Agenda:

9:00 - 9:10 am Introduction

9:10 - 9:30 “Agilent QPCR Portfolio and Introducing the New AriaMx”

Ray DiDonato, Ph.D., Genomics Specialist , Agilent Technologies, Inc.

9:30 - 10:00 am “MxPro Analysis and Visualization of QPCR Data and New Features of the AriaMx

Software”

Scott D. Leppanen, Ph.D., Field Application Scientist , Agilent Technologies, Inc.

10:00 - 10:10 am Coffee Break

10:10 - 10:35 am "Multiplex Real-Time PCR for the Passive Surveillance of Ticks, Tick-bites, and Tick-

borne Pathogens”

Guang Xu, Ph.D., Manager of Genomics Resource Laboratory (GRL) , UMass Amherst

10:35 - 11:05 am “Optimization and Validation of QPCR Results”

Scott D. Leppanen, Ph.D., Field Application Scientist , Agilent Technologies, Inc.

11:05 - 11:15 am Closing Remarks

Agilent Solutions for Real-time

PCR

Ray DiDonato, Ph. D. Genomics Specialist, Northern New England Agilent Technologies, Inc.

A COMPLETE REAL-TIME PCR PORTFOLIO

Bravo Automation

TapeStation Electrophoresis

SureCycler PCR

BioAnalyzer AriaMx and Mx3000 series qPCR System

SW Informatics Microarrays

Agilent Genomics Workflow Solutions

NGS Target Enrichment

The Mx is a flexible platform and can accommodate multiple chemistry options.

Agilent Mx3000P® and Mx3005P®

Detected dyes

Excitation range: 350 to 750 nm

Emission range: 350 to 700 nm

Standard filter set on Mx3005P® , choose any mix of 5 excitation filters Available filters can be used with alternative dyes

Alx350 FAM HEX Cy5 ROX

Cy3 TxRd JOE

TET TAM

Cy7

Designed for Multiplexing

Single vs Multiplex Standard curves

Each assay/dye separate

Performance - Uniformity of results SYBR Green I™ detection

96 wells

Mean Ct = 18.1

Std Dev = 0.05

CV = 0.3%

Ct Range = 0.26

cycles

Performance - Dynamic Range 3 copies to 3x1010 copies of target DNA

Standard curve

R2 = 0.999

Slope = -3.366

Amplification

efficiency=98.2%

Performance - Sensitivity

2-times serial dilution (20,000 to 1.2 copies hgDNA -Actin Mol. Beacon)

Std Curve

R2 = 0.996

Slope = -3.183

Amplification Efficiency

=106.1%

CtY = 35.13 (Ct

value at y-intercept,

theoretical point detect

<1 copy)

MxPro Software: Flexible, Easy to Use

A COMPLETE REAL-TIME PCR PORTFOLIO

AriaMx AMPLIFIES YOUR PRODUCTIVTY

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UPSTREAM EFFICIENCY | EASIER WORKFLOWS

• Detect bias in NGS runs before you

run all of your samples

• Confirm expression changes in gene expression

data and

alleles in genotyping arrays

• Thermal precision and uniformity enable

you to detect alleles better than other systems

• Easy reporting that exports in multiple

formats

• Track experiments across multiple sites

or collaborators

ROBUST VALIDATION

100,000 samples, 14 assays and chemistries across

14

Factory calibrated:

Each instrument is calibrated as it leaves the factory so you have easier first time set up

Blazing scan times:

All channels, less than 2 seconds irrespective of plex number/optical channels used

Ultra-fast reagents:

Supports SYBR, Probe and HRM reagents

STATS

R2 = 0.999

Slope = -3.484

E = 93.7

Run Time = 42 minutes

Brilliant III SYBR Chemistry

This assay shows 8 orders of magnitude change, but

instrument can perform10 orders of magnitude measurements

READY, GO!

