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Sample to Insight Critical Factors for Successful Real-Time PCR: Multiplex PCR Laura Alina Mohr, MSc, Global Market Manager Assoc. Cover Page 2 1 Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

Critical Factors for Successful Real-Time PCR: Multiplex PCR

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Page 1: Critical Factors for Successful Real-Time PCR: Multiplex PCR

Sample to Insight

Critical Factors for Successful Real-Time PCR: Multiplex PCR

Laura Alina Mohr, MSc, Global Market Manager Assoc.

Cover Page 2

1Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

Page 2: Critical Factors for Successful Real-Time PCR: Multiplex PCR

Sample to Insight

Maximizing Real-Time PCR Results: Sample to Insight

2

Two-part webinar series

Part 1: Practical Hints and New Solutions for Successful Real-

Time PCR Studies

Part 2: Critical Factors for Successful Real-Time PCR, Multiplex

PCR

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

Page 3: Critical Factors for Successful Real-Time PCR: Multiplex PCR

Sample to Insight

Maximizing Real-Time PCR Results: Sample to Insight

3

Two-part webinar series

Part 1: Practical Hints and New Solutions for Successful Real-

Time PCR Studies

Part 2: Critical Factors for Successful Real-Time PCR, Multiplex

PCR

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

Page 4: Critical Factors for Successful Real-Time PCR: Multiplex PCR

Sample to Insight

Legal disclaimer

4

QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis, prevention, or

treatment of a disease.

For up-to-date licensing information and product-specific disclaimers, see the

respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and

user manuals are available at www.QIAGEN.com or can be requested from

QIAGEN Technical Services or your local distributor.

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Sample to Insight

Agenda

5

Principles and advantages of real-time, multiplex PCR

Critical factors for successful real-time, multiplex PCR

Application data – QuantiFast Multiplex Kits

General considerations for real-time, multiplex PCR

Checklist for successful multiplex, real-time PCR

1

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Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Sample to Insight

Agenda

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Principles and advantages of real-time, multiplex PCR

Critical factors for successful real-time, multiplex PCR

Application data – QuantiFast Multiplex Kits

General considerations for real-time, multiplex PCR

Checklist for successful multiplex, real-time PCR

1

3

4

5

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Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Principles and advantages of real-time, multiplex PCR

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Principles of real-time, multiplex PCR:

Simultaneous quantification of several targets in the same reaction

Sequence-specific probes labeled with a distinct fluorescent dye and a quencher moiety

Can be performed as a one-step or two-step reaction

Benefits:

Conserve precious samples – more data per sample

Coamplification of internal controls – increased data reliability

Increase throughput – more targets analyzed per run

Efficient use of real-time cycler capacities – more results per run

Save on reagent costs – targets are amplified together instead of separately

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Sample to Insight

Agenda

8

Principles and advantages of real-time, multiplex PCR

Critical factors for successful real-time, multiplex PCR

Application data – QuantiFast Multiplex Kits

General considerations for real-time, multiplex PCR

Checklist for successful multiplex, real-time PCR

3

4

5

1

2

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Critical factors for successful real-time, multiplex PCR

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Prerequisites for successful multiplexing

Successful multiplexing requires careful experimental design and optimization. Multiplex assay optimization can be tedious and time consuming as several factors need consideration:

Primer concentration

Mg2+concentration

DNApolymerase

dNTP concentration

Buffer composition

Multiplex reactions must run with the same efficiency as their singleplex reactions, even with varying targets' abundance.

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Critical factors for successful real-time, multiplex PCR

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QuantiFast Multiplex PCR and RT-PCR Kits – for instant multiplex qPCR

success

QuantiFast Multiplex RT-PCR protocolQuantiFast Multiplex PCR protocol

Accurate results Accurate results

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Critical factors for successful real-time, multiplex PCR

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Proprietary QIAGEN PCR Buffer technology

QIAGEN dual cation buffer for increased specificity. Uniquely

balanced combination of two cations,

the buffer provides stringent primer-annealing conditions over a wider

range of annealing temperatures

and Mg2+ concentrations.

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Critical factors for successful real-time, multiplex PCR

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Synthetic Factor MP: An innovative PCR additive

Factor MP supports macromolecular crowding:

Displaces H2O at the template

Increases the local concentration of primers and probes on the template

Leads to more efficient hybridization of primer/probes to the template

Supports the binding of polymerase to the primer–template complex

Stabilizes specifically bound primers

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Critical factors for successful real-time, multiplex PCR

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Fast cycling facilitated by Q-Bond

Q-Bond mediated fast cycling. A) Q-Bond the DNA polymerase and primer to bind as a

single complex, reducing the annealing time to a few seconds. In addition, the unique buffer

composition supports the melting of DNA, reducing denaturation and extension times.

B) Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing

annealing time.

B

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

A

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Critical factors for successful real-time, multiplex PCR

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Well proven HotStarTaq Plus enzyme and blend of Omniscript and Sensiscript

HotStarTaq Plus DNA Polymerase

Unique chemical modification of recombinant Taq DNA polymerase

Fast 5 minute polymerase activation by initial heat incubation step

Robust reactivation independent of PCR environment (pH, salts)

Special blend of RT enzymes for efficient and sensitive multiplex RT-PCR

Optimized combination of Omniscript and Sensiscript

High sensitivity

Efficient cDNA synthesis in just 20 minutes

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Sample to Insight

Agenda

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Principles and advantages of real-time, multiplex PCR

Critical factors for successful real-time, multiplex PCR

Application data – QuantiFast Multiplex Kits

General considerations for real-time, multiplex PCR

Checklist for successful multiplex, real-time PCR

4

5

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Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Application data – QuantiFast Multiplex Kits

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Wide linear range

Reliable duplex PCR Duplex, real-time two- step RT-PCR was

carried out using the QuantiFast Multiplex

PCR Kit and self-designed TaqMan assays

for IL8 (interleukin 8) and ACTB (β-actin).

Analysis of ten-fold dilutions of leukocyte

cDNA template from 100 ng to 1 pg

provided high PCR efficiencies of around

95%. The Cq values were comparable with

those achieved in control singleplex

reactions, demonstrating the reliability of

the duplex assay.

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Application data – QuantiFast Multiplex Kits

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Highest sensitivity

Sensitive duplex PCR, down to 10 copies of template. Duplicate reactions were run on the Applied

Biosystems 7500 Fast System using a DNA template mix providing 108 copies of β-actin (data shown in

insets) and 106 to 10 copies of RPS27A (a ribosomal protein). The QuantiFast Multiplex PCR +R Kit showed

higher sensitivity than the duplex PCR kit from Supplier AII, enabling the cycler in fast-cycling mode to detect

10 copies of target and quantify over 6 log dilutions of template.

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Application data – QuantiFast Multiplex Kits

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Efficient detection of targets varying greatly in abundance

Efficient, sensitive duplex analysis of targets

varying greatly in abundance. Duplex, real-

time one-step RT-PCR using in vitro transcripts of

HSP90AA1 (109, 107, 105, 103 or 10 copies) and

GAPDH (109 copies) was performed (colored

curves). For comparison, singleplex RT-PCR was

also carried out (gray curves). Duplicate

reactions were run on the QuantiFast Multiplex

RT-PCR Kit and self-designed TaqMan assays.

Reliable duplex RT-PCR is demonstrated by the

evenly spaced curves for HSP90AA1 and

overlapping curves for GAPDH. The efficiency of

HSP90AA1 amplification was in an optimal

range.

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Application data – QuantiFast Multiplex Kits

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Precise discrimination of small differences in template amount in singleplex and duplex PCR

Linear Cq values over twofold decreases in template. Duplex and singleplex PCR were carried out using the

QuantiFast Multiplex PCR +R Kit and assays for the t(8;14) chromosomal translocation and for GAPDH.

Quadruplicate reactions were run using genomic DNA from the Ramos cell line as template (two-fold dilutions

from 10 ng to 0.625 ng). Cq values increased linearly by 1 Cq value with decrease in template dilution for both the

singleplex and duplex reactions, demonstrating the ability of the kit to precisely discriminate between small

differences in template amount.

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Sample to Insight

Agenda

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Principles and advantages of real-time, multiplex PCR

Critical factors for successful real-time, multiplex PCR

Application data – QuantiFast Multiplex Kits

General considerations for real-time, multiplex PCR

Checklist for successful multiplex, real-time PCR5

1

2

3

4

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General considerations for real-time, multiplex PCR

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Designing primers and probes

Keep amplicon size small: ideally 60-150 bp

Use specialized software to design primers and probes

Use the same settings for all assays to ensure they work optimally under

the same cycling conditions

• Amplicons should be ±5 bp and with similar GC content

• Primers and probes should have a Tm within ±5°C

Check primer specificity using a BLAST search

To avoid gDNA detection, design intron/exon spanning primers

Design of intron/exon spanning primers.

