Touchscreen (standalone capabilities, PIN-protected user accounts, and dye calibration/rnasep functionality)

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QuantStudio 3 & 5 Real-Time PCR Systems: The Basics Touchscreen (standalone capabilities, PIN-protected user accounts, and dye calibration/rnasep functionality) USB ports Overview of the QuantStudio

e Duangdao, Ph.D. Real-Time PCR Field Applications Scientist USB port for template upload and data download Motorized block drawer (controlled by touchscreen) RS232 port (Service only) WiFi adapter

1 QuantStudio 3 & 5 Real-Time PCR Systems: The Basics Touchscreen (standalone capabilities, PIN-protected user accounts, and dye calibration/rnasep functionality) USB ports Overview of the QuantStudio TM 3 & 5 Real-Time PCR System Dee Duangdao, Ph.D. Real-Time PCR Field Applications Scientist USB port for template upload and data download Motorized block drawer (controlled by touchscreen) RS232 port (Service only) WiFi adapter port (optional use) Ethernet port : RJ45 (10/100Mbps) Fuse cover Power port: 100/240 VAC The world leader in serving science 2 QuantStudio 5 Real-Time PCR Systems: The Basics White LED Technical Specifications Block configurations ml QuantStudio 3 (96 well block 0.1ML) QuantStudio 5 (384-well block) 5-20 ml Excitation filter wheel 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 mirror 21 CFR p11 enablement No Yes excitation lens emission Emission filter wheel CMOS 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) 384 well plate Note: Data file is always saved to the instrument! 3 4 1

A Flexible Optical Technology : OptiFlex System What is VeriFlex Block?

high/low temps 1 2 3 4 5 FAM, SYBR, SYTO 9, Fluorescein, PTS Dye 470 ± 15nm 520 ± 15nm VIC, JOE, TET, HEX 520 ± 10nm 558 ± 12nm TAMRA, NED,, ABY, BODIPY TMR-X 550 ± 10nm 586 ± 10nm ROX, JUN, Texas

for optimization or running multiple assays at the same time 60 o C 65 o C 60 o C 61 o C 62 o C 63 o C 64 o C 65 o C 5 6 QuantStudio 3 Real-Time PCR Systems: The Basics VeriFlex TM Blocks 3

2 A Flexible Optical Technology : OptiFlex System What is VeriFlex Block? Channel Dye Examples Excitation Filter Emission Filter QuantStudio 3 (96-well) QuantStudio 5 (384-well) Gradient Unclear temperature setting in each well Lack of flexibility, limited by gradient high/low temps FAM, SYBR, SYTO 9, Fluorescein, PTS Dye 470 ± 15nm 520 ± 15nm VIC, JOE, TET, HEX 520 ± 10nm 558 ± 12nm TAMRA, NED,, ABY, BODIPY TMR-X 550 ± 10nm 586 ± 10nm ROX, JUN, Texas Red 580 ± 10nm 623 ± 14nm LIZ, Cy 5, Mustang Purple 640 ± 10nm 682 ± 14nm 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 60 o C 65 o C 60 o C 61 o C 62 o C 63 o C 64 o C 65 o C 5 6 QuantStudio 3 Real-Time PCR Systems: The Basics VeriFlex TM 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 Veriflex Blocks: Temperature Optimization 60 o C 63 o C 65 o C 60 o C 63 o C 65 o C 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 60 C 61 C 62 C Data courtesy of Verification and Validation TCF3 Assay 4 replicates for each temperature 7 8 2

3 Interactive Touch Screen Use touchscreen to edit reagent info, destination, and plate setup Open/Close door Help Sign In or Create a Local User Account Connection type Settings: - Calibrations - Runs - Logs - Ship Prep 2D reagent barcodes supported for Applied Biosystems reagents! Data destination: None- file is saved to computer by default 9 10 Edit run protocol Monitor Progress During the Run Time Remaining Thermal Protocol Status Live Amplification Curves Full method editing capabilities on the touch screen, including VeriFlex, Pause, and Melt

4 Review amp plots in real time New Feature: Run Pause Review well details and select amplification plots to view by Target, Sample, Task, or Master Mix Program a Pause into the run: define which step and at what temperature to pause Or pause a run on the fly Enhanced Instrument Touchscreen 10-Log Dynamic Range Sensitivity Power Failure Mode On-going run resumed automatically within 30 On screen notifications, Run log & instrument log 16 Data courtesy of System Verification and Validation 22 4

1.5-Fold Discrimination: TaqMan Fast Advanced Master Mix KAZ plasmid amplified with PE2 TaqMan assay Offers Superb

Quantity C T 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.

