The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

This article has been cited by other articles:

				I. G. Yulug and B. Gur-Dedeoglu Functional genomics in translational cancer research: focus on breast cancer Brief Funct Genomic Proteomic, March 7, 2008; (2008) eln009v1. [Abstract] [Full Text] [PDF]

				M. Kratz, J. Q. Purnell, P. A. Breen, K. K. Thomas, K. M. Utzschneider, D. B. Carr, S. E. Kahn, J. P. Hughes, E. A. Rutledge, B. Van Yserloo, et al. Reduced Adipogenic Gene Expression in Thigh Adipose Tissue Precedes Human Immunodeficiency Virus-Associated Lipoatrophy J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 959 - 966. [Abstract] [Full Text] [PDF]

				M. Band, I. Shams, A. Joel, and A. Avivi Cloning and in vivo expression of vascular endothelial growth factor receptor 2 (Flk1) in the naturally hypoxia-tolerant subterranean mole rat FASEB J, January 1, 2008; 22(1): 105 - 112. [Abstract] [Full Text] [PDF]

				E. W. Dickson, C. P. Hogrefe, P. S. Ludwig, L. W. Ackermann, L. L. Stoll, and G. M. Denning Exercise enhances myocardial ischemic tolerance via an opioid receptor-dependent mechanism Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H402 - H408. [Abstract] [Full Text] [PDF]

				V. A. Lira, Q. A. Soltow, J. H. D. Long, J. L. Betters, J. E. Sellman, and D. S. Criswell Nitric oxide increases GLUT4 expression and regulates AMPK signaling in skeletal muscle Am J Physiol Endocrinol Metab, October 1, 2007; 293(4): E1062 - E1068. [Abstract] [Full Text] [PDF]

				S. S. Lakhman, X. Chen, V. Gonzalez-Covarrubias, E. G. Schuetz, and J. G. Blanco Functional Characterization of the Promoter of Human Carbonyl Reductase 1 (CBR1). Role of XRE Elements in Mediating the Induction of CBR1 by Ligands of the Aryl Hydrocarbon Receptor Mol. Pharmacol., September 1, 2007; 72(3): 734 - 743. [Abstract] [Full Text] [PDF]

				H. Muller, N. Schweitzer, O. Johren, P. Dominiak, and W. Raasch Angiotensin II stimulates the reactivity of the pituitary-adrenal axis in leptin-resistant Zucker rats, thereby influencing the glucose utilization Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E802 - E810. [Abstract] [Full Text] [PDF]

				D. Gnanashanmugam, M. S. Falkovitz-Halpern, A. Dodge, M. Fang, L. J. Wong, M. Esparza, R. Hammon, E. E. Rivas-Merelles, J. I. Santos, and Y. Maldonado Shedding and Reversion of Oral Polio Vaccine Type 3 in Mexican Vaccinees: Comparison of Mutant Analysis by PCR and Enzyme Cleavage to a Real-Time PCR Assay J. Clin. Microbiol., August 1, 2007; 45(8): 2419 - 2425. [Abstract] [Full Text] [PDF]

				L. Serrano, K. M. Halanych, and R. P. Henry Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crab Callinectes sapidus J. Exp. Biol., July 1, 2007; 210(13): 2320 - 2332. [Abstract] [Full Text] [PDF]

				X. Jiang, K. M. Saw, A. Eaves, and C. Eaves Instability of BCR-ABL Gene in Primary and Cultured Chronic Myeloid Leukemia Stem Cells J Natl Cancer Inst, May 2, 2007; 99(9): 680 - 693. [Abstract] [Full Text] [PDF]

				N. A. Janovick-Guretzky, H. M. Dann, D. B. Carlson, M. R. Murphy, J. J. Loor, and J. K. Drackley Housekeeping Gene Expression in Bovine Liver is Affected by Physiological State, Feed Intake, and Dietary Treatment J Dairy Sci, May 1, 2007; 90(5): 2246 - 2252. [Abstract] [Full Text] [PDF]

				P. Hou, Z. Chen, M. Ji, N. He, and Z. Lu Real-time PCR Assay for Ultrasensitive Quantification of DNA-Binding Proteins Clin. Chem., April 1, 2007; 53(4): 581 - 586. [Abstract] [Full Text] [PDF]

				C. L.H. Lo, S. P. Yip, P. K.C. Cheng, T. S.S. To, W. W.L. Lim, and P. H.M. Leung One-Step Rapid Reverse Transcription-PCR Assay for Detecting and Typing Dengue Viruses with GC Tail and Induced Fluorescence Resonance Energy Transfer Techniques for Melting Temperature and Color Multiplexing Clin. Chem., April 1, 2007; 53(4): 594 - 599. [Abstract] [Full Text] [PDF]

				D. Greenwood, D. J. Jagger, L.-C. Huang, N. Hoya, P. R. Thorne, S. S. Wildman, B. F. King, K. Pak, A. F. Ryan, and G. D. Housley P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea Development, April 1, 2007; 134(7): 1407 - 1417. [Abstract] [Full Text] [PDF]

				M. Zeitlinger, C. Marsik, I. Steiner, R. Sauermann, K. Seir, B. Jilma, O. Wagner, and C. Joukhadar Immunomodulatory effects of fosfomycin in an endotoxin model in human blood J. Antimicrob. Chemother., February 1, 2007; 59(2): 219 - 223. [Abstract] [Full Text] [PDF]

				K. Thamatrakoln and M. Hildebrand Analysis of Thalassiosira pseudonana Silicon Transporters Indicates Distinct Regulatory Levels and Transport Activity through the Cell Cycle Eukaryot. Cell, February 1, 2007; 6(2): 271 - 279. [Abstract] [Full Text] [PDF]

				P. Cheng, Y. Nefedova, C. A. Corzo, and D. I. Gabrilovich Regulation of dendritic-cell differentiation by bone marrow stroma via different Notch ligands Blood, January 15, 2007; 109(2): 507 - 515. [Abstract] [Full Text] [PDF]

				W. Cui, D. D. Taub, and K. Gardner qPrimerDepot: a primer database for quantitative real time PCR Nucleic Acids Res., January 12, 2007; 35(suppl_1): D805 - D809. [Abstract] [Full Text] [PDF]

				C. K. Tipsmark, J. A. Luckenbach, S. S. Madsen, and R. J. Borski IGF-I and branchial IGF receptor expression and localization during salinity acclimation in striped bass Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R535 - R543. [Abstract] [Full Text] [PDF]

				O. J. Jabado, G. Palacios, V. Kapoor, J. Hui, N. Renwick, J. Zhai, T. Briese, and W. I. Lipkin Greene SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments Nucleic Acids Res., December 2, 2006; 34(22): 6605 - 6611. [Abstract] [Full Text] [PDF]

				E. G. Grubbs, Z. Abdel-Wahab, D. S. Tyler, and S. K. Pruitt Utilizing Quantitative Polymerase Chain