Table Of ContentCARL WITTWER MEINHARD HAHN KAREN KAUL (Eds.)
Rapid Cycle Real-Time PCR - Methods and Applications
Quantification
EM
Springer-Verlag Berlin Heidelberg GmbH
Carl Wittwer Meinhard Hahn Karen Kaul (Eds.)
Rapid Cycle
Real-Time PCR -
Methods and Applications
Quantification
With 78 Figures and 108 Tables
Springer
EU
Professor Dr. CARL WITTWER
Director of Flow Cytometry & New Technology
Department of Pathology
University of Utah
School of Medicine
Salt Lake City, UT 84132
USA
Dr. MEINHARD HAHN
Deutsches Krebsforschungszentrum
Abt. Molekulare Genetik
Im Neuenheimer Feld 280
69120 Heidelberg
Germany
Dr. KAREN KAUL
Evanston Northwestern Healthcare
Department of Pathology
2650 Ridge Avenue
Evanston, IL 60201
USA
ISBN 978-3-642-62317-2
We thank Mr. Olfert Landt, TIB MOLBIOL Syntheselabor, Berlin, Germany, for carefully reviewing the
sequence and technical information and for his valuable comments.
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at <http://dnb.ddb.de>.
ISBN 978-3-642-62317-2 ISBN 978-3-642-18840-4 (eBook)
DOI 10.1007/978-3-642-18840-4
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Table ofContents
PartI
Methods
HousekeepingGenes:AGoldStandard? 3
ONNOBAKKER,DAPHNE C.TIMMER
TheChoiceofHouseKeepingGenesinMRD-Quantification
ofAMLl-ETOPositiveAcuteMyeloidLeukemia 11
MARTINWEISSER,CLAUDIA SCHOCH,TORSTEN HAFERLACH,
WOLFGANG HIDDEMANN,SUSANNE SCHNITTGER
QuantificationofmRNAUsinglinearRegressionofLog-linearPCRData-Points
asanAlternativefortheStandardCurveApproach 21
CHRISTIANRAMAKERS,DAPHNETIMMER,ONNOBAKKER,
RONALD H.LEKANNE DEPREZ,JAN M.RUIJTER,ANTOON EM.MOORMAN
MeasuringGenomeSizesbyAbsoluteQuantification 31
JOCHENWILHELM,MEINHARD HAHN
Part11
Applications
RegulationandDevelopment
RelativeQuantificationofInsulinGeneExpressiononthelightCycler
UsingSYBRGreenI 45
NICOLE NEUBAUER
QuantificationofBRec-'!'JaSignalJointT-CellReceptorExcisionCircleDNA
inPatientsafterAutologousandAllogeneicStemCellTransplantation 53
JUERGEN LOEFFLER,HOLGER HEBART,LUTZ LOCHMANN,
THOMAS DAIKELER,PETERBADER,RALF BAUER,KATHRINSCHMIDT,
HERMANN EINSELE
171
TableofContents
ExpressionAnalysisofMitochondrialComponentsinaVarietyofPlantSpecies
UsingReal-TimeQuantitativePCR 61
KATHARINEA. HowELL,RYANLISTER,JAMES WHELAN
QuantificationofIkarosSpliceVariantsbyReal-TimePCR 73
ELLIVEISTINEN,KALLE-PEKKA NERA,JUKKA ALINIKULA,OLLI LASSILA
MethodstoQuantifyCytokineGeneExpressionbyReal-TimePCR 83
PATRICKSTORDEUR
Oncology
ProfilingBreastCancerUsingReal-TimeQuantitativePCR 95
SCOT G.FRANK,PHILIP S.BERNARD
HER-2/neuGeneCopyNumberQuantifiedbyReal-TimePCRinCelllines
andBreastCancerTissue 107
MELANIE KONIGSHOFF,JOCHENWILHELM,MEINHARD HAHN
QuantificationofSeveralComponentsofFibrinolytic
andMatrixMetalloproteinaseSystemsinPrimaryBreastCancer 117
REMEDIOS CASTELLO,FRANCISCOESPANA,JUSTO AZNAR,
AMPARO ESTELLES
ARapidCycleReal-TimeQuantitativeP-9lobinPCRforQuantification
ofHumanDNAinFeces 125
CORNE H.W.KLAASSEN,CLEMENSEM.PRINSEN,
FREDERIKB.J.M.THUNNISSEN
QuantificationofTumorLoadforFollicularLymphomabyQuantifyingBCL2/IGH
UsingReal-TimeQuantitativePCRbylightCycler™ 131
CHUNG-CHE CHANG,BERNARD C.SCHUR,B.S.
Real-TimeQuantificationoftheAMLRearrangements,AML1-ETOandTEL-AML1,
inAcuteLeukemia 141
EVABARRAGAN,PASCUAL BOLUFER,MIGUELANGELSANZ
Genetics
RapidDetectionofGeneDuplicationsinCharcot-Marie-Tooth1ADisease
bySNPGenotypingUsingReal-TimePCR 159
C.RUIZ-PONTE,A.VEGA,A. CARRACEDO,E BARROS
TableofContents QD
CytochromeP4S02D6DeletionGenotypingUsingDerivativeCurveAnalysis
ontheLightCycler 171
ALISONMILLSON,ELIZABETH L.FRANK,ELAINE LYON
Trisomy21 DetectedbySNPAlleleRatios 179
GENEVIEVE PONT-KINGDON,ELAINE LYON
QuantitativeChimerismAnalysisbyAllele-SpecificReal-TimePCRofa10bp
Insertion/DeletionPolymorphismwithinthePromotorRegionofFactorVllc 187
HENDRIK REUTER,BJORNTEWS,JOCHENWILHELM,MEINHARD HAHN
Microbiology
DetectionofSARS-CoronavirusintheLightCycler
byS'-NucleaseReal-TimeRT-PCR 199
CHRISTIANDROSTEN
NormalizedQuantitativeRapid-CycleReal-TimePCRfortheAssessment
ofCMV-DNACopies 211
MARKUS STOCHER,JORG BERG
Methods
HousekeepingGenes:AGoldStandard? 3
ONNOBAKKER,DAPHNE C.TIMMER
TheChoiceofHouseKeepingGenesinMRD-Quantification
ofAML'-ETOPositiveAcuteMyeloidLeukemia 11
MARTINWEISSER,CLAUDIASCHOCH,TORSTEN HAFERLACH,
WOLFGANG HIDDEMANN,SUSANNE SCHNITTGER
QuantificationofmRNAUsingLinearRegressionofLog-LinearPCRData-Points
asanAlternativefortheStandardCurveApproach 21
CHRISTIAN RAMAKERS,DAPHNETIMMER,ONNO BAKKER,
RONALD H.LEKANNE DEPREZ,JAN M.RUIJTER,ANTOON EM.MOORMAN
MeasuringGenomeSizesbyAbsoluteQuantification 31
JOCHENWILHELM,MEINHARD HAHN
•
Housekeeping Genes:AGold Standard?
ONNO BAKKER*,DAPHNE C.TIMMER
Introduction
Geneexpression studies that aim atprecision require normalization to an inter
nalcontrol or"housekeeping"gene.Thegreatest challengeinchoosing an appro
priate housekeeping gene is maintaining expression consistency during treat
ment.
Manygenes can,in principle, be used ashousekeepinggenes [1];however,the
two most commonly used are glyceraldehyde-3-phophate dehydrogenase
(GAPDH) and ~-actin. Unfortunately, these genes are not as constant in their
expression asone would think (or hope) and this particularproblem has result
ed in many recent publications [2-10]. ~-actin shows a diurnal rhythm and the
expression levelchanges when treating with certain hormones [4].In addition,
thepresence ofpseudogenescanbeconfoundingwhen there is,evenalittle,DNA
contamination in the RNA preparation [11].Similar difficulties can occur with
the well-known housekeeping genes GAPDH,elongation factor 1alpha (EFla),
and cyclophilin [11].
Usually,an adequate housekeepinggene can be found in acontrolled system
(e.g.,cellculture) using acommercialkit or the"trial anderror"method. How
ever, more challenges arise when working with samples from patients who
undergo avarietyoftreatments [12],because it isimpossibletomanipulatecon
ditions orrepeat the experiment.Similardifficultiesarise whenthe expressionof
aspecificgene isstudied in animals throughout the day.Therefore, this chapter
focusesontwospecificexperimentaldesigns:(1)the expressionoffour different
housekeepinggenes studied in patienttissue samples, and (2) the expression of
three housekeepinggenesstudiedduringa12-hlight and a12-hdarkcycleinrat
liver.
" O.Bakker,Endocrinology&Metabolism,FS-l71,Meibergdreef9,llOSAZAmsterdam,
TheNetherlands,E-mail:o.bakker@amc.uva.nl
C. Wittwer et al. (eds.), Rapid CycleReal-TimePCR - Methodsand Applications Quantification
© Springer-Verlag Berlin Heidelberg 2004
.. Methods
Materials
Equipment RNase-free glasswareanddisposables
MagNAPure LCinstrument (Roche Diagnostics,Mannheim,Germany)
LightCycler®instrument(Roche Diagnostics,Mannheim,Germany)
Reagents Amplification primers (BioLegio,Nijmegen, The Netherlands)
High Pure Tissue mRNAkit (Roche MolecularBiochemicals, Germany)
MagNA Pure LCRNAIsolation Kit 11 (tissue) (Roche Molecular Biochemicals,
Germany)
First StrandcDNASynthesis Kit(Roche MolecularBiochemicals, Germany)
LightCycler- FastStart DNAMaster SYBR Green I (Roche Molecular Biochemi
cals,Germany)
LightCycler- DNAMasterSYBRGreenI(RocheMolecularBiochemicals,Germany)
Procedure
PrimerDesign The primers are listed in Table 1 and have been previously published. Primer
specificitywascheckedbycomparison to the Genbankdatabase.
SamplePreparation TotalRNAwaspurified from 58human liver biopsies using the TriPure reagent.
From this, cDNAwas prepared using the First Strand cDNASynthesis Kitwith
Table lA. Humanprimersequences
: I
Forward TGA ACGGGAAGCTCA CTGG(728-746)
Reverse TCCACCACCCTGTTGCTGTA(1015-1034)
MgC1 mM Length:306bp
2:4
Forward GAA CCATCCAGGCCAAAT AA(1059-1078)
Rever e CCGTTC TTC CACCACTGA TT (l440-1459)
MgC1 mM Length:400bp
2:4
• 11
Forward GGGTCA GAA GGATTC CTATG(202-221)
Rever e GGTCTCAAACATGATCTGGG(420-439)
MgC1 :5mM Length:237bp
2
.1
Forward GAGACTTCA CCAGGGG(302-317)
Reverse CTGTCT GTCTTG GTGCTCTCC(534-554)
MgC1 mM Length:252bp
2:4