068 Data courtesy of System Verification and Validation 23 24 Complete Multiplexing Solution TaqMan MultiPlex Master

(C T ) FZD1 FAM 27.72 0.051 APOE VIC 24.47 0.071 CD44 ABY 25.58 0.101 GAPDH JUN 17.61 0.

5 1.5-Fold Discrimination: TaqMan Fast Advanced Master Mix KAZ plasmid amplified with PE2 TaqMan assay Offers Superb Uniformity in Less Than 40 Minutes Duplex reaction Standard Fast run conditions TaqMan Fast Advanced Master Mix Quantity C T Std. Dev Target Dye C T Std. Dev. (C T ) 18S VIC TGFß FAM Data courtesy of System Verification and Validation Complete Multiplexing Solution TaqMan MultiPlex Master Mix and built-in dye Consistent Uniformity from One Zone to Full Block RNaseP from hgdna Targets Dye Ave C T Std. Dev. (C T ) FZD1 FAM APOE VIC CD44 ABY GAPDH JUN Temperature 60 C C T C T Std. Dev. Tm Tm Std. Dev. Replicates Zone Whole block Data courtesy of System Verification and Validation

Temp oc Ct 10/31/2017 QuantStudio 5 Verification &

Mean Tm SD 25.737 0.077 84.169 0.071 27.

5 E 1 2 3 C 4 5 6 7 8 9 A 10 11 12 27-27.5 26.5-27 26-26.

QuantStudio 5 96-Well 0.2ml SYBR Green Tm 85.5 85 84.

5 E 27 1 2 3 4 5 6 7 8 9 C 10 A 11 12 28 The world leader

6 Temp oc Ct 10/31/2017 QuantStudio 5 Verification & Validation data: SYBR Green QuantStudio 5 Ct Mean Ct SD Tm Mean Tm SD QuantStudio 5 96-Well 0.2ml SYBR Green Ct G 25.5 E C A QuantStudio Design and Analysis Software QuantStudio 5 96-Well 0.2ml SYBR Green Tm G E C 10 A The world leader in serving science Experiment Setup Experiment Properties Create your experiment or start it from a template

7 Run Method Define Samples and Targets Programmable Pause Advanced Settings Veriflex AutoDelta Import and Save to Library Optional passive reference Assign plate information Standard Curve: Define and Setup Standards Wizard Tools> Preferences> Defaults Select wells and type names Set your decimals

8 Start your run Analysis Settings: Defaults Save your experiment as template Start Run from touchscreen or desktop Analysis Settings: Changing threshold and baseline for single target Analysis Settings: Changing thresholds and baselines for all targets

Export Settings Where can I find technical help?

Gene Expression Support SNP Genotyping Support mirna and

Detection Support High Resolution Melt (HRM) Support Digital PCR

9 Export Settings Where can I find technical help? Instrument and Application Support Centers 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 Training Services

LabCoat Live - A uniquely interactive virtual training program Support Hotlines Technical support: 800.955.6288 option 4, option 1, option 2 techsupport@thermofisher.

Not for use in diagnostic procedures. 2017 Thermo Fisher Scientific Inc. All rights reserved.

10 LabCoat Live - A uniquely interactive virtual training program Support Hotlines Technical support: option 4, option 1, option 2 techsupport@thermofisher.com Course fee: $300 Includes reagents needed to perform protocol. Instrument support: option 3, option 1 instrumentservices@thermofisher.com For more information, visit For Research Use Only. Not for use in diagnostic procedures 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. QUESTIONS? Note: For Research Use Only. Not for use in diagnostic procedures. The world leader in serving science 48 10

QuantStudio 3 and QuantStudio 5 Real-Time PCR Systems. Connect to your data anytime, anywhere

QuantStudio 3 and QuantStudio 5 Real-Time PCR Systems Connect to your data anytime, anywhere Connect with your future The modern laboratory is undergoing a technological revolution. Today s scientific