15

Next generation optical cartridges

Each cartridge has 8 excitation and emission read points,

one for each row minimizing edge and row effects

8 Photodiode detectors and dichromatic mirrors at each positions to reflect emitted light to photodiode

Instrument always saves data for all wells and for all the optical cartridges that are inserted

Lower instrument life-time costs

On-board diagnostics mean less on-site field engineer visits – in-lab upgrades and in-lab

solutions for instrument maintenance

Intuitive interface

Touch-screen makes the instrument easy to learn

CHOICES TODAY & TOMORROW

16

Uniform performance across all channels

Consistent performance provides you with confidence in routine testing

No reference channel needed

Maximize the productivity of your instrument

Customize your instrument’s plex level

With modular optical modules you can customize the instrument to your exact needs today and add modules when

needed

Ct difference between single-plex and multiplex assays is less than 0.7

SUPERIOR DATA, MULTIPLEXING

Standard Curve for all five channels Target information table

17

SUPERIOR DATA, HRM REAGENTS

Class 3, 76bp amplicon Class 4, 142bp amplicon

Class 1, 220bp amplicon Class 2, 114bp amplicon

Coming in November

EVA GREEN HRM

fastest in the industry

Early Access Available

18

Copy number detection

AriaMx has the ability to detect down to a two fold change in copy number on various dye chemistries

Relative or quantitative applications

AriaMx can run a wide variety of applications – gene expression and miRNA detection in relative or quantitative

assays

SUPERIOR DATA, 2 FOLD DETECTION

STATS

R2 = 0.999

Slope = -3.503

Efficiency = 92.9

Chemistry: TaqMan

Channel: FAM

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SUPERIOR DATA, THERMAL PROFILE

Amplify your sensitivity

AriaMx has little well to well variation. Our

thermal uniformity assay standard of deviation is

less than 0.2Cq

Superior thermal accuracy at target

AriaMx can reach target temperatures less than

0.2C discrimination.

Temperature Uniformity

Tight validation standards had well to well

variability within 0.2C 97% of the time for

thousands and thousands of samples run

Six peltier elements

2 Ceramic plates with semi-conductor elements

contribute to accuracy and

robust instrumentation.

FAM

ROX

HEX

CY5 CY3

Consistent Performance with Multiple Dyes

Intuitive Software and Networkable Instrument for Remote

Experiment Set-Up

Agilent QPCR and QRT-PCR Kits

These kits are used to sensitively quantify RNA expression levels (QRT-PCR) or to determine the DNA concentration (QPCR) of a sample

•Brilliant® was our line of traditional QPCR and QRT-PCR kits. All of these kits with the exception of the Brilliant Multiplex Master Mix were discontinued in 2011

•Brilliant® Multiplex kit is designed specifically for multiple amplification reactions in a single tube

•Brilliant® II kits are formulated for higher sensitivity

•Brilliant® III kits are formulated for fast cycling allowing shorter run times

•Brilliant® Sidestep and Sidestep II kits allow for QPCR and QRT-PCR directly from cell lysate, with no purification step necessary

•miRNA, FFPE and NGS Library Quantification

Brilliant® II QPCR & QRT-PCR Reagent Kits

• Consistent results and versatility

• Compatible with multiple fluorescent detection chemistries, including:

-SYBR™ Green I dye

-TaqMan probes

-Molecular Beacons

-Others...

• Several different kit configurations allow customers to choose the one that is right for them,

master mix and core reagent kits, multiplexing kits

•Brilliant II kits contain chemically modified hotstart Taq (SureStart) -Reduces nonspecific primer annealing and extension

•Brilliant II kits include a higher concentration of Taq polymerase for improved sensitivity

compared to the original Brilliant kits

Agilent Brilliant II

Agilent Brilliant

Competitor A

Competitor Q

Earliest Ct value

Amplification plot of cyclophillin target in 8 replicates at 100-fold dilutions from 100ng down to 0.01ng (100pg) of template. The improved sensitivity of

Brilliant II is evident in earlier Ct values compared to original Brilliant, Competitor A and Competitor Q. In addition, replicate reproducibility is better

with Brilliant II compared to most others , particularly at 0.01ng. cDNA from Human Reference RNA used as template.

Brilliant® II

Reference Dye (free ROX)

All the QPCR and QRT-PCR kits are available with Passive Reference dye

• Included for normalization

• Controls for pipetting variability during assay setup

• Required in some instruments to correct for well to well signal variation, but

optional in the Mx instruments

Brilliant II and Brilliant III High and Low ROX kits have the reference dye pre-

mixed in the Master Mix so less aliquoting is necessary and you can order

the catalog number that is right for their instrument.

