Scott Reierstad Field Applications Scientist · Expression Suite Software • Free data analysis software from AB for ddCt data analysis. • Accepts run files (one or multiple runs),

Real-time PCR TrainingReal-time PCR TrainingScott Reierstad

Field Applications Scientist

Topics we’ll cover

• Quick introduction to real-time PCR

• ABI-supported real-time chemistries

• Instrumentation

• Quantitation via Standard Curves

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• Quantitation via Standard Curves

• Relative Gene Expression workflow

• Protein Thermal Shift

• Live Software Demo

ViiA™ 7 Real-Time PCR System

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96, 96 FAST, 384, TLDA thermal blocks• Block change in less than 1 min

− Heated Cover (96 vs 384)− Plate Adaptor (96 vs 384 vs TAC)

• 384-well and TAC Blocks at launch

Validated Programmable

384 5-20uL 1-30uL

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• 96-well and Fast 96-well followed

384 5-20uL 1-30uL

TAC 1uL 1uL

Validated Programmable

96 10-100uL 1-200uL

Fast 96 5-30uL 1-100uL

Touchscreen: Main Screen• Run experiments directly from touchscreen

• Collect ran experiments

• Set shortcuts from the home screen

In summary: you do not need to have a PC* connected to the


have a PC* connected to the instrument to run an experiment

*provided

User Experience

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• 100+ plate studies without a database

• Streamlined Importing sample info and exporting run results

• Intuitive software (similar to StepOnePlus and 7500 v2.0

• Upload 7900 Protocols / Export 7900 formats

• Monitor run remotely

ViiA 7-Twister IIFlexible: multiple ViiA 7s to 1 robot

All Plates


Able to use FAST plates

2 input, 1 output

80 plate input

The Optics: OptiFlex™ System• 6 Excitation and 6 Emission filters (455nm to 722nm)

• Excitation and Emission Filters can be decoupled for optimal flexibility


FAM, SYBR, SYTO9 (MeltDoctor), Fluorescein,

Sypro Orange, LC Green, Eva Green, Pulsar 650

VIC, JOE, TET, HEX

TAMRA, NED, CY3, Bodipy TMR-X

ROX, Texas Red

Liz, CY5 Cy5.5, Alexa, Joda-4

Expanded Multiplex Capabilities

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Multiplex on cDNA with 5 probes

Dynamic Range, Fast Run

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Amplification of plasmid DNA (7-7E10 total copies/rxn)

using the 384-well block.

9 logs of linear dynamic range in 35 minutes.

ViiA 7: 1.5 Fold Sensitivity

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RNaseP copies:- 10,000- 6,667- 4,500- 3,000- 1,500- 1,000

Calibration

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Calibration• Hardware: Requires Calibration plates

− ROI, Background, Uniformity, Pure Dyes, Normalization, HRM (optional), − Verify instrument performance using RNaseP

• Calibrations: Takes about 10 min/ per plate

• Performed by FSE @ install

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• User recommended to re-calibrate every 6 months. • How to calibrate:

− Access via instrument console, select instrument from “My Instruments”

− Click on instrument to access Instrument Manager

Custom Dye Calibration• Dye MUST be added to dye library before performing a custom

calibration

• Default temperature = 60°C

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Setting Calibration Reminders• Select the instrument from the Instrument Console

• Go to Calibration Reminders

• Email Address Settings

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Maintenance Schedule

Frequency Action

Weekly 1. Power cycle instrument controlling computer

2. Perform instrument self test

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2. Perform instrument self test

Monthly 1. Check lamp status with software

2. Perform background calibration

Every 6 mos 1. Run instrument calibration kit (ROI, Background, Dye, Normalization, RNaseP

System normalization

• Following each real-time run, we will carry out normalization steps to compensate for two different variables:− Non-PCR fluctuations in fluorescence.− Background signal.

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− Background signal.

Common sources of fluorescent variation

Light source

optics

18

cover

condensation

ROX™ Passive Reference Dye

Greatly improves precision of replicates.

(Reporter)

10 ng Sample A10 ng Sample A

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Normalized reporter = Reporter / Rox � Rn

(Rox)

Well 1Well 1 Well 2Well 2

ROX™ dye �� better precision

36 replicates analyzed with ROX™ passive reference dye.

