real-time PCR  &  qPCR  literature
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  Web based Dictionaries & Glossaries:

  • This tutorial will provide a understamding of the following key points:
  • Limitations of traditional PCR
  • Introduction to Real-Time PCR
  • Advantages of Real-Time chemistries over traditional PCR methods


A list of useful and available books on Polymerase Chain Reaction Technique since 1990 to 2002 as well as PCR Books that will be published in 2003. This section contain 74 links with Title, Publication year, and Edition number to vendors of these books as well as additional information. PCR Books are sorted by publishing year, and then alphabetically.  Check out this section at

some full text articles are in Adobe Acrobat Format ( PDF ) Get Acrobat Reader

Book cover A-Z of qPCR

A-Z of quantitative PCR    ( Editor:  S.A. Bustin )
International University Line (IUL), La Jolla, CA, USA

Flyer for the book   =>  PDF

Search inside  @

Chapter 3:        Quantification strategies in real-time PCR
by Michael W. Pfaffl          
Download  =>  Chapter 3  PDF

This chapter analyzes the quantification strategies in real-time RT-PCR and all corresponding markers of a successful real-time RT-PCR. The following aspects are describes in detail: RNA extraction, reverse transcription (RT), and general quantification strategies - absolute vs. relative quantification, real-time PCR efficiency calculation, data evaluation, automation of quantification, data normalization, and statistical comparison. The discussion turns into practical considerations with focus on specificity and sensitivity.

DNA Amplification: Current Technologies and Applications

Real-Time PCR: An Essential Guide

Publisher: Horizon Bioscience
Editor: Kirstin Edwards, Julie Logan and Nick Saunders
Genomics Proteomics and Bioinformatics Unit, Health Protection Agency, London


PCR  BioBook

  • PCR and real-time PCR (in general):
T.B. Balla F.A. Plummerb K.T. HayGlassa (2003)
Improved mRNA Quantitation in LightCycler RT-PCR
Int Arch Allergy Immunol 2003;130:82–86

Bernard PS, Wittwer CT.  (2000)
Homogeneous amplification and variant detection by fluorescent hybridization probes.
Clin Chem. 2000 Feb;46(2):147-8.

Belgrader P, Young S, Yuan B, Primeau M, Christel LA, Pourahmadi F, Northrup MA.  (2001)
A battery-powered notebook thermal cycler for rapid multiplex real-time PCR analysis.
Anal Chem. 2001 Jan 15;73(2):286-9.

Boulay JL, Reuter J, Ritschard R, Terracciano L, Herrmann R, Rochlitz C.  (1999)
Gene dosage by quantitative real-time PCR.
Biotechniques. 1999 Aug;27(2):228-30, 232.

Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S.  (1997)
The elimination of primer-dimer accumulation in PCR.
Nucleic Acids Res. 1997 Aug 15;25(16):3235-3241

Burkardt HJ (2000)
Standardization and quality control of PCR analyses
Clin Chem Lab Med  2000 Feb;38(2):87-91 

Bustin SA.   (2000)
Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.
J Mol Endocrinol. 2000 Oct;25(2):169-93. Review.

Bustin SA.   (2002)
Quantification of mRNA using real-time reverse transcription PCR: trends and problems
J Mol Endocrinol. 2002 29: 23-29   Review.

Chandler DP, Wagnon CA, Bolton H Jr. (1998)
Reverse transcriptase (RT) inhibition of PCR at low concentrations of template and its implications 
for quantitative RT-PCR.
Appl Environ Microbiol. 1998 Feb;64(2):669-77.

de Silva D, Wittwer CT.  (2000)
Monitoring hybridization during polymerase chain reaction. 
J Chromatogr B Biomed Sci Appl. 2000 Apr 28;741(1):3-13. Review.

de Kok, J.B., Hendriks, J.C., van Solinge, W.W., Willems, H.L., Mensink, E.J., and Swinkels, D.W.  (1998)
Use of real-time quantitative PCR to compare DNA isolation methods. Clin.Chem. 44(10):2201-2204, 1998.

DNA / RNA  real-time quantitative PCR  (????)
PE Biosystems

Elenitoba-Johnson KS, Bohling SD, Wittwer CT, King TC.  (2001)
Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis.
Nat Med. 2001 Feb;7(2):249-53.

Enard et al.  (2002)
Intra- and Interspecific Variation in Primate Gene Expression Patterns 
Science 296 (5566): 340-343

Ferré, F (1992)
Quantitative or semi-quantitative PCR: Reality versus Myth. 
PCR Methods and Applications 2, 1-9.

Fitzgerald DA,   (2000)
The Scientist 14 (23):31, Nov. 27, 2000

Freeman, WM, Walker, SJ, and Vrana, KE (1999) 
Quantitative RT-PCR: pitfalls and potential.   Biotechniques 26, 112-122.

Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. (2001)
An overview of real-time quantitative PCR: applications to quantify cytokine gene expression.
Methods  2001 Dec;25(4):386-401 

Gibson, UE, Heid, CA, and Williams, PM (1996)
A novel method for real time quantitative RT-PCR. Genome Res 6, 1095-1001.

Ginzinger DG. (2002)
Gene quantification using real-time quantitative PCR.  An emerging technology hits the mainstream
Exp Hematol  2002 Jun;30(6):503-12

Higuchi, R, Fockler, C, Dollinger, G, and Watson, R (1993)
Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. 
Biotechnology 11, 1026-1030.

Jung R, Soondrum K, Neumaier M. (2000)
Quantitative PCR. 
Clin Chem Lab Med. 2000 Sep;38(9):833-6. Review.

Kainz P.  (2000)
The PCR plateau phase - towards an understanding of its limitations.
Biochim Biophys Acta  2000 Nov 15;1494(1-2):23-7 

Klein D. (2002)
Quantification using real-time PCR technology:  Applications and limitations.
Trends Mol Med  2002 Jun;8(6):257-60 

Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM, Moorman AF. (2002)
Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions.
Anal Biochem. 2002 307(1): 63-69.

Lie, Y. S. and C. J., Petropoulos.  (1998)
"Advances in quantitative PCR technology: 5' nuclease assays." 
Curr Opin Biotechnol 9 (1998): 43-48.

Liss B. 
Improved quantitative real-time RT-PCR for expression profiling of individual cells (2002)
Nucleic Acids Res  2002 Sep 1;30(17):e89 

Liu W & Saint DA.  (2002) 
A new quantitative method of real time reverse transcription polymerase chain reaction 
assay based on simulation of polymerase chain reaction kinetics. 
Anal Biochem. 2002  302(1): 52-59. 

Liu W & Saint DA (2002) 
Validation of a quantitative method for real time PCR kinetics.
Biochem Biophys Res Commun. 2002 294(2): 347-353

Mackay IM, Arden KE, Nitsche A. (2002)
Real-time PCR in virology.
Nucleic Acids Res. 2002 Mar 15;30(6):1292-305. Review.

Mannhalter, C, Koizar, D, and Mitterbauer, G (2000)
Evaluation of RNA isolation methods and reference genes for RT-PCR analyses of 
rare target RNA. Clin Chem Lab Med 38, 171-177.

Morrison TB, Weis JJ, Wittwer CT.   (1998)
Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification.
Biotechniques. 1998 Jun;24(6):954-8, 960, 962.

Nath K, Sarosy JW, Hahn J, Di Como CJ.  (2000)
Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems.
J Biochem Biophys Methods. 2000 Jan 3;42(1-2):15-29.

Orlando, C, Pinzani, P, and Pazzagli, M  (1998)
Developments in quantitative PCR. Clin Chem Lab Med 36, 255-269.

Peccoud J  &  Jacob C.  (1998)
Gene Quantification (eds. Francois Ferre)
Statistical estimations of PCR amplification rates.

Ramakers C, Ruijter JM, Deprez RH, Moorman AF. (2003)
Assumption-free analysis of quantitative real-time PCR data
Neurosci Lett  2003 Mar 13;339(1): 62-66

Schmittgen TD.    (2001)
Real-time quantitative PCR.    Methods. 2001 Dec;25(4): 383-385.

Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. (2000)
Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.
Anal Biochem. 2000 Oct 15;285(2):194-204.

Sequence detection systems quantitative assay design and optimization.   (????)
PE Biosystems

Souazé, F, Ntodou-Thomé, A, Tran, CY, Rostene, W, and Forgez, P (1996)
Quantitative RT-PCR:  Limits and accuracy. BioTechniques 21, 280-285.

Svanvik N., G. Westman, W. Dongyuan & M. Kubista. (2000)
Light-up Probes Thiazole Orange Conjugated PNA for Detection of Nucleic Acid in Homogeneous Solution. 
Anal. Biochem. 281, 26-35 (2000).

Svanvik N., A. Stålberg, U. Sehlstedt, R. Sjöback & M. Kubista. (2000)
Detection of PCR Products in Real-time Using Light-up Probes.
Anal. Biochem. 287, 179-182 (2000).

Svanvik N., J. Nygren, G. Westman & M. Kubista. (2001)
Free-probe Fluorescence of Light-up probes. 
J. Amer. Chem. Soc., 123, 803-809 (2001).

Ales Tichopad, Anamarija Dzidic, Michael W. Pfaffl (2002)
Improving quantitative real-time RT-PCR reproducibility 
by boosting primer-linked amplification efficiency
Biotechnology Letters 24: 2053-2056 (2002)

Ales Tichopad, Michael Dilger,  Gerhard Schwarz  &  Michael W. Pfaffl (2003)
Standardized determination of real-time PCR effciency from a single reaction set-up.
Nucleic Aids Research 31(20): e122  (2003)

ERRATUM of Nucleic Aids Research 31(20): e122  (2003) Figure 2

Ales Tichopad, Andrea Didier, Michael W. Pfaffl (2003)
Inhibition of real-time RT–PCR quantification due to tissue-specific contaminants

Molecular and Cellular Probes (2003)

Vandesompele J, De Paepe A, Speleman F.  (2002)
Elimination of Primer-Dimer Artifacts and Genomic Coamplification Using a
Two-Step SYBR Green I Real-Time RT-PCR. Anal Biochem. 2002 Apr 1;303(1):95-98

Nigel J. Walker (2002)
A Technique Whose Time Has Come

Teo IA, Choi JW, Morlese J, Taylor G, Shaunak S. (2002)
LightCycler qPCR optimisation for low copy number target DNA
J Immunol Methods  2002 Dec 1;270(1):119-33

Wang, AM, Doyle, MV, and Mark, DF (1989)
Quantitation of mRNA by the polymerase chain reaction. PNAS 86, 9717-9721.

Weis JH, Tan SS, Martin BK, Wittwer CT.   (1992)
Detection of rare mRNAs via quantitative RT-PCR.
Trends Genet. 1992 Aug;8(8):263-4.

Whitcombe, D., Theaker J., Guy, S.P., Brown, T., Little, S. (1999)
Detection of PCR products using self-probing amplicons and flourescence. 
Nature 17, 804-807

Wilson IG. (1997)
Inhibition and facilitation of nucleic acid amplification.
Appl Environ Microbiol. 1997 Oct;63(10):3741-51. Review.

Wittwer CT, Herrmann MG, Gundry CN, Elenitoba-Johnson KS.   (2001)
Real-Time Multiplex PCR Assays.
Methods. 2001 Dec;25(4):430-442.

Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ.   (1997)
The LightCycler: a microvolume multisample fluorimeter with rapid temperature control.
Biotechniques. 1997 Jan;22(1):176-81.

Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP.   (1997)
Continuous fluorescence monitoring of rapid cycle DNA amplification. 
Biotechniques. 1997 Jan;22(1):130-1, 134-8.

Wittwer CT, Garling DJ.   (1991)
Rapid cycle DNA amplification: time and temperature optimization.
Biotechniques. 1991 Jan;10(1):76-83.

Wittwer CT, Fillmore GC, Hillyard DR.   (1989)
Automated polymerase chain reaction in capillary tubes with hot air.
Nucleic Acids Res. 1989 Jun 12;17(11):4353-7.

Wong, L, Pearson, H, Fletcher, A, Marquis, CP, and Mahler, S (1998) 
Comparison of the efficiency of M-MuLV reverse transcriptase, Rnase H—M-MuLV reverse transcriptase 
and AMV reverse transcriptase for the amplification of human immunglobulin genes. 
Biotechnology Techniques 12, 485-489.

Zhijian J. Chen, Hongxie Shen & Kenneth D. Tew ( 2002)
Gene expression profiling using a novel method: amplified differential gene expression (ADGE)
Nucleic Acids Res. 2001; 29: e46


  • LightCycler specific technical notes:
Publications by Roche Molecular Biochemicals

Link  ->  LightCycler Literature Request Form

Link  ->  References & Literature: LightCycler Articles Published in Biochemica


Pfaffl, MW  (2001)
Development and validation of an externally standardised quantitative Insulin like growth factor-1 (IGF-1) RT-PCR using LightCycler SYBR ® Green I technology. 
In: Meuer, S, Wittwer, C, Nakagawara, K, eds. Rapid Cycle Real-time PCR, Methods and Applications Springer Press, Heidelberg, ISBN 3-540-66736-9
LightCycler Relative Quantification Software, Version 1.0 (2001)
Technical Note No. LC 1/1999 
How to Reduce Primer Dimers in a LightCycler PCR
Technical Note No. LC 4/1999 
Adaptation Protocol for Sequence-Independent Detection of RNA with SYBR Green I 
Technical Note No. LC 2/1999
Adaptation Protocol for Sequence-Independent Detection of DNA with SYBR Green I 
Technical Note No. LC 9/2000
Optimization Strategy
Technical Note No. LC 10/2000
Overview of LightCycler Quantification Method
Technical Note No. LC 11/2000
Absolute Quantification with External Standards
Technical Note No. LC 12/2000
Absolute Quantification with External Standards and an Internal Controls
Technical Note No. LC 13/2001
Relative Quantification
Technical Note No. LC 15/2002
Selection of Housekeeping Genes

Technical Support: Literature Series on Technical Aspects of the LightCycler System

Soong, R, Ruschoff, J, and Tabiti, K (2000) 
Detection of colorectal micrometastasis by quantitative RT-PCR of cytokeratin 20 mRNA.  (Roche Molecular Biochemicals Internal Publication)
LightCycler Softwares
LightCycler Manuals

  • absolute quantification:
Berisha B,  Pfaffl MW,  Schams D. (2002)
Expression of estrogen and progesterone receptors in the 
bovine ovary during estrous cycle and pregnancy.
Endocrine  2002 Apr;17(3):207-214

Bustin SA.    Absolute quantification of mRNA using real-time reverse transcription  (2000)
polymerase chain reaction assays.
J Mol Endocrinol. 2000 Oct;25(2):169-93. Review.

Fronhoffs S, Totzke G, Stier S, Wernert N, Rothe M, Bruning T, Koch B, Sachinidis A, Vetter H, Ko Y.  (2002)
A method for the rapid construction of cRNA standard curves in quantitative real-time RT-PCR.
Mol Cell Probes  2002 Apr;16(2): 99-110

Georgiev I P, T M Georgieva, M Pfaffl1, H M Hammon and J W Blum (2003)
Insulin-like growth factor and insulin receptors in intestinal mucosa of neonatal calves.
Journal of Endocrinology, 176: 121-132

Inderwies T., M. W. Pfaffl, H. H. D. Meyer, J. W. Blum & R. M. Bruckmaier (2003)
Detection and quantification of mRNA-expression of alpha- and beta-adrenergic receptor subtypes
in the mammary gland of dairy cows.
Domestic Animal Endocrinology (24): 123-135

T. Inderwies, M. W. Pfaffl & R. M. Bruckmaier (2003)
Milking characteristics and their relation to adrenergic receptor mRNA
expression and ligand binding in the mammary gland of dairy cows.

Domestic Animal Endocrinology 2003 (25):

Ke LD, Chen Z, Yung WK. (2000)
A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards.
Mol Cell Probes. 2000 Apr;14(2):127-135.

Reidun Ovstebo,* Kari Bente Foss Haug, Knut Lande, and Peter Kierulf (2003)
PCR-based Calibration Curves for Studies of Quantitative Gene Expression in Human Monocytes: Development and Evaluation.
Clinical Chemistry 49:3 425–432 (2003)

LightCycler Software ®, Version 3.5 (2001) 
Roche Molecular Biochemicals.

Pfaffl, MW  (2001)
Development and validation of an externally standardised quantitative Insulin like growth factor-1 (IGF-1) RT-PCR using LightCycler SYBR ® Green I technology. 
In: Meuer, S, Wittwer, C, Nakagawara, K, eds. Rapid Cycle Real-time PCR, Methods and Applications Springer Press, Heidelberg, ISBN 3-540-66736-9

Pfaffl, MW  (2001) 
Development and validation of an externally standardised quantitative Insulin like growth factor-1 (IGF-1) RT-PCR using LightCycler SYBR ® Green I technology.
Biochemica 3/2000  - ROCHE Diagnostics

M. W. Pfaffl & M. Hageleit  (2001)
Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration 
curves in real-time RT-PCR.  Biotechnology Letters (2001) 23, 275-282

Pfaffl MW, Lange IG, Daxenberger A, Meyer HH.  (2001)
Tissue-specific expression pattern of estrogen receptors (ER): quantification of ER alpha and ER 
beta mRNA with real-time RT-PCR. APMIS. 2001 May;109(5):345-55.

M.W. Pfaffl, I.G. Lange, H.H.D. Meyer (2003)
The gastrointestinal tract as target of steroid hormone action:   Quantification of steroid receptor mRNA expression (AR, ER-alpha, ER-beta and   PR) in 10 bovine gastrointestinal tract compartments by kinetic RT-PCR.
Journal of Steroid Biochemistry & Molecular Biology (2003) 84 (2-3): 159-166

M.W. Pfaffl, T. Mircheva Georgieva, I. Penchev Georgiev, E. Ontsouka, M. Hageleit & J. W. Blum  (2002)
 Real-time RT-PCR quantification of insulin-like growth factor (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor, insulin receptor, growth hormone receptor, IGF-binding proteins 1, 2 and 3 in the bovine species.
 Domest Anim Endocrinol, 22(2): 91-102.

M.W. Pfaffl, A. Daxenberger, M. Hageleit & H.H.D. Meyer (2002)
Effects of synthetic progestagens on the mRNA Expression of Androgen Receptor, Progesterone Receptor, Estrogen Receptor a and b, Insulin-like Growth Factor (IGF)-1 and IGF-1 receptor in Heifer tissues
J Vet Med A 49: 57-64

M. W. Pfaffl, S. L. Wittmann, H. H. D. Meyer  &  R. M. Bruckmaier (2003)
Gene Expression of Immunologically Important Factors
in Blood Cells, Milk Cells and Mammary Tissue of Cows
Jornal of Dairy Science 86(2): 538-545

Ramakers C, Ruijter JM, Deprez RH, Moorman AF. (2003)
Assumption-free analysis of quantitative real-time PCR data
Neurosci Lett  2003 Mar 13;339(1): 62-66

S. Sauter, E. Ontsouka, B. Roffler, Y. Zbinden, C. Philipona, M. W. Pfaffl, B. H. Breier, J. W. Blum & H. M. Hammon (2003)

Effects of dexamethasone and colostrum intake on the somatotropic axis in neonatal calves
Am J Physiol Endocrinol Metab 285: E252-261

Schams D, Kohlenberg S, Amselgruber W, Berisha B, Pfaffl MW, Sinowatz F. (2003)
Expression and localisation of oestrogen and progesterone receptors in the bovine mammary
gland during development, function and involution
J Endocrinol 2003 May;177(2): 305-317

Ales Tichopad, Michael W. Pfaffl, Andrea Didier (2003)
Tissue-specific expression pattern of bovine prion gene:
quantification using real-time RT-PCR
Mol Cell Probes  17: 5-10

Vandenbroucke II, Vandesompele J, Paepe AD, Messiaen L. (2001)
Quantification of splice variants using real-time PCR. Nucleic Acids Res. 2001 Jul 1;29(13): E68

Wittmann SL, Pfaffl M., Meyer HHD & Bruckmaier RM  (2002)
5-Lipoxygenase, cyclo-oxygenase-2 and tumor necrosis factor alpha gene expression in somatic milk cells
 Milk Science International, 57(2): 63-66.


  • relative quantification:
ABI Prism 7700 Sequence detection System User Bulletin #2 (2001)
Relative quantification of gene expression.

A. Hartel, A. Didier, M.W. Pfaffl, Heinrich H.D. Meyer (2003)
Characterisation of gene expression patterns in 22RV1 cells for  determination of environmental androgenic/antiandrogenic compound
Journal of Steroid Biochemistry & Molecular Biology (2003) 84 (2-3):  231-238

Horgan, GW, and Rouault J  (2000)

Introduction to randomisation tests, Biomathematics and Statistics, Scotland

LightCycler Relative Quantification Software, Version 1.0 (2001) 
Roche Molecular Biochemicals.

Livak KJ, Schmittgen TD.  (2001)
Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 
2(- delta deltaC(T))  Method.
Methods. 2001 Dec;25(4): 402-408.

Pfaffl MW.   (2001)
A new mathematical model for relative quantification in real-time RT-PCR.
Top  NAR  Paper 2001 Methods
Nucleic Acids Res. 2001 May 1; 29(9):E45

Michael W. Pfaffl, Graham W. Horgan & Leo Dempfle   (2002)
Relative Expression Software Tool (REST©) for group wise comparison and statistical analysis
of relative expression results in real-time PCR
Nucleic Acids Research 2002 May 1; 30(9):E36

Rasmussen, R  (2001) 
Quantification on the LightCycler. In: Meuer, S, Wittwer, C, Nakagawara, K, eds. 
Rapid Cycle Real-time PCR, Methods and Applications Springer Press, Heidelberg; page 21-34.

Soong R, Beyser K, Basten O, Kalbe A, Rueschoff J, Tabiti K.  (2001)
Quantitative reverse transcription-polymerase chain reaction detection of
cytokeratin 20 in noncolorectal lymph nodes.
Clin Cancer Res. 2001 Nov;7(11): 3423-3429.

Anders Ståhlberg, Pierre Åman, Börje Ridell, Petter Mostad, and Mikael Kubista (2003)
A quantitative real-time PCR method for detection of B-Lymphocyte Monoclonality by comparison of kappa and lambda Immunoglobulin Light Chain Expression.
Clinical Chemistry 49(1): 51-59

Technical Note No. LC 13/2001 - Relative Quantification


  • classical block RT-PCR  &  competitive RT-PCR:
Brandstetter AM, Pfaffl MW, Hocquette JF, Gerrard DE, Picard B, Geay Y, Sauerwein H. ( 2000)
Effects of muscle type, castration, age, and compensatory growth rate on androgen 
receptor mRNA expression in bovine skeletal muscle. J Anim Sci. 2000 Mar;78(3):629-37.

Competitive quantitative RT-PCR  (11 / 1998)
by Ambion Inc.

Elsaesser, F., Pfaffl, M.W., Meyer, H.H.D., Serpek, B. and Sauerwein, H.  (2002)
Differences in the somatotropic axis, in blood cortisol, insulin and thyroid hormone concentrations between two pig genotypes with markedly divergent growth rates and the effects of growth hormone treatment. 
Animal Science, 74 (3): 423-430

Malucelli A, Sauerwein H, Pfaffl MW, Meyer HH.  (1996)
Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a 
template in reverse transcription-polymerase chain reaction.
J Steroid Biochem Mol Biol. 1996 Aug;58(5-6):563-8.

Pfaffl MW   (1997)   GERMAN

Analytische Untersuchungen zum allometrischen Muskelwachstum beim Rind: Quantifizierung der
Expression des insulinähnlichen Wachstumsfaktors-1 und rekombinante Herstellung des

ISBN 3-89675-198-0 · 1997,   20,5 x 14,5 cm · broschiert;   200 Seiten · 50 Abb. · 24 Tab.;  49,50 EUR
Endokrine Regulationsmechanismen bieten zur Erklärung allometrischer Muskelwachstumsvorgänge keine
ausreichende Erklärung. Das individuelle Muskelwachstum unterliegt offenbar auch einer lokalen Regulation. Als ein hier in Frage kommender Faktor wurde IGF-1 untersucht, für dessen mRNA eine kompetitive RT-PCR entwickelt wurde zur präzi-sen und sensitiven Quantifizierung der IGF-1 mRNA in Rindergeweben.
Kastration und Ernährung hatten unterschiedliche, gewebespezifische Effekte auf die IGF-1 Expression. Zur
Untersuchung der Regulation des Wachstumshormon-Rezeptors (GHR) und des Bindungsproteins (GHBP), wurden mit der rekombinanten Darstellung von GHBP in E. Coli die methodischen Voraussetzungen (Nutzung des Proteins als Antigen zur Antikörperdarstellung und als Standardsubstanz) geschaffen. Die Überexpression wurde optimiert, eine Reindarstellung sowie Charakterisierung des GHBP auch in Hinblick auf seine biologische Aktivität ist erfolgt.

Pfaffl M, Meyer HH, Sauerwein H.  (1998)
Quantification of insulin-like growth factor-1 (IGF-1) mRNA: development and validation of 
an internally standardised competitive reverse transcription-polymerase chain reaction. 
Exp Clin Endocrinol Diabetes. 1998;106(6):506-13.

M. Pfaffl; F. Schwarz & H. Sauerwein (1998)
Quantification of the insulin like growth factor-1 (IGF-1) mRNA: Modulation of growth intensity by feeding results in inter- and intra-tissue specific differences of IGF-1 mRNA expression in steers
Experimental and Clinical Endocrinology & Diabetes 106: 513-520.

Quantitative end-point RT-PCR gene expression measurement using the Agilent 2100    (2001)
Bioanalyzer and stndardised RT-PCR.  (2001)  "StaRT-PCR" technology is a standardized, 
quantitative method used to measure gene expression at the mRNA level in human tissues. 

Raeymaekers L. (2000) Basic principles of quantitative PCR.    (2000)
Mol Biotechnol. 2000 Jun;15(2):115-22.

Sauerwein H.; Pfaffl M.; Hagen-Mann K.; Malucelli A. & Meyer H.H.D. (1995)
Expression of estrogen and androgen receptor in the bovine gastrointestinal tract.
Deutsche tierärztliche Wochenschrift 1995, 102: 164-168.

SauerweinH., A. Brandstetter, M.W. Pfaffl, H.H.D. Meyer, E. Möstl, J. Handler & K. Arbeiter (1998)
Uterine Androgen Receptor mRNA Expression in Metestrous and Anestrous Bitches being healthy or suffering from pyometra.
Deutsche tierärztliche Wochenschrift 105: 173-208.

Wall SJ, Edwards DR. (2002)
Quantitative reverse transcription-polymerase chain reaction (RT-PCR): a comparison of primer-dropping, competitive, and real-time RT-PCRs
Anal Biochem  2002 Jan 15;300(2):269-73

Y. Cao, Houren Wei, Da Banghong, Michael W. Pfaffl  & Li Zhongyu (2002)
Insulin-like growth factor-1 gene cloning and protein expression in bovine trabecular Meshwork tissue and cells Department of Ophthalmolgy, Union Hospital of Tongji Medical University, Wuhan, China, and *Institute of Physiology, München Technical University, Freising-Weihenstephan, Germany
Journal of Huazhong University of Science and Technology 22(1): 69-72.

Y. Cao, M. W. Pfaffl, B. Da, H. Wei (2002)
Insulinartige Wachstumsfaktor-1- (IGF-1-)mRNA und IGF-1-Protein Expression in den Zellen des Trabekelwerks im Rinderauge
Bovine trabecular meshwork cells express insulin-like growth factor-1 (IGF-1) mRNA and protein
Der Ophthalmologe 2002, 99(7): 555-558


  • reference genes - housekeeping genes:
Bhatia, P, Taylor, WR, Greenberg, AH, and Wright, JA  (1994)
Comparison of glyceraldehyde-3-phosphate dehydrogenase and 28S-ribosomal 
RNA gene expression as RNA loading controls for northern blot analysis of cell lines of 
varying malignant potential. Anal Biochem. 216, 223-226.

Bereta, J, and Bereta, M   (1995) 
Stimulation of glyceraldehyde-3-phosphate dehydrogenase mRNA levels by endogenous
nitric oxide in ytokine-activated endothelium. Biochem Biophys Res Commun. 217, 363-369.

Bonini JA and Hofmann C.   (1991)
A rapid, accurate, nonradioactive method for quantitating RNA on agarose gels.
Biotechniques. 1991 Dec;11(6):708-710.

Chang, TJ, Juan, CC, Yin, PH, Chi, CW, and Tsay, HJ (1998) 
Up-regulation of beta-actin, cyclophilin and GAPDH in N1S1 rat hepatoma. 
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