520 ± 10nm 558 ± 12nm ✔ ✔

3 TAMRA™, NED™,,

ABY®, BODIPY® TMR-X 550 ± 10nm 586 ± 10nm ✔ ✔

4 ROX™, JUN®, Texas

Red® 580 ± 10nm 623 ± 14nm ✔ ✔

5 LIZ™, Cy®5, Mustang

Purple® 640 ± 10nm 682 ± 14nm ✔

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60oC 65oC

60oC 61oC 62oC 63oC 64oC 65oC

Gradient

• Unclear temperature setting in

each well

• Lack of flexibility, limited by

gradient high/low temps

VeriFlex™ Blocks • Precise temperature control in each

zone

• Can program at will, including

multiple zones with same temp

• Great for optimization or running

multiple assays at the same time

What is VeriFlex™ Block?

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QuantStudio™ 3 Real-Time PCR Systems: The Basics

• VeriFlexTM Blocks

• 3 programmable zones

• Independent temperature control in each zone (more precise than gradient)

• Can program at will, including multiple zones with same temp

• Great for optimization and also running multiple assays at the same time

60°C 61°C

62°C

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Veriflex™ Blocks: Temperature Optimization

TCF3 Assay

4 replicates for each temperature Data courtesy of Verification and Validation

60oC 63oC 65oC

• Power SYBR™ Green Master Mix, with

primers designed using Primer Express™

Software

• Secondary peak appears at 90°C when

annealing temperature goes above 63°C

60oC 63oC 65oC

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Interactive Touch Screen

Open/Close

door

Help

Settings:

-

Calibrations

- Runs

- Logs

- Ship Prep

Sign In or

Create a

Local User

Account

Connection

type

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Use touchscreen to edit reagent info, destination, and plate setup

2D reagent

barcodes

supported for

Applied

Biosystems

reagents!

Data destination:

None- file is saved

to computer by

default

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Edit run protocol

Full method editing capabilities on the touch screen,

including VeriFlex, Pause, and Melt

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Monitor Progress During the Run

Live Amplification Curves Thermal Protocol Status Time Remaining

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Review amp plots in real time

Review well details and

select amplification plots to

view by Target, Sample,

Task, or Master Mix

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New Feature: Run Pause

Program a Pause into the run:

define which step and at what

temperature to pause

Or pause a run on the fly

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Enhanced Instrument Touchscreen

• Power Failure Mode

• On-going run resumed automatically within 30’

• On screen notifications, Run log & instrument log

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10-Log Dynamic Range Sensitivity

Data courtesy of System Verification and Validation

10/31/2017

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1.5-Fold Discrimination: TaqMan™ Fast Advanced Master Mix

Quantity CT Std. Dev.

1000 27.9 0.063

1500 27.45 0.059

3000 26.40 0.060

4500 25.80 0.047

6667 25.20 0.049

10000 24.50 0.041

• KAZ plasmid amplified with PE2

TaqMan assay

• Standard Fast run conditions

• TaqMan Fast Advanced Master Mix

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Offers Superb Uniformity in Less Than 40 Minutes

Data courtesy of System Verification and Validation

Duplex reaction

Target Dye CT Std. Dev. (CT)

18S VIC® 12.85 0.086

TGFß FAM™ 25.067 0.068

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Complete Multiplexing Solution

Data courtesy of System Verification and Validation

TaqMan MultiPlex Master Mix and built-in dye

Targets Dye Ave CT Std. Dev. (CT)

FZD1 FAM™ 27.72 0.051

APOE VIC® 24.47 0.071

CD44 ABY® 25.58 0.101

GAPDH JUN® 17.61 0.106

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Consistent Uniformity from One Zone to Full Block

Temperature

60°C CT

CT

Std. Dev. Tm

Tm

Std. Dev. Replicates

Zone 2 25.687 0.074 84.117 0.035 16

Whole block 25.737 0.077 84.169 0.071 98

RNaseP from hgDNA

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QuantStudio 5 Verification & Validation data: SYBR™ Green

QuantStudio 5

Ct Mean Ct SD Tm Mean Tm SD

25.737 0.077 84.169 0.071

A

C

E

G

25.5

26

26.5

27

27.5

1 2 3 4 5 6 7 8 9 10 11 12

Ct

QuantStudio 5 96-Well 0.2ml SYBR Green Ct

27-27.5

26.5-27

26-26.5

25.5-26

A

C

E

G

83

83.5

84

84.5

85

85.5

1 2 3 4 5 6 7 8 9 10 11 12

Tem

p o

C

QuantStudio 5 96-Well 0.2ml SYBR Green Tm

85-85.5

84.5-85

84-84.5

83.5-84

83-83.5

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The world leader in serving science

QuantStudio™ Design and Analysis Software

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Experiment Setup

• Create your experiment or start it from a template

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Experiment Properties

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Advanced Settings

• Veriflex™

• AutoDelta

Programmable

Pause

Run Method

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Optional passive reference

Import and Save to

Library

Define Samples and Targets

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Select wells and type names

Assign plate information

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Standard Curve: Define and Setup Standards Wizard

Tools> Preferences> Defaults

Set your decimals

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Start Run from touchscreen or desktop

Save your experiment as template

Start your run

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Analysis Settings: Defaults

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Analysis Settings: Changing threshold and baseline for single target

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Analysis Settings: Changing thresholds and baselines for all targets

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Export Settings

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Where can I find technical help?

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Instrument and Application Support Centers www.thermofisher.com/qpcrsupport

12 Applications:

• Real-Time PCR Basics Support

• Gene Expression Support

• SNP Genotyping Support

• miRNA and pri-miRNA Support

• Copy Number Variation (CNV) Support

• Mutation Detection Support

• High Resolution Melt (HRM) Support

• Digital PCR Support

• Protein Thermal Shift Support

• Pharmacogenomics Support

• TaqMan Protein Assay Support

• TaqMan Primers and Probes Support

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Training Services

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LabCoat Live™ - A uniquely interactive virtual training program

For more information, visit www.thermofisher.com/labcoatlive

Course fee: $300

Includes reagents needed to perform protocol.

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Support Hotlines

Technical support: 800.955.6288 option 4, option 1, option 2

techsupport@thermofisher.com

Instrument support: 800.955.6288 option 3, option 1

instrumentservices@thermofisher.com

The world leader in serving science

QUESTIONS?

Note: For Research Use Only. Not for use in diagnostic procedures. 48

For Research Use Only. Not for use in diagnostic procedures.

© 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the

property of Thermo Fisher Scientific and its subsidiaries unless otherwise

specified.

TaqMan is a registered trademark of Roche Molecular Systems, Inc., and is used

with permission and under license.