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36 replicates analyzedwithout ROX™.

Two points about ROX™

• All ABI real-time PCR master mixes contain ROX™.

• ABI software automatically performs normalization to ROX™, unless you specifically tell it not to.

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to ROX™, unless you specifically tell it not to.− If you do not use ROX™, you must disable the passive

reference in software for accurate analysis.

Amplification plot graphs cycle vs. fluorescence

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Fluorescent units

?

After we account for non-PCR variables properly, we obtain Ct values…

• BUT, we still need some way to convert Cts into numbers we can make sense of.

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Two ways . . .

Can use a standard / dilution curve

104 103 102 101 100

Do serialdilutions

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At the end of your real-time run

Standard /

Qty = 25010,000

1000

100

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Standard /dilution amt.

Cycle (Ct)

1

100

10

Unknown sample Ct

Two curve types

• Absolute standard curves− Actual copy number of standard is known.− Ex., microbial quantification.

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Absolute curve �� absolute copies

Standard /

Qty = 25,054106

105

104

50

Standard /dilution amt.

Cycle (Ct)

102

10

103

Unknown sample Ct

Two curve types

• Absolute standard curves− Actual copy number of standard is known.− Ex., microbial quantification.

• Dilution (a.k.a., relative standard) curve

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• Dilution (a.k.a., relative standard) curve− Only dilution factor is known.− Ex., Gene expression, copy number.

What if you hate standard / dilution curves?

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Reasons some don’t like standards

• Take up too much room on reaction plate.

• Extra reagent cost.

• Time-consuming to prepare.

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• Difficult to pipet accurately.

Example experiment

• I have 3 servings of bean sprouts of equal mass.

• My concern: sprouts contaminated with E. coli.

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• My concern: sprouts contaminated with E. coli.

• I isolate DNA from each.

• Next, I design an E. coli-specific TaqMan® Assay.

• Now, I amplify DNA from all three in real-time.− Note: There are no curves on this reaction plate.

Real-time results

28 29 30

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Sample 1

Cycle number

Sample 2

Sample 3

Q: Can I say anything about starting amounts?

28 29 30

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Sample 1

Cycle number

Sample 2

Sample 3

Relatively speaking . . .Yes!

PCR should double product after each cycle in geometric phase

28 29 30

∆∆Ct=1Ct=1

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Sample 1

Cycle number

Sample 2

Sample 3

∆∆Ct=1Ct=1

TwoTwo--fold differencefold difference

Handy relationship for calculating relative starting amounts

∆Ct of 1 = 2-fold difference

∆Ct of 2 = 4-fold difference

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∆Ct of 2 = 4-fold difference8-fold difference∆Ct of 3 =

10-fold difference∆Ct of =3.3

However, math makes an assumption . . .

Math makes an assumption

• Namely, that we really are doubling the amount of product with each cycle.

-Doubling = 100% amplification efficiency*

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*Efficiency: the percentage of target molecules used as template for each round of geometric-phase amplification.

When we have 100% efficiency

100%10-fold = 3.3 cycles

28 31.3

60

28 31.3

1000

100

However, if we reduce efficiency . . .


28 31.3


33 36.9

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28 31.3

1000

100

33 36.9

Relative Quantification (gene expression):

Relative quantification is used to determine

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Relative quantification is used to determine fold differences of a target nucleic acid (usually RNA) between two samples.

Ex: Gene expression

Most common method for Relative Quantification is the ∆∆Ct method (does

not require standard cuves):

• At least two samples: Test sample and a calibrator/reference sample.This is a sample to which unknown samples are compared (example -untreated sample, control, timepoint “zero”).

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-untreated sample, control, timepoint “zero”).

• At least two genes: Target gene and Endogenous Control. This is a transcript that is present at a constant amount in total RNA (housekeeping gene). Levels are used to normalize differences in the amount of total RNA loaded in each reaction.

