Performance of Applied Biosystems™ consumables on Bio-Rad CFX™ Real-Time PCR Detection System February 15, 2017.

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Performance of Applied Biosystems™ consumables on Bio-Rad CFX™ Real-Time PCR Detection System February 15, 2017

Applied Biosystems™ Consumables on Bio-Rad CFX™ Demonstrate performance of Applied Biosystems consumables on the Bio- Rad CFX (96 & 384 well) instruments. TaqMan® Fast Advanced Master Mix with TaqMan® Gene Expression Assays Singleplex vs Duplex Performance Reproducibility Dynamic Range Power Up™ SYBR™ Green Master Mix Specificity

Materials TaqMan® Fast Advanced master mix Assays: 18S Hs99999901_s1 VIC-Primer limited GPR6 Hs00920605_s1 FAM PPIA Hs99999904_m1 FAM YWHAZ Hs03044281_g1 FAM cDNA: UHR PowerUp™ SYBR™ Green Master Mix Targets (all primers run at final concentration of 300nM) A2M APOA1 ABCC2 ARL1 ADAM10 C11orf30 AOF1 PGK1 cDNA: Liver All reactions run in quadruplicates CFX96 – 20ul rxns CFX384 – 10ul rxns

FAMM protocol on Bio-Rad CFX96™ or CFX384™ Protocol run with FAMM is boxed in black. No changes need to be made to run Applied Biosystems™ master mix and assays on the Bio-Rad CFX 96/384 instruments.

Singleplex vs Duplex Performance 3 genes + 18S Average dCq between singleplex & duplex are comparable. T-Test shows no significant difference between singleplex & duplex There are no setting for ROX – so data is not normalized to ROX Only difference in graphs is that one has target labels next to data points, and the other has target names on X axis GPR6 PPIA YWHAZ

Reproducibility Data shows excellent reproducibility From dynamic range 18S, PPIA, GAPDH From singleplex data 18S PPIA GPR6 YWHAZ

FAMM 6 log dynamic range PPIA 18S Dynamic range is likely wider, but didn’t run higher concentrations of cDNA

FAMM – 18S (VIC Primer limited)

FAMM – PPIA

FAMM - GAPDH

PowerUp™ SYBR™ Green Master Mix 95 deg / 2min 95 deg/15sec x40 cycles 60 deg/ 1 min Dissoc curve

Dynamic Range - PowerUp™ SYBR™ Green Master Mix Excellent reproducibility 3 targets 2 targets (PGK1& ARL1) – 4 logs 1 target –(ABCC2) - 2 logs

Dynamic Range - PowerUp™ SYBR™ Green Master Mix Same data as previous slide, with ABCC2 data removed

PowerUp™ SYBR™ Green Master Mix - PGK1

PowerUp™ SYBR™ Green Master Mix – ARL1

PowerUp™ SYBR™ Green Master Mix – ABCC2

PowerUp™ SYBR™ Green Master Mix -6 targets - Reproducibility A2M, AOF1, ARL1 ADAM10, APOA1, C11orf30

PowerUp™ SYBR Green Master Mix – Excellent Specificity – Dissociation Data APOA1 ARL1 C11orf30 A2M ADAM10 AOF1 No non-specific amplification or primer dimer

PowerUp™ SYBR™ Green Master Mix – Excellent Reproducibility

PowerUp™ SYBR™ Green Master Mix – Excellent Reproducibility All data from all 6 genes plus 3 dil curves

Conclusions and Next Steps Applied Biosystems™ consumables perform well on the Bio-Rad CFX™ (96 & 384 well platforms) TaqMan® Gene Expression Assays run with TaqMan® Fast Advanced Master Mix shows excellent reproducibility, dynamic range and equivalent results in singleplex vs duplex. PowerUp™ SYBR™ Green Master Mix shows excellent reproducibility and sensitivity. To learn more about Applied Biosystems qPCR master mixes or to request a free sample, please visit www.thermofisher.com/qpcrmm To download the ‘Design and Optimization of SYBR Green Assays’ and/or ‘Protocol for Comparing SYBR Green Master Mixes’ please visit www.thermofisher.com/sybr Did you know TaqMan assays are guaranteed to perform? To learn more about TaqMan assays or purchase an extra small size, please visit www.thermofisher.com/taqman PowerUp PGK1 – 4 logs ARL1 – 4 logs ABCC2 – 2 logs © 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., used under permission and license. For research use only. Not for use in diagnostic procedures.