Brilliant III Ultra-Fast

-Utilizes mutant Taq that is capable of fast extension (runs completed in

under 40 minutes)

-Enhanced chemically-mediated hotstart allows for shorter hotstart

activation step

-QRT-PCR kits use a Moloney-based RT for 1st strand synthesis with

optimal performance at a synthesis temperature of 50°C.

-Protocols optimized to run on fast-cycling QPCR instruments such as

the ABI StepOnePlus and BioRad CFX96

32

Improved Specificity over the leading competitor master mix

Brilliant III Ultra-

Fast QPCR

Master Mix

Competitor A

Eff.=85%

Eff.=100%

32

Brilliant III Ultra-Fast SYBR Green QPCR Master Mix exhibits precise detection of 2-fold

differences from 1536 copies down to ~3 copies

33

Efficiency = 90%, R2 = 0.988

33

34

10-fold serial dilution of plasmid from 1x 109 to 10 copies

34

y = -3.4377x + 39.411 R2 = 0.999 Eff: 95%

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12

avg

Ct

(n=

4)

10X dilution series of plasmid (1x109 - 1x100 copies per reaction)

Brilliant III Ultra-Fast QPCR Master Mix delivers Superior Sensitivity and Precision to Detect

Target Across 9 Orders of Magnitude

35

Novel Hot Start of Brilliant III Delivers Minimal Primer-Dimer Formation

Brilliant III Ultra-Fast SYBR

Green QPCR Master Mix

Competitor Q

R2: 0.941; Eff: 84%

R2= 0.996 Eff: 90%

10X dilution series of human gDNA of

Numb-1 target (305 bp) – known to

generate primer-dimers

36

Competitor T

R2: 0.979; Eff: 95%

Competitor R

R2: 0.994; Eff: 98%

Novel Hot Start of Brilliant III Delivers Minimal Primer-Dimer Formation

Other QPCR Related Mixes

•Sidestep

•Absolutely RNA® FFPE Kit

•High-Specificity miRNA QRT-PCR Detection Kit

•NGS Library Quantification

Sidestep

Allows for QPCR or QRT-PCR directly from cells with no nucleic acid

purification step necessary. Results are equivalent compared to purified RNA.

•Brilliant® SYBR® Green SideStep™ QPCR Master Mix (Cat #400904)

•SideStep™ mRNA Enrichment Kit (Sidestep Buffer plus Absolutely mRNA kit)

(Cat #400902)

•SideStep™ Lysis and Stabilization Buffer (Cat #400900)

SideStep Product Features

•Quantitate gene expression in cells — without isolating RNA

•Immediate cell lysis and nucleic acid stabilization for up to 20 months at -80°C

•Single tube “In-solution” format

•Easier to use than other “in solution” kits

•Prepare lysates from as few as 100 cells or up to 1 million cells, can dilute down to one cell-equivalent for QPCR

•10 minute lysis and RNA stabilization

•Specific kits for RNA, mRNA, cDNA and DNA directly from cells for use in real time PCR

•Non-toxic, no organic or ethanol precipitations

Sidestep- Time Savings

Typical RNA isolation: ~ 30 minutes SideStep Lysis: ~ 10 minutes

QRT-PCR

Absolutely RNA® FFPE Kit

For purification of RNA from formalin fixed, paraffin embedded tissue samples

Results in highly pure DNA-free total RNA that is suitable for QRT-PCR

Method removes modifications to the RNA that occur during the fixation process that would prevent amplification

Phenol-free, non-hazardous method

High-Specificity miRNA

QRT-PCR Detection Kit

• The first step of the kit involves elongation with with E. coli poly A polymerase (PAP) to generate a poly-A tail at the 3´ end of each RNA molecule.

• A 1st strand synthesis is then performed using AffinityScript™ RT.

• The cDNA synthesis reaction is primed using an RT adapter primer containing a polyT sequence with the addition of a universal reverse primer binding site at the 5’ end.

• QPCR is then carried out using a Universal Reverse Primer (included with the kit) that will anneal to the tag added in cDNA synthesis, and an miRNA-specific forward primer which provides the target specificity.

• The dsDNA-binding dye EVA Green is used for detection (detected with FAM filter set).

miRNA polyA

miRNA polyA

miRNA

miRNA polyA

adapter primer

universal reverse

primer binding site

cDNA

QPCR NGS Library Quantification Kits

• Product offers a means to accurate quantify libraries prior to Next

Generation Sequencing.