Sample experiment: IL-4 expression change following drug treatment

Calibrator/Ref sample

time


t=0 t=12 t=24 t=48time

total RNA

cDNA

total RNA

cDNA

total RNA

cDNA

total RNA

cDNA

∆Rn

Comparison of Target Gene and Endogenous Control


CyclesCt =14 Ct = 24

Endogenous control (18S)Target gene (IL-4)

∆Rn

CycleCt=22 Ct=27

t=12 h∆Rn

CycleCt=23 Ct=30

t=0

Comparative Ct Method


Cycles

Ct=22 Ct=27

∆Rn

Cycles

Ct=24 Ct=26

t=24 h ∆Rn

Cycles

Ct=23 Ct=33

t=48 h

Cycles

Ct=23 Ct=30

Endogenous control Target gene

Fold change calculation-Comparative Ct Method

step 2: Normalization to calibrator sample

step 1: Normalization to endogenous control

Ct Target gene – Ct Endogenous control = ∆Ct


∆Ct Sample – ∆Ct Calibrator = ∆∆Ct

step 3: use the formula

22--∆∆∆∆CtCtFold Change =

The software does this automatically

Best part about ViiA 7™ software?

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It does all the math for you!

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ddCt method (fold changes) is fast and easy but works only if:

22--∆∆∆∆CtCtEfficiencies (E) are

equal!

Target gene

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Efficiencies (E) of genes are 100%

Qf = Qs (1+Eff)C

Target gene

Endogenous

In ∆∆∆∆∆∆∆∆Ct method efficiencies MUST be equal (+/- 10%)!!!

∆∆∆∆∆∆∆∆Ct Efficiency test

Ct v

alue

∆ Ct

∆ Ct

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Log of Input

Ct v

alue

18S

IL-4Log of Input

slope + 0.1∆Ct allowed

What if efficiencies are not equal?

Relative standard curve method (long, tedious)-Must use a standard curve on each plate for all genes

-Fold changes are calculated using values calculated on a standard

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-Fold changes are calculated using values calculated on a standard curve

Efficiency correction* (ViiA 7™ can do this)-Must use a standard curve one time only for all genes

-Fold changes are calculated using a mathematical formula

factoring different efficiencies*Nucleic Acids Research, 2001, Vol. 29, N.9

Efficiency correction: alternative to relative standard curve method (Pfaffl efficiency correction method*)


*A new mathematical model for relative quantification in real-time RT–PCR

Nucleic Acids Research, 2001, Vol. 29, N.9

Multiple endogenous control normalization (gNorm algorithm*)


Vandesompele J, et. al: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 2002, June 18

Expression Suite Software

• Free data analysis software from AB for ddCt data analysis.

• Accepts run files (one or multiple runs), then calculates RQ data.

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calculates RQ data.

• Numerous plots and features: heat maps, volcano plots, endogenous control selection, etc.− To locate and download, go to AB’s website and search

for “Expression Suite.”

Protein Thermal Shift™ SolutionShift™ Solution

Using Applied Biosystems Real-Time PCR Systems

Training Outline

• Application Overview

• Product Offering Overview

• Protein Thermal Shift™ workflow:− Set up a run− Assay setup and optimization

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− Assay setup and optimization

• Data Examples

• Live Software Workflow Demo: Setup, analysis, export

Protein Thermal Shift™ Software

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Protein Thermal Shift™ Software and Reagents

Application Overview

Protein Thermal Shift

Is a technique used to study the thermal stability of a protein using melt curves in Real Time PCR. Many applications…

• Protein Stability Screen:− improving protein preps (pH, salt, excipients)− profiling crystallization conditions− protein formulation and storage buffers− effect of mutations or modifications

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− effect of mutations or modifications− Protein prep QC

• High-Throughput Ligand Screening:− Small-molecule and fragment screens− Antibody-target specificity− Protein-protein interaction− Inhibitor binding

� The protein unfolds as it is heated.

� An environmentally-sensitive dye binds exposed hydrophobic regions and fluoresces.

� The Tm (melting F

luor

esce

nce

How does Protein Thermal Shift work?

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� The Tm (melting temperature) is calculated from the melt curve.

� Changes in Tm are correlated to changes in protein stability.

