Practical hints and new solutions for successful real-time PCR studies

Sample to Insight

Practical Hints and New Solutions for Successful Real-Time PCR Studies

Dr. Ina Scheuerpflug, Director LS Foundation, Strategic Marketing

Cover Page 2

1Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017

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

Practical Hints and New Solutions for Successful Real-Time PCR Studies, 15.09.2017

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: Improving Accuracy and Reproducibility in your Real-Time

PCR Results: Novel In-Process Monitoring Tools


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Legal disclaimer

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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.


http://www.qiagen.com/

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Agenda

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RNA: Critical steps for real-time PCR analysis

Improved methods for cDNA synthesis

Real-time PCR essentials

New real-time PCR solutions

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Cells /Tissues

RNAStabilization

RNAPurification

ReverseTranscription

Real-TimeDetection


Sample to Insight

Agenda

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1.1 Stabilization of RNA patterns and RNA quality

1.2 RT enzyme efficiency

1.3 Priming strategy

1.4 Genomic DNA contamination




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4

Cells /Tissues

RNAStabilization

RNAPurification


Real-TimeDetection


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What happens to RNA ex vivo?


Sample to Insight

Principles of RNA stabilization

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Title only (a)

Requirements

• Stop induction of new RNA transcription (e.g., stress response)

• Prevent regulated turnover of mRNA

• Prevent nonspecific degradation of RNA, either enzymatically (due to release of nucleases

from specific cell types or compartments), or chemically (pH, temperature, etc. dependent)

Approaches

Agents that bind directly to nucleic acids

Agents that lyse cells

Agents that inhibit, denature and/or precipitate nucleases (proteins in general)

Fixation

Preserve morphology (usually with no consideration of effects on RNA)


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Requirements for stabilization of blood and tissue samples

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Blood and tissue samples have different stabilization technology

requirements

Blood contains only solitary cells but has a high protein content

Diffusion rates of stabilizing agent no problem

Agents that precipitate or cross-link proteins inappropriate

Blood volume is not limited in principle (container is limited)

One-step stabilization technology required

Tissue contains connected cells

Diffusion rates of the stabilizing agent are critical

Thickness of the tissue sample is limited

Multi-step technology applicable as tissue piece transferrable

Protein precipitation or cross-link is not a problem in principle


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Collection and stabilization solutions for different sample types


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Critical factors for RNA purification

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Quality of the purified RNA: integrity and purity Quality of starting sample material

Inactivation of RNases

Co-purification of potential inhibitors e.g., for the RT-step

Protein (nuclease) contamination

gDNAcontamination

RNA yield Efficient cell/tissue lysis

mRNA content of total RNA is only 1–5%

Purification of total RNA and small RNA (e.g.,miRNA)


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Different lysis steps for different material

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Lysis: Protocol dependent on sample type


• Mechanical disruption of cells/tissue (e.g., TissueLyser)

• Lysis of cells and nuclei by chaotropic salts

• Inactivation of RNases by chaotropic salts in lysis buffer

• Proteinase for lysis of tough samples (i.e. muscle)

◦ requires conditions where proteinase is active but RNases are not

• QIAzol (phenol-guanidine based):

◦ lysis of tough samples, and removal of contaminants by organic extraction very

versatile, but more work, hazardous reagent

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Silica absorption protocol

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Lysis: Protocol dependent on the sample type

Bind nucleic acids to silica: High concentrations of chaotropic salts, low pH,

+ alcohol

Wash the silica membrane: Removal of proteins, inhibitors and chaotropic

salts

Elute nucleic acids from silica membrane: Water or low-salt buffer pH 7–8


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Choosing the right RNA purification product / protocol

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Is the RNA purification procedure appropriate for the amount of starting material?

Overloading usually results in lower purity, lower yields

With small samples it is important to use an efficient RNA purification method

RNA purification from large samples:

Provides a pool of RNA that can be used for multiple experiments

May be required to get sufficient amounts of RNA from samples that have low RNA

content

High binding capacity – usually also implies higher dead volume


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Guidance: RNA extraction kit targeted to your starting material

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If you would like to order the wheel for your lab just send us an email

Download the selection wheel at: www.qiagen.com


http://www.qiagen.com/

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New RNeasy Kit development: RNeasy Plus Universal Kits

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Purification of total RNA from any tissue type using gDNA Eliminator solution

• One-for-all tissue solution

• Time-saving integration of RNeasy and QIAzol technologies

• Fast non-enzymatic removal of genomic DNA

• Pure RNA for use in all downstream applications

• Optimized protocols enable purification of high-quality RNA from any type of tissue,

even difficult-to-lyse tissues.

• QIAzol Lysis Reagent included for lysing fatty tissue and other types of tissue, and RNeasy

spin columns for purifying high-quality RNA.

Kits are available in two formats:

RNeasy Plus Universal Mini Kit enables purification of RNA from up to 50 mg tissue

RNeasy Plus Universal Midi Kit enables purification of RNA from up to 250 mg tissue.

The RNeasy Plus Universal Mini Kit can be automated on the QIAcube.


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Effect of phenol on UV absorbance

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Phenol contamination imitates higher RNA content of the sample


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Agenda

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1.1 Stabilization of RNA patterns and RNA quality

1.2 RT enzyme efficiency

1.3 Priming strategy

1.4 Genomic DNA contamination




1

4


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Critical factors for an efficient RT step


• Quality of the RNA starting material

◦ Phenol contamination

◦ Alcohol contamination

◦ Salt contamination

◦ Protein inhibitors

• Affinity to the RNA to avoid secondary structure effects

• Choice of priming method

◦ Random priming vs. Oligo dT priming

• Genomic DNA contamination

• Sequences near the 5' end effect the cDNA yield

• Use of internal controls is critical

RT enzyme efficiency is influenced by:

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High efficiency and sensitivity – Why?

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QuantiNova Reverse Transcriptase

Highly efficient transcription for sensitive detection even of low abundance targets

Novel Reverse Transcriptase - use of a wide range of RNA amounts (10 pg – 5 µg)

High affinity for RNA - leads to high sensitivity

Up-scaling option for larger input volumes (up to double volume)

Successful use even for difficult templates

Plus RNase inhibitor

gDNA removal buffer

Efficient removal of gDNA (>1000 fold reduction) is integrated in the protocol

RT-Primer Mix

Optimized mix of oligo-dT and random primers

Internal control

Optional use of internal control included to monitor cDNA synthesis efficiency


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Agenda

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Cells /Tissues

RNAStabilization

RNAPurification


Real-TimeDetection

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2


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QuantiNova Reverse Transcription Kit – Protocol

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• With unique Internal Control RNA

• With gDNAremoval

Fast and convenient protocol:

1. RNA (potentially contaminated with gDNA)

Add gDNA Removal Buffer (incl. RNase Inhibitor)

Optionally spike in Internal Control RNA

2 min 45°C

2. Synthesize cDNA

Add 5 µl RT-Master Mix

3 min 25°C, 10 min 45°C, 5 min 85°C

cDNA (incl. IC cDNA, if applicable)

Stable storable, colorable with QN Yellow Template Dye

3. Stop reaction (95°C, 3 min)

Get cDNA without gDNAcontamination!!!

cDNA synthesis procedure in only 20 min


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QuantiNova Reverse Transcription Kit – Internal Control

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QuantiNova IC reliably indicates inhibition or failure of RT or qPCR reaction

Reliable in-process monitoring of RT and qPCR performance


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QuantiNova Reverse Transcription Kit – gDNARemoval

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Efficient, optional gDNA removal prevents CT shifts caused by DNA

contamination

Precise mRNA quantification even if exon spanning primers cannot be used


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Effect of primer choice

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10 kb transcript (amplicon 2kb and 6kb away from 3´end)


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Summary: QuantiNova Reverse Transcription Kit

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High sensitivity due to novel enzyme

Synthesize cDNA and remove gDNA in 20min

Integrated gDNA removal step

Reverse transcription from all regions due to primer mix

(5’region)

Internal control


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Agenda

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• Different quantification strategies

• Probe technology

• Factors influencing PCR specificity


Cells /Tissues

RNAStabilization

RNAPurification


Real-TimeDetection

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3

1


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PCR kinetics

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Little or no increase in PCR product yield

Half-life of PCR enzyme

Inhibition of polymerase by end products

Starvation of PCR substrate

Generation of PCR product is

directly proportional to input

target molecules/PCR cycle

Start of PCR


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CT = thresholdcycle

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Cycle number at which the fluorescent signal reaches the detection threshold

Time point when the fluorescence signal can be detected by the real-time cycler

Start of PCR

DNA conc./fluorescent signal

has reached the fluorescence

detection threshold of the real-

time cycler


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Quantification methods in real time PCR

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Comparison of differential gene expression in different samples:

Quantification

Relative Quantification Absolute Quantification

Target mRNA copies relative to an

internal RNA, e.g., GAPDH or rRNATarget mRNA copies relative to an external

standard and per cell or unit mass


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Absolute quantification

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Determines absolute copy number of sample

CT value of unknown sample compared to standard curve of known standard

Important: Amplification efficiencies of standard and target are equivalent

Log amount of standard

Initial concentration of unknown sample


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Relative quantification using ∆∆CT

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Direct comparison of CT values

Important: both PCR reactions have same PCR efficiency

Compares ∆CT of unknown sample with ∆CT of calibratorsample

∆∆CT = ∆CT (calibrator) - ∆CT (sample)

2 – ∆∆CT = x-fold expression level of GOI in comparison to calibrator

Livak, K.J. and Schmittgen, T.D. (2001) Methods 25, 402.


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Relative quantification using ∆∆CT

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∆CT1 (control) - ∆CT2 (sample)=

∆∆CT

2 –∆∆C

T = Expression level of TNFin

comparison to HPRT

HPRT = Hypoxanthin Phosphoribosyltransferase


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Endogenous reference gene

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Choose a suitable gene for use as a reference or endogenous control.

An endogenous control gene‘s expression level should not differ between samples.

Comparison of CT values of a target gene and endogenous control gene allows the

target gene expression level to be normalized to the amount of input RNA or cDNA

Use of an endogenous control gene corrects for variation in RNA content and reverse-

transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA

sample, variation in nucleic acid recovery and differences in sample handling


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Endogenous reference gene

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More information at:

http://normalisation.gene-quantification.info/

https://www.qiagen.com/de/spotlight-pages/newsletters-and-magazines/articles/endogenous-controls/


http://normalisation.gene-quantification.info/

http://www.qiagen.com/de/spotlight-pages/newsletters-and-magazines/articles/endogenous-controls/

Sample to Insight

Agenda

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Cells /Tissues

RNAStabilization

RNAPurification


Real-TimeDetection

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3

1


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Different amplicon detection methods

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Sequence-nonspecific detection with fluorescing dyes

SYBR Green

Sequence-specific probes

HybProbe

TaqMan Probes

Molecular Beacons, MGB Probes, Scorpions...


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SYBR Green chemistry

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Detection of all double-stranded DNA molecules

Detection of specific and nonspecific PCR products

dsDNA + free dye

(weak fluorophore)

Binds minor groove

(fluorescence ,000x)

dsDNA binding dye shows increased fluorescence when binding to the minor

groove of the DNA

This dye is excited at 497 nm and emits at 520 nm


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Annealing – the most critical step for high PCR specificity

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Specific annealing

Specific product

High yield

High sensitivity

Due to high primer concentration

Nonspecific product (e.g., primer-dimer)

Low yield

Low sensitivity

Inaccurate quantitation


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The melting curve shows unspecific product

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Fluorescence drops when DNA melts

Melting point is determined as the maximum in the negative 1st derivative

negative 1st derivative


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Factors influencing PCR specificity

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Amount of starting template

Primer design

Cations contained in reaction buffer

Initial generation of artifacts by Taq DNApolymerase


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Primer design

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Sequence:

Length of PCR product smaller than 150 bp (ideal 100–150 bp)

Avoid complementary sequences

Avoid mismatches at the 3´–end

Avoid a 3´–end T has a greater tolerance of mismatch

Avoid G/C runs at 3’ end results in stable mismatched primers

Length:

18–30 nucleotides

GC content:

40–60% – High GC content increases the stability of primer/template

Tm = 2°C x (A+T) + 4°C x (C+G)

Concentration:

0.3 µM (e.g. ABI systems, iCycler), 0.5 µM Lightcycler


Sample to Insight

Effect of target length

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Amplification efficiency and sensitivity drops significantly the longer the amplicon

Improved PCR efficiency and sensitivity with shorter products (Optimal length: 100–150 bp)


Sample to Insight

Effects of different cations


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Effects of different cations

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Mg2+

Stabilize the annealing process

Low Mg2+ concentration leads to specific annealing

But: The more Mg2+ ions the more nonspecific product is seen

Using NH4+ and K+

Leads to PCR results which are not influenced by Mg2+


Sample to Insight

NH4+ buffers destabilize non-specifically bound primers

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Comparison of two types of PCR buffer with respect to the Mg2+ concentration:

PCR: 150 ng human genomic DNA, 0.75 kb of single-copy gene for prion protein


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Effect of PCR specificity

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NH4+ Buffer Buffer without NH4

+

Gene:

cDNA:

Cycler:

Human BAX

100 pg – 10 ng

Rotor-Gene 3000

Primer–dimer in the NTC showing nonspecific reaction


Sample to Insight

Agenda

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Cells /Tissues

RNAStabilization

RNAPurification


Real-TimeDetection

4

3

1


Sample to Insight

Target-specific detection

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Hydrolysis probes:

Hydrolyzed by the Taq polymerase results in separation of the reporter and the

quencher fluorochrome

TaqMan probes

Hybridization probes:

Probe is labelled with a donor fluorochrome and an acceptor fluorochrome, but not

hydrolyzed during PCR

FRET probes, Molecular Beacons, Scorpions


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TaqMan chemistry


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Which dyes can be combined?

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Check the technical specifications of your real-time PCR cycler!

Excitation spectra:

Stephen A. Bustin, A–Z of Quantitative PCR


Sample to Insight

Agenda

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3

4

Cells /Tissues

RNAStabilization

RNAPurification


Real-TimeDetection

1


Sample to Insight


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QuantiNova: High efficiency and sensitivity – Why?

Novel antibody-mediated hot-start mechanism

Novel additive QuantiNova Guard stabilizes a binding complex between

DNA Polymerase and the Antibody. The DNA Polymerase remains inactive in this

configuration.

Precise reaction setup with a built-in visual indicator to minimize pipetting errors.

Accurate results with robust detection of rare targets down to a single copy

Magnified throughput and speed with ultrafast cycling

Seamless performance when combined with QuantiTect Primer Assays


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Novel additive QuantiNova Guard

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Unmatched specificity due to a novel antibody-mediated hot-start mechanism

QuantiNova Guard stabilizes the DNA Polymerase – Antibody binding complex. DNA

Polymerase remains inactive in this configuration. Within 2 minutes at 95°C, the

antibody and QuantiNova Guard are denatured and the QuantiNova DNA

Polymerase is activated, enabling PCR amplification: https://vimeo.com/87778004


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The new built-in control minimizes pipetting errors

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Accurate reaction setup indicated by the new built-in pipetting control:

QuantiNova SYBR Green PCR Master Mix contains an inert blue dye,

when combined with QuantiNova Yellow Template Dilution Buffer the

resulting solution turns green – indicating correct reaction setup.

Does not interfere with the reaction


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Summary: QuantiNova PCR Kit Chemistry

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QuantiNova SYBR Green and Probe Kits streamline qPCR by delivering:

High specificity due to a novel antibody-mediated hot-start mechanism

Precise reaction setup with built-in visual indicator to minimize pipetting errors

No need for optimization (e.g., no search for the optimal annealing temperature

or a Mg2+ titration)

Accurate results with robust detection of rare targets down to a single copy

Magnified throughput and speed with ultrafast cycling

Seamless performance when combined with QuantiTect PrimerAssays


Sample to Insight

For additional information

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You can find all product information at:

https://www.qiagen.com/de/products/life-science-

research/cancer-research/gene-expression-analysis/


http://www.qiagen.com/de/products/life-science-

Sample to Insight

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

Ina Scheuerpflug, Ph.D.

[email protected]

Dirk Schacht, Ph.D.

[email protected]

Questions?


http://www.support.qiagen.com/

mailto:[email protected]

mailto:[email protected]

Practical hints and new solutions for successful real-time PCR studies

Health & Medicine

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