• When preparing DNA libraries for next generation sequencing, accurate

quantification of the library molarity is required to ensure efficient data

generation.

• Overly concentrated libraries will result in data that is often impossible to

resolve.

• Conversely, concentrations that are too low can reduce sequencing

coverage and read-length.

-Both Illumina and AB SOLiD library quantification kits available

Bravo Automation

TapeStation Electrophoresis

SureCycler PCR

BioAnalyzer AriaMx and Mx3000 series qPCR System

SW Informatics Microarrays

Agilent Genomics Workflow Solutions

NGS Target Enrichment

Thank You!

Questions?

Note: Any of the QPCR Master

Mixes can be used in combination

with the AffinityScript QPCR

cDNA Synthesis Kit (600559) for

performing 2-step QRT-PCR.

Master Mix

Performing QRT-PCR

SYBR Green

Brilliant II

SYBR Green

QRT-PCR

AffinityScript

2-Step Master

Mix

Cat# 600834

High

Sensitivity

2-step

Brilliant

III Ultra

Fast SYBR

Green QRT-

PCR Master

Mix

Cat# 600886

10 pack

Cat# 600887

1-step

High

Sensitivity

Separate

ROX

High

ROX

Low

ROX

Brilliant II

SYBR

Green QRT-

PCR High

ROX Master

Mix, 1-step

Cat# 600836

10 pack

Cat# 600840

Brilliant II

SYBR

Green QRT-

PCR Low

ROX Master

Mix, 1-step

Cat# 600835

10 pack

Cat# 600839

Fast

Brilliant II

SYBR

Green QRT-

PCR Master

Mix, 1-step

Cat# 600825

10 pack

Cat# 600826

Note: Any of the QPCR Master

Mixes can be used in combination

with the AffinityScript QPCR

cDNA Synthesis Kit (600559) for

performing 2-step QRT-PCR.

Core

Reagents

Single-Step

Core Kit

Brilliant II

QRT-PCR

Core Kit, 1-

step

Cat# 600810

Performing QRT-PCR

Probe Based

10 pack

Cat# 600819

Master Mix

Sidestep II

QRT-PCR

Master Mix,

1-step

Cat# 400917

Direct from

Cells

2-step

Brilliant II

QRT-PCR

AffinityScript

2-step

Master Mix

Cat# 600827

1-step

Brilliant II

QRT-PCR

Master Mix,

1-step

Cat# 600809

10 pack

Cat# 600818

Sidestep II

QRT-PCR

Master Mix,

2-step

Cat# 400918

Direct from

Cells

High

Sensitivity

Separate

ROX

High

ROX

Low

ROX

High

Sensitivity

Brilliant II

QRT-PCR

High ROX

Master Mix,

1-step

Cat# 600838

10 pack

Cat# 600842

Brilliant II

QRT-PCR

Low ROX

Master Mix,

1-step

Cat# 600837

10 pack

Cat# 600841

Fast

Brilliant

III Ultra

Fast QRT-

PCR Master

Mix

Cat# 600884

10 pack

Cat# 600885

Master Mix

Performing QPCR

SYBR Green

High

Sensitivity

10 pack

Cat

#600831

Brilliant II

SyBr

Green

QPCR

Master

Mix

Cat #

600828

Separate

ROX

High

ROX

Low

ROX

10 pack

Cat

#600832

Brilliant II

SyBr

Green

QPCR

High Rox

Master

Mix

Cat #

600829

Brilliant II

SyBr

Green

QPCR

Low Rox

Master

Mix

Cat #

600830

10 pack

Cat

#600833

Brilliant

III Ultra Fast

SYBR Green

QPCR

Master Mix

Cat# 600882

10 pack

Cat# 600883

Fast

Master Mix

Performing QPCR

Probe Based

Brilliant

II QPCR

Master

Mix

Cat#

600804

10 pack

Cat

#600815

High

Sensitivity

Separate

ROX

High

ROX

Low

ROX

Brilliant

II QPCR

High

ROX

Master

Mix

Cat #

600805

10 pack

Cat #

600816

Brilliant

II QPCR

Low

ROX

Master

Mix

Cat #

600806

10 pack

Cat #

600817

Brilliant

Multiplex

QPCR

Master Mix

Cat #

600553

Multiplex

Fast

Brilliant

III Ultra

Fast QPCR

Master Mix

Cat# 600880

10 pack

Cat# 600881