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General considerations for real-time, multiplex PCR

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Choice of reporter dyes

Probes must be labeled with reporter dyes whose fluorescence spectra are well

separated or exhibit only minimal overlap

Reporter dyes and quenchers must be compatible with the detection optics of

your cycler

Refer to the instrument user manual for which reporter dyes can be used

Dyes commonly used in multiplex, real-time PCR

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General considerations for real-time, multiplex PCR

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Handling and storing primers and probes

Primers and probes should be purchased from an established manufacturer

Upon receipt, resuspend the lyophilized primers and probes and check

their concentrations by spectrophotometry

Dissolve primers and probes in TE buffer to make a 100 µM stock solution

Prepare small aliquots to avoid repeated freezing and thawing

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General considerations for real-time, multiplex PCR

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Evaluating the performance of a real-time, multiplex PCR assay

Check each set of primers and probe in individual PCR assays before

combining in a multiplex assay

Test assay performance: Assay serial dilutions of a sample containing the

target nucleic acids (n.a.)

Test dynamic range: Make dilutions of one target n.a. keeping the

concentration of the others constant (Target n.a. cloned in a plasmid or

prepared as a PCR product can be used)

Test for linearity : Perform reactions with ten-fold dilutions of template, and

check if the Cq values are similar to those of the corresponding single PCR

assays. A standard curve can be used to evaluate the linear range and the

PCR efficiency of the assay.

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General considerations for real-time, multiplex PCR

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Programming the real-time cycler

Activate filters or detectors for the reporter dyes used in the multiplex PCR assay

Follow the optimized cycling protocols in the handbooks, even for assays where

cycling conditions have already been established using a different kit or reagent.

Rotor-Gene Q real-time cycler

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General considerations for real-time, multiplex PCR

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Analyzing data from a real-time, multiplex PCR assay

Optimal analysis settings for each reporter dye are prerequisite for

accurate quantification data:

Adjust the analysis settings for every reporter dye channel in every run

The instrument software default analysis settings may not provide

accurate results and may need to be adjusted

Save the multiplex reactions after amplification to check the PCR

products on a gel or on the QIAxcel

QIAxcel system

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Sample to Insight

Agenda

27

Principles and advantages of real-time, multiplex PCR

Critical factors for successful real-time, multiplex PCR

Application data – QuantiFast Multiplex Kits

General considerations for real-time, multiplex PCR

Checklist for successful multiplex, real-time PCR

1

2

3

5

4

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Checklist for successful multiplex, real-time PCR

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Design primers and probes

Choose predesigned assays (predesigned or published in literature)

Carefully design assays yourself reporter dyes and quenchers

Choose reporter dyes and quenchers

Choose appropriate combination of reporter dyes

Use non-fluorescent quenchers

Choose reagents

Optimize reaction conditions

Use dedicated Master Mix (e.g., QuantiFast Multiplex PCR and RT-PCR kits)

Reconstitute primers and probes

Use TE to prepare a 100 µM stock solution

Store frozen in small aliquots away from light

Set up your real-time cycler

Check if your cycler needs to be calibrated for the reporter dyes and activate the filters

Adjust the analysis setting for each reporter dye channel in each run

Evaluate the performance of the multiplex assay

Check that primer-probe set works in singleplex PCR

Compare the performance in Multiplex PCR

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Versatility of QIAGEN's QuantiFast Multiplex kits

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Optimized ROX concentration, universal protocol

Rox Dye Kits Compatible cycler

In Master Mix

QuantiFast Multiplex PCR Kit (80) 204652QuantiFast Multiplex PCR Kit (400) 204654

QuantiFast Multiplex PCR Kit (2000) 204656

QuantiFast Multiplex RT-PCR Kit (80) 204852

QuantiFast Multiplex RT-PCR Kit (400) 204854

All cyclers from Applied Biosystems

except Applied Biosystems 7500,ABI

ViiA7

Separate tube

QuantiFast Multiplex PCR +R Kit (80) 204752QuantiFast Multiplex PCR +R Kit (400) 204754

QuantiFast Multiplex PCR +R Kit (2000) 204756

QuantiFast Multiplex PCR +R Kit (4000) 204757

QuantiFast Multiplex RT-PCR +R Kit (80) 204952

QuantiFast Multiplex RT-PCR +R Kit (80) 204954

QuantiFast Multiplex RT-PCR +R Kit (80) 204956

Applied Biosystems 7500,ABI

ViiA7†

Bio-Rad, Cepheid, Eppendorf,

Roche and Stratagene/Agilent‡

QIAGEN

*ROX dye must be added to master mix. †

ROX dye not required.

One for all protocol: optimized cycling conditions for 2-plex/3-plex/4-plex and universal

primer/probe concentrations!

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017

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Thank you for attending

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Contact QIAGEN Technical Service

Call: 1-800-426-8157 for US

Call: +49 2103-29-12400 for EU

www.support.qiagen.com

Laura Alina Mohr, MSc.

[email protected]

Questions?

Critical Factors for Successful Real-Time PCR: Multiplex PCR , 28.09.2017