Applied Biosystems™ Consumables on Bio-Rad CFX™

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Presentation transcript:

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

Applied Biosystems™ Consumables on Bio-Rad CFX™ Demonstrate performance of Applied Biosystems consumables on the Bio- Rad CFX96 and CFX384 Real-Time PCR detection systems*. TaqMan® Fast Advanced Master Mix with TaqMan® Gene Expression Assays Reproducibility Dynamic Range Power Up™ SYBR™ Green Master Mix Specificity *Bio-Rad, CFX96 and CFX384 are trademarks of Bio-Rad Laboratories, Inc., which is not affiliated with Thermo Fisher. The statements herein have not been reviewed or endorsed by Bio-Rad.

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

TaqMan Fast Advanced Master Mix protocol on Bio-Rad CFX96™ or CFX384™ (Optional UNG step) https://tools.thermofisher.com/content/sfs/brochures/cms_087006.pdf Note: Use an anneal temperature appropriate for your assay or primer pair design

TaqMan Fast Advanced Master Mix: Reproducibility From dynamic range data with 4 replicates GAPDH GPR6 PPIA YWHAZ At high Ct’s detection is near single copy, therefore precision becomes less reliable. Cq < 32

TaqMan Fast Advanced Master Mix: Six log dynamic range GAPDH Dynamic range is likely wider, but didn’t run higher concentrations of cDNA PPIA YWHAZ

PowerUp™ SYBR™ Green Master Mix An optional UNG treatment step at 50 ºC may be added at the start of the run. Use an annealing temperature appropriate for the primer pair. 50 ºC / 2 min (optional UNG step) 95 ºC / 2 min 95 ºC / 3 sec x40 cycles 60 ºC / 30 sec Melt curve

PowerUp™ SYBR™ Green Master Mix: Dynamic Range Excellent efficiency and reproducibility Cq < 32

PowerUp™ SYBR™ Green Master Mix: PGK1 R2 value of 99.9% shows excellent linearity over a wide input range Single peak showing high target specificity Steep curves show high efficiency

PowerUp™ SYBR™ Green Master Mix: ARL1 R2 value of 99.8% shows excellent linearity over a wide input range Low peak (1 pm) is equivalent to the low plateau Single peak showing high target specificity Steep curves show high efficiency

A2M AOF1 ARL1 ADAM10 APOA1 C11orf30 PowerUp™ SYBR™ Green Master Mix: Excellent Reproducibility, Six targets 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

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 - 2018 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.