Flu

ores

cenc

e

Temperature ( oC)

Calculate the inflection point of

the curve

Complete Protein Thermal Shift ™ Solution

Applied Biosystems Applied Biosystems PTS ≤1 µg Protein/Well

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Applied BiosystemsReal-Time Instrument

PTS Advantages :• High Throughput: 384 assays in < 30 minutes, robotics available• <1 ug protein / assay• No protein or ligand structure information necessary• Minimal upfront optimization

Applied Biosystems PTS Analysis Software

≤1 µg Protein/Well + PTS Dye




Product Offering Overview

• Optimized Protein Thermal Shift™ reagents:− Starter Kit: Control Protein + Ligand (100 rxns), Protein Thermal Shift™

Dye and Buffer (2000 rxns)− Dye Kit: Protein Thermal Shift™ Dye and Buffer (2000 rxns)

• Streamlined software workflow− Accepts *.eds files from StepOne™, StepOnePlus™, 7500 Fast, ViiA™7

Applied Biosystems Total Protein Thermal Shift™ Solution

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− Accepts *.eds files from StepOne™, StepOnePlus™, 7500 Fast, ViiA™7 and QuantStudio™ 12K Flex Systems.

− Multi-plate study-based software (>100 x 384-well plates/study)− No special calibration required, uses ROX™ dye calibration− Calculate ∆Tm with respect to a Reference Sample across plates

Protein Thermal Shift™ Software v1.1

• Life Technologies owns the exclusive rights to the Protein Thermal Shift™ application.

• Stand-alone, Protein Thermal Shift™ analysis software

• Compatible with *.eds Melt run files from:− StepOne™ Software v2.2 (or later)− 7500 Software v2.0.4 (or later)− ViiA™7 Software v1.0 (or later)

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− QuantStudio™ 12K Flex Software v1.0 (or later)

• Brand new analysis application− Multi-plate study-based analysis for High Throughput experiments− Define assay conditions and setup within the software− Single/Multiple peak and Auto/Manual analysis options− Boltzmann fit and Derivative Analysis results− Calculate Tm and Delta Tm (∆Tm)




Workflow

Protein Thermal Shift™ basic workflow

Protein Thermal Shift™ software will only accept analyzed *.eds files

Use ROX™ Detector No Passive Reference

Run melt experiment onAB qPCR Instrument

Analyze the experiment on the qPCR instrument sw

Open or Start Study in Protein Thermal Shift™ software

A Study is a collection of runsfrom a single instrument platform

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Each Analysis Group containsa single Reference sample group

Analyze melt curves in Protein Thermal Shift™ software

Protein Thermal Shift™ software

Import .eds file(s) into Study

from a single instrument platform

Studies can contain >100 *.edsrun files

Protein Thermal Shift™ Software Basic Workflow – Run Setup

134 | Life Technologies | 4/10/2013

The ramp rate can be made faster for shorter runs, but some detail for the melt will be lost. Researchers need to optimize their melt conditions for

individual proteins and screens.

Assay Setup and Optimization

• Assay Optimization can be performed using the PTS Dye (1X-20X) and various amounts of protein

• Use 10-50 ul total volume per reaction

• Use ~0.05-5µg (on average 1ug) of protein per reaction

• Setup reaction on ice: protein, Protein Thermal Shift™ Dye, buffer and

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• Setup reaction on ice: protein, Protein Thermal Shift™ Dye, buffer and compound.

• Start run as soon as convenient. Dye stability for the assay at 4°°°°C as been tested to 24 hrs, at Room Temperature in the dark also 24 hrs.

• Perform continuous dissociation with 1-6% ramp rate (obtain results in 12-30 minutes) or 0.05-0.3°C/second

Assay Optimization Workflow• Conditions to vary:

− Protein: 0.05-5 ug per well− Dye: 1X-20X− pH: pH 2.0 – pH 9.0− Salt: protein dependent

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− Buffers: We have successfully tried HEPES, Tris, KPO4, NaCitrate, Glycine HCl

− Ligands: small molecules and fragment libraries.

Guidelines to consider• Use a protein that has Tm < 98°C.

• In the native state, ensure that the protein has no external hydrophobic sites and that it has sufficient internal hydrophobic residues.

• Multi-domain proteins, or proteins that form oligomers may undergo a multi-state unfolding and multiple melt phases in a melt curve.

• All of these conditions may be tweaked by performing a buffer/additive

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• All of these conditions may be tweaked by performing a buffer/additive screening study so that the protein unfolds in a 2-state model.

• Titration study of PTS dye and Protein may be necessary to assess optimal protein:dye ratio

• Ramp speed and ramp rate can be optimized to achieve optimal data resolution

• Recommended that each plate has a reference group

• Recommended that each protein melt reaction is repeated 4 times on a plate to ensure statistical significance.

Other guidelines to consider• The PTS software is used to simply get Tm of a reverse melt curve data, and

have ability to create melt curve “studies”.

• Keep things simple; 95% of the software is simply assigning wells (color-coding/sorting/grouping melt curve data).

• The final summary of results is captured in the “Replicate Results” table.

• The Tm’s that are provided are the Tm B (Boltzmann; based on a sigmoidalcurve of raw data) and Tm D (derivative).

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Other guidelines to consider• 2 Tm’s are provided

− Tm B (Boltzmann; based on a sigmoidal fit of raw data)− Tm D (derivative)

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− X1 – M3 filter (Viia7 only)

Guidelines and Recommendations cont.

• NPC: Contains only buffer, water, and dye

• LOC: Contains only ligand, buffer, water, and dye. Ligands are capable of skewing data if ligand interacts with dye.

• Prepare a fresh dilution of Protein Thermal Shift™ Dye (1000✕) to 8✕.

• Keep reaction cold at all times prior to putting it on the machine.

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• Keep reaction cold at all times prior to putting it on the machine.

• PTS software runs an algorithm that reduces the noise in the primary raw melt curve data file. So curves may look a little different.




Example Data

Protein-Ligand Binding:Increase in Protein Thermal Stability with Bound Li gand

Protein + Ligand

Protein

No Protein Control41oC

48oC

Melt Curve

Derivative Curve

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48 C

~ 7oC delta Tm

ViiATM 7 Real Time PCR System

Effect of Buffer Conditions on Protein Thermal Stab ility

Higher Tm

Protein Stability

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-Na citrate pH 5.5 + 150 mM NaCl-KPO4 pH 6.0 + 150mM NaCl

-KPO4 pH 7.0 + 150mM NaCl-Hepes. pH 7.5 + 150mM NaCl

Stability

Better Buffer!

Examine effects of point mutations & ligandbinding on protein stability

WT, Mut1, Mut2 WT, Mut1, Mut2 + Ligand

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57.0oC51.1oC

46.3oC55.5oC

49.2oC

43.8oC

PTS data from StepOnePlusTM instrument showing the Normalized Reporter and Derivative Melt profiles

Conclusions• The Protein Thermal Shift™ Assay is a rapid, inexpensive, and straight-

forward high-throughput tool for screening conditions that maximize protein stability or libraries of ligands.

• PTS has been performed on many Applied Biosystems Real-Time PCR Instruments, expanding the flexibility of these systems to protein research.

• Applied Biosystems’ complete Protein Thermal Shift™ solution:

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dye reagent � instrumentation � analysis software

www.lifetechnologies.com/proteinmelt

Where can I find technical help?

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Getting Started Guides

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ABI online support

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ABI online support

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Technical Support Hotline

11--800800--762762--40014001

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11--800800--762762--40014001

Scott Reierstad, Field Applications Scientist

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[email protected]

TrademarksFor Research Use Only. Not for diagnostic procedures.

The PCR process and 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.

Applied Biosystems, ABI (Design), and VIC are registered trademarks , and Applera and FAM are registered trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.

TaqMan and AmpliTaq Gold are registered trademarks of Roche Molecular Systems, Inc.

SYBR Green is a registered trademark of Molecular Probes, Inc.

All other trademarks are the sole property or their respective owners.

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All other trademarks are the sole property or their respective owners.

©2006 Applied Biosystems. All rights reserved.

Documents

Scott Reierstad Field Applications Scientist · Expression Suite Software • Free data analysis software from AB for ddCt data analysis. • Accepts run files (one or multiple runs),