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RealtimePCR

Classicpolymerase chain reaction

• Low sensitivity • Poor precision • Results are not expressed as numbers • Ethidium bromide staining is not quantitative •Post-PCR processing required •Narrow dynamic range (<2 logs)

Traditional End-Point PCR


Classicpolymerase chain reaction

Copynumber ofPCRtemplate

PCRconditions:32cycles

• has a narrow dynamic range (<2 logs) à SEMI-QUANTITATIVE

Amplification indynamic range:increase intemplate number results anincreasedamountofPCRproduct,visibilebygelelectrophoresis à semi-quantitativeinformation

Amplification outofdynamic range:increaseintemplate number does not result anincreased amount ofPCRproduct,visibilebygelelectrophoresis –à Endpoint arrived already with100template-copiesà Noquantitativeinformation

Templatecopy

numbers

Amou

ntofPCR

produ

ct

PCRCycle number301

Copynumber oforiginalPCRtemplate:

1000300100301031

10and30copies:Different amout

ofamplified product

Saturation

chosen endpoint ofPCR

Backgroundofendpoint PCR

End-point PCR:à ideal togive qualitative information(forexampe amplification ofamutation using specific

primers)à Only limited quantitative informationpossible

Template copynumbertoo low


Amou

ntofPCR

produ

ct

PCRCycle number301


10003001003010

10and30copies:Different amout

ofamplified product

Saturation


Get better quantitative informationfromclassic PCR:- Optimize PCRconditions:

A. Testforideal end-point ofPCR(example 25)B. Optimize theamount oforiginal template used forPCR

25

Optimizingquantitive information fromclassic PCR

TimeWasteofprimary materialCostsVariability

Adjust ideal number of cycles

Amou

ntofPCR

produ

ct

PCRCycle number301


1000


25

Real-timePCR

Follow theamplification ofPCRamplicons in“REAL-TIME”=REALTIMEPCR

Measureamount ofPCRproductafter eachcompletedPCRcycle

Obtaining QUANTITATIVEinformationfromPCR

Follow PCR product amplification in real-time (RT-PCR)

♦Eliminate use of gel electrophoresis ♦Increase reproducibility♦Enable use of internal controls/standards ♦Reduce turnaround time ♦Increase throughput♦Reduce sample amount usage♦Results expressed as numbers

Real-timePCR

Real-Time PCR Chemistries

Strategies tofollowPCRproduct generation

1.SYBR®GreenIDye Assay Chemistry

ClassicPCRsetupwithaddition ofSYBRGreen:SYBRGreenis agreenfluorescent cyanine dye that has highaffinity fordouble-strandedDNA.Themodeofbinding isbelieved tobeacombination ofDNAintercalation andexternalbinding.When bound,SYBRabsorbs at awavelength around497nmandemits fluorescence around 520nm.

DenaturationAnnealing

DNAsynthesisDetection ofemission offluorescence

Fluorescence emmission is increasing withincreasing ofPCRcycles

www.biorad.com

2a.excitationfilters

2b.emissionfilters

1.halogentungstenlamp

4.sampleplate

3.intensifier5.ccd detector350,000pixels

Basicsofreal-timePCRmeasurements

Every PCRcycle:ExitationofSYBRgreen(497nm)+measurmentofemission fromSYBRgreen(520nm)


02000000004000000006000000008000000001000000000120000000014000000001600000000

0 5 10 15 20 25 30 35PCR CYCLE NUMBER

AM

OU

NT

OF

DN

AMeasuredem

ission

AMPLIFICATIONBLOT

Quantitativeinformation

METLTINGCURVEANALYSISThetemperature-dependent dissociationbetween two DNA-strands canbemeasuredusing aDNA-intercalating fluorophoresuchas SYBRgreen,or fluorophore-labelled DNAprobes.InthecaseofSYBRgreen(whichfluoresces 1000-foldmoreintensely whileintercalated intheminorgroove oftwostrandsofDNA),thedissociation oftheDNAduring heating is measurable bythelargereduction influorescence that results.

Thetemperatureat which 50%ofDNAisdenatured is known as themeltingtemperature.

Temperature

dissociatio

nofdsD

NA

MeltingcurveisdeterminedafterthelastcycleofPCR:à PCRmachineheatsuo PCRproductsfrom0°Cto100°Cà DissociationofDNAfilamentsismeasuredà IFPCRHASAMPLIFIEDSPECIFICALLYAMPLIFIEDASPECIFICREGIONALLDNAMOLECULESWILLMELTATASPECIFIC

TEMPERATUREàmeltingtemperatureisdeterminedbyDNAsequence!!!à IFYOURUNPCRPRODUCTONAGAROSEGEL,ONLYONE BANDWILLBEVISIBLE


Agarose gel

AREAUNDERPEAK=SYBRincorporation

METLTINGCURVEANALYSIS


dissociatio

nofdsD

NA

MeltingcurveisdeterminedafterthelastcycleofPCR:à PCRmachineheatsuo PCRproducts

from0°Cto100°Cà DissociationofDNAfilamentsis

measuredà IFPCRHASAMPLIFIEDMULTIPLE

FRAGMENTSINANON_SPECIFICMANNERTHEMELTINGCURVEANALYSISWILLIDETIFYMORETHENONEPEAK(PCRprimersarenotsepcific!!)

à Example:IFYOURUNPCRPRODUCTONAGAROSEGEL,FIVEBANDsWILLBEVISIBLE

METLTINGCURVEANALYSISGIVEQUALITATIVEINFORMATIONOFTHEREAL-TIMEPCRREACTION(without necessarily requiring anagarose gelrun)

PCRproduct 1

orprimer dimers

PCRproduct 2

Agarose gel

AREAUNDERPEAKs=SYBRincorporation

Here:signals fromdifferent types ofdsRNA(not only targetamplicon)

Real-Time PCR Chemistries

Strategies tofollowPCRproduct generation

PCRtargetregionamplifiedusingprimer1and2

TaqmanprobePrimer 1

Primer 2

2.Real-TimePCRChemistries based onFluorogenic 5’Nuclease assay

ClassicPCRsetupwithaddition ofamplicon-specific,modified ssDNA oligonucleotide

Taqman probe:- Oligonucleotide- PCRamplicon sitespecific- Hybridizes withone strand ofthePCRproduct- Carries afluorophor (R)- Carries aQuencher that absorbes lightemited fromfluorophor

=“FRET”FRET:Fluorescence ResonanceEnergyTransfer Important:FRETonly works when Q is inclose proximity toR

FRET

Forexample:Cycle 5during PCR

- Denaturation at 95°C- Annealing ofPCRprimers andTaqmanprobe


FRET

NOFRET

- DNASYNTHESISBYTaqpolymerase

Taq has 5’à 3’exonucleaseactivity:Taqman probeesdegraded

Loss ofFRET:lightfromR is not chechedandcanbedetected in“real-time”duringPCRFluoresence increases withevery cycle ofPCRuntil reaching saturation inPCRplateauphase


FRET

NOFRET

- DNASYNTHESISBYTaqpolymerase

Taq has 5’à 3’exonucleaseactivity:Taqman probeesdegraded

ADVANTAGE:HIGHLYSPECIFICDETECTIONOFAMPLIFIEDDNAREGIONs:1.Seqeunce specific PCRprimers forPCR2.Amplicon specific DNAprobeenables selective detectionofregionofinterest!!!


Terminology ofamplification blots

2parallel PCRreacationsshown inblot:BLUE:PCRwithtemplateRED:PCRwithout template(negativecontrol)

Rn:reportersignal obtained fromdetector


CtVALUE:Most important value fortheanalysis ofreal-timePCRdata

Ct = threshold cycle: è il ciclodella reazione diamplificazione in cui ilsegnale di fluorescenza delcampione è maggiorerispetto a quello dellaThreshold


Y =N (1+E)n Y = resa di amplificazione/amount amplifidN = numero di molecole di DNA di partenza (number of starting DNA molecules)E = efficienza di reazione (efficieny of reaction)n = numero di cicli di amplificazione (number of PCR cycles)

WITHEVERYCYCLEOFPCR,THEAMOUNTOFAMPLIFIEDDNADOUBLES

Theamount ofinitial DNAis reverseproportional tothenumber ofcicles required toovercomethethreshold (arrive at Ct)

Sample1(dublicate1)Sample1(dublicate2)




Ct Ct

Ct=26,2

Ct=28,0

Ct=28,5

Ct=30,5

Amount ofDNAof

interest indifferentsamples

Threshold

Basics fortheanalysis ofreal-timePCRdata:Ct andΔCt

WITHEVERYCYCLEOFPCR,THEAMOUNTOFAMPLIFIEDDNADOUBLES





Ct Ct

Ct=26,2

Ct=28,0

Ct=28,5

Ct=30,5

Amount ofDNAof

interest indifferentsamples

Ct=28,5 Ct=30,5

ΔCt=2

Fold change sample3tosample4=2ΔCt

22=4

Ct (sample4)appears 2cycles later thatCt (sample3)à note:inevery cycle ofPCRtheamout ofamplifiedDNAdoublesà 2cycles difference=concentrationoftargetDNAis 4times lower insample4comparedtosample3

Analysisofreal-timePCRdata:Ct andΔCt

Analysisofreal-timePCRdata:Ct andΔCt

Provides absolute measurement of starting copy number –Requires standards of known quantity (Mol or ng) –e.g. Forensic science: Is there DNA and how much DNA (copy number) is there for forensics purposes- e.g. Diagnostics: Virus titer in blood: is there virus DNA and how much is there?


Cts derived fromreal-timePCRusing andincreased copynumber oftargetsite:PCRTARGETREGIONMUSTBEAVAILABLEAT(forexample cloned into aplasmid)DIFFERENTDILLUTIONSAREUSEDFORPCRTOGENERATEASTANDARDCURVE

Biological samples withunknowncopynumber ofPCRtargetsite

ABSOLUTEQUANTITATION

Biological sampleCt=21

Defined amounts oftargetsiteused forPCR(standards)

Defined amounts oftargetsiteused forPCR(standards)

Ct ofall standards areused togenerateastandardcurve:y=-2,9966x+15.311

Ct ofBiological sample=2121=-2,9966x+15.311x=-1,89848logng


ABSOLUTEQUANTITATION


Provides accurate discrimination between relative amounts of starting material –e.g. Comparing expression levels of wildtype vs. mutated alleles –e.g. Comparing expression levels of a gene across different tissues or between different biological conditions –e.g. Validating array results

RELATIVEQUANTITATION

Basics fortheanalysis ofreal-timePCRdata:relativequantitation

Cells stimulated forseveral hours(0-48h)withretinoic acidQUESTION:Howaregenes ofinterest(forexample Hox Agene)regualtedduring this time

Used prepared cDNA forreal-timePCRtodetermine thelevels ofmRNAs ofinterest indiffernet experimentalsamples

Hox genes

REFERENCEGENETHATISNOTAFFECTEDBYRETINOICACIDTREATMENT


Basics fortheanalysis ofreal-timePCRdata:relativequantitation

Cells stimulated forseveral hours(0-48h)withretinoic acid

t=0 Ct Hox Agene=30

Ct reference gene=15

ΔCt


REFERENCE GENE:- no altered expression in relevant

biological- normally expressed at high levels- Serves to control of sample quantity- Serves to control for pipetting errors - Examples:18S rRNA, GAPDH, β-

actin, tubilin, RNA polymerase II, histone H3

REALTIMEPCR:performed inparallel (t=0– 48)at thesame time.

Very important:precisepipetting!

Gene t=0 t=12 t=24 t=36

Ct Reference 15 15 15 15

Ct Hox gene 30 28 26 24

ΔCt 15 13 11 9

ΔΔCt 15-13=2(cicli) 15-11=4 15-9=6

2ΔΔCt 22=4 24=16 26=64

0 12 24 36

10203040506070

1

416

64

timefoldexpressio

nchange

of

Hoxg

eneΔΔC t(t=

0)se

t“1”

Basics fortheanalysis ofreal-timePCRdata:Ct andΔCt andΔΔCt

1

Real-Time PCR has become a cornerstone of molecular biology:

• Gene expression analysis• Cancerresearch• Drugresearch

• Disease diagnosis and management• Viralquantification

• Food testing• PercentGMOfood

• Animal and plant breeding• Genecopynumber

WhatisReal-TimePCRusedfor?

Forallapplications thatrequirethequantification ofRNA/DNAsequences

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