Real-Time quantitative PCR: Choices and Decisions Dr Sandrine Javorschi-Miller R&D program Manager European Functional Genomic Seminar May 2005.

Real-Time quantitative PCR:Choices and Decisions

Dr Sandrine Javorschi-MillerR&D program Manager

European Functional Genomic SeminarMay 2005

Invitrogen Proprietary and Confidential

Why using real-time PCR?


Gene Expression Analysis Technologies

High-densityArrays

Real-time PCR

Nu

mb

er

of

sam

ple

s

Number of genes

1

10

100

>10,000

2-10 100-1000 >10,000

SAGE

Low-densityArrays

RPANorthern

To consider: Ratio samples / genes, needs for accuracy


Comparison of Quantitative Assays (RNA/DNA):

Real-Time PCR

Amplicor/TMA NASBA

bDNA

XPLORE

Microarrays

RPA

Northern

100

101

102

103

104

105

106

107

108

108

107

106

105

104

103

102

101

100

Dynamic RangeSensitivity

NASBA: nucleic acid seq based amplification bDNA: branched DNA assay

Xplore: based on Invader technology

TMA: transcription mediated amplificationRPA: RNAse protection assay

Advantage: Real-Time PCR


Real-time PCR in more details


Phases of amplification

• Exponential phase• Plateau phase


Exponential phase vs. plateau

• At some time or another, all reactions regardless of initial amount reach the same plateau!– Plateau is not quantitative– Exponential phase is quantitative

Cycles

Amplicon amount

Plateau information is not quantitative


Before the Real-Time era: End-point PCR

• Examples of semi-quantitative PCR– End point analysis after reduce number of cycles (exponential

phase)• Not accurate• Not sensitive

– Competitive PCR• Time and reagent consuming

GOI (pg) 0 0.1 1 10?

PCR condition 95oC for 2 min# of cycles 30

95oC for 15 sec 62oC for 30 sec 72oC for 30 sec

End Point is at best semi-quantitative


Choices and Decisions


One Step or Two Step RT-PCR?



RNAOne Step RT-PCR

(One tube)Two Step RT-PCR

(Two tubes)

Amplicon Amplicon

cDNA

GS primersOligo dT

Random Primers(GS Primers)

1 target

1 amplicon

X targets

X amplicons

Target pool


Two-Step RT-PCR• Separate conditions for cDNA

synthesis & PCR• Flexible choice of primers• Ideal for quantification of

multiple genes from a limited number of RNA samples

One-Step RT-PCR• Highly defined conditions to

support RT and Taq• Requires gene specific primer• Ideal for quantification of 1 or 2

messages from a large number of RNA samples


Perfect for:- Lot of samples- Small amount of targets

Perfect for:- Few samples- Large amount of targets

Two-step RT-PCR is more convenient and cost effective


Which Reverse Transcriptase?


RNAse H reduced Thermostable

ReverseTranscriptase:

SuperScript III ™ RT

• No RNA template degradation > Higher cDNA yield– Improved sensitivity

• Reduced 5’ / 3’ bias due to mispriming– Increased reliability

• Greater percentage of full-length cDNA– complete sequence representation

• Improved thermostability– high temperature cDNA synthesis– relaxes secondary structure– improved primer specificity

• Consistent cDNA synthesis efficiency– wide template diversity– wide range of template amount

What RT enzyme?

Good Reverse transcriptase is essential!


B-Actin TaqMan primers (RNA concentrations from 100ng to 0.1pg)

1.00E-01

1.00E+00

1.00E+01

0 5 10 15 20 25 30 35 40

Cycle

Nor

mal

ized

Flu

ores

cenc

e

Company A one step kitSSIII one step qRT-PCR

SuperScript™ III Platinum® One-Step RT-PCR


Which Polymerase?


Antibody Hot Start

- Fast activation

- Maximum activity in early cycles

Hot Start

- Increased specificity

- Reduced artifacts (less Primer Dimers)

Which Taq polymerase?

Platinum® Taq DNA Polymerase

Hot Start Taq is a must!


PLATINUM® Taq automatic hot start

PCR Assembly

InactiveTaq DNA Polymerase

Fully ActiveTaq DNA Polymerase

Temperature CyclingInitial TemplateDenaturation

94oC, 30s - 3 min


UDG or no UDG?


Carry-over protection/comparison

Uracil DNA Glycosylase DecontaminationDone by Nested PCR with pCR2.1 Plasmid

0 5 10 15 20 25 30 35 40 45 50

Cycle Number

I-with UDG I-without UDG Q-with UDG Q-without UDG

20 cycles, ~1,000,000 fold difference

DNA Amplicon with dUTP

Mix with UDG and dUTP

XAmplicon with dUTP

Not all UDG kits are built equal!


Which Detection system?


• dsDNA specific stains– Ethidium bromide (used in first experiments)– SYBR Green I (most widely used today)

• Probe-Based Systems (unlabeled primers plus probes)– TaqMan and TaqMan MGB probe system (5’ nuclease assay)– Dual Probe System, uses FRET between probes hybridized side by side to the

amplicon– Molecular Beacon probe (hairpin probe, quencher and fluorophore are

separated when hybridized to the amplicon)– Epoch probe (second structure probe, quencher and fluorophore are

separated when hybridized to the amplicon)

• Primer/oligo labeled systems– Amplifluor: hairpin primer with fluorophore and quencher– LUX™: primer with one fluorophore and no quencher, proprietary to

Invitrogen

Detection chemistry?

Decision, Decision…


Detection chemistry?

TaqMan®

Quantiprobe®

SYBR Green I®

Amplifluor™

Q

Q

LUX™


Which detection system?

- Multiplexing

- High Specificity

- High Sensitivity

Probe based

or

LUX™ primers

- Monoplexing- Cost saving- Fast initial screening

Sybr Green I®

A detection system for every applications!


LUXTM (Light Upon eXtension)

The fluorescent intensity is modulated due to the proximity of the fluorophore to specific primary & secondary structures of the oligonucleotide. The design rules have been incorporated into the LUX Designer™. The labeled primer exhibits low fluorescence but incorporation during PCR produces a large increase in fluorescence

LUX™

Light Upon eXtension!


Quenching effect

• The fluorogenic LUX primer has an attached fluorophore at the

3’end and a short sequence tail on the 5’ end that is

complementary to the 3’end of the primer. The resulting hairpin hairpin

secondarysecondary structure structure provides optimal quenching of the attached

fluorophore.

• The quenching of fluorescence in duplex (ds DNA) is provided by

the terminal dG-dC or dC-dG base pairthe terminal dG-dC or dC-dG base pair when the dye is attached to

a thymidine / cytosine within three nucleotides from the 3΄-end.


Competitive Audit Data

• Certified LUX primers were compared to TaqMan® Gene Expression Assays from ABI and QuantiTect Gene Expression Assays from QIAGEN using Platinum Super-Mix UDG (1X ROX).– Standard curve generated from ten-fold serial dilutions of ORF clone

(107 –102 copies).ABI 7700

PCR Efficiency: 93%R2:0.998


Quantitect IL6


LUX IL6 TaqMan IL6


Comparison of LUX™ to TaqManComparison of LUX™ to TaqMan

LUX™ sensitivity advantages


Melting curve analysis

Method:

1. Real-time PCR2. After amplification: 60°C to 95°C (slow ramp)3. Fluorescence signal is recorded continuously during the slow temperature ramp4. Melting curve : Fluorescence signal vs. Temperature5. Melting curve is converted to melting peaks by plotting –dF/dT vs Temperature

LUX™ Built-in control feature!


0

0.05

0.1

0.15

0.2

0 5 10 15 20 25 30 35 40 45 50Cycle

No

rm.F

luo

ro.

Sample 1

Sample 2

Sample 3

Positive Control

NTC

NTC

0

0.2

0.4

0.6

0.8

60 65 70 75 80 85 90 95Temperature

-dF

/dT

Sample 1

Sample 2

Sample 3

Positive Control

NTC

NTC

Fast control of the PCR product specificity without:- opening the tube and - running a gel

Provides accurate real time assessment of quality:

- eliminates risk of false positives

- reduces risk of contamination

Specificity – Melting Curve

Advantages LUX™ versus TaqMan®



Alexa 546

TET

FAM

All detectors

Triplex amplification using standard protocol:

- 10,000 copies of Gene X are amplified using a LUX primer set labeled with Alexa Fluor 546- 1,000 copies of Gene Z are amplified using a LUX primer set labeled with TET - 100 copies of Gene Y are amplified using a LUX primer set labeled with FAM

Excitation (nm)

Emission (nm)

FAM 492 517

JOE 520 548

TET 521 536

HEX 533 550

Alexa Fluor 546 554 570

Data from Molecular Probes spectra

Easy multiplexing!


Simple Primer Design with the new D-LUX™ Designer


www.invitrogen.com/dluxdesigner


3 Choices to access LUX™ primers

Scientific project

D-LUX™ Designer Self Service

Certified LUX™ primers- HSKG

-Virus /Bacteria- Human genes

EvoQuest™ LUX™ Custom Service

Flexibility!


Other convenient formats?


Cells Direct kit

No RNA isolation step required.

The cDNA synthesis is performed by SuperScript™ III RNase H- RT.

DNase treatment eliminates genomic DNA so you can be confident that results are due to cDNA amplification.

Cell Lysis

DNase Treatment

cDNA Synthesis

Application: qPCR

All done in 1

tube!

From cells to cDNA in ONE TUBE!


From 1 cell to 10,000 cells without NAP!

Cells Direct kit


RTS kits

Proprietary Polymer Mix

Optimized PCR SuperMix

Temperature-controlled Lyophilization

Packaging

Vialing

Complete Cycle within 3 days

Shelf-life = 1 year

Room Temperature Stable


Standard Curve RTS one step y = -3.548(x) + 37.1

R2 = 0.998

10

15

20

25

30

35

40

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06Starting Concentration

Cyc

le T

hre

sh

old

Standard Curve aqueous (SuperScript III one step)y = -3.505(x) + 36.8

R2 = 0.999

10

15

20

25

30

35

40

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06Starting Concentration

Cyc

le T

hre

sh

old

RTS and regular mixes have equal performances

RTS kits


What mix for what instrument?


Invitrogen qPCR Reagent Selection Guide

SYBR Green Detection Fluorescent Probes/Primers(LUX™, TaqMan®, etc.)

ABI 7000, 7700, 7900

Roche LightCycle

r

Any Instrumen

t

ABI 7000, 7700, 7900

Roche LightCycle

r

Any Instrument

DNA or

cDNA

Platinum SYBR Green

qPCR SuperMix-

UDG w/ROX

Platinum SYBR Green

qPCR SuperMix-

UDG w/BSA

Platinum SYBR Green

qPCR SuperMix-

UDG

Platinum Quantitative

PCR SuperMix-

UDG w/ROX


PCR SuperMix-

UDG


PCR SuperMix-

UDG

RNA, 1-

Step

SuperScript III Platinum SYBR Green

One-Step qRT-PCR Kit

w/ROX



w/BSA



SuperScript III Platinum One-Step

qRT-PCR Kit w/ROX


qRT-PCR Kit


qRT-PCR Kit

RNA, 2-

Step


Two-Step qRT-PCR Kit

w/ROX



w/BSA



SuperScript III Platinum Two-Step

qRT-PCR Kit w/ROX


qRT-PCR Kit


qRT-PCR Kit

A solution for each problem!


SYBR Green LC qPCR versus Roche qPCR

HPRT primers were used with plasmid standards from 1x107 to 1x101 copies. Invitrogen SYBR Green LC (blue) quantified all 7 logs with a slope of –3.644 and an R-value of -1.00. No template controls (NTC’s) were negative with the SYBR LC kit. Roche’s FastStart DNA Master Plus (green) quantified 5 of 7 logs with a slope of –3.700 and an R-value of -1.00. The NTCs along with the last two dilutions showed contamination.


What method of quantitation?


What Method and When

http://www.gene-quantification.de/strategy.html


Absolute quantitation


Relative Quantitation: ΔΔCt Method

CtGOIs – Ctnorm

s = ΔCtSample

CtGOIc – Ctnorm

c = ΔCtCalibrator

ΔCtSample – ΔCtCalibrator = ΔΔCt

Fold Induction = 2- ΔΔCt

GOI = Gene of InterestNorm = Normalizer (Housekeeping) gene


Where to find more info?


Invitrogen qPCR Central

www.invitrogen.com/qpcr


Invitrogen qPCR Central



Invitrogen multidisplinary qPCR group

• R&D qPCR group in Carlsbad, CA• Enzymologists in Carlsbad, CA• Chemists from Molecular Probes in Eugene, OR• Custom Primer manufacturing facility in Frederick, MA• EvoQuest service group in Carlsbad, CA• And more…


The end


Real-Time quantitative PCR: Choices and Decisions Dr Sandrine Javorschi-Miller R&D program Manager European Functional Genomic Seminar May 2005.

Documents

invitrogen proprietary

realtime pcr slide

confidential realtime

confidential udg

decisions slide

platinum onestep rtpcr

realtime quantitative

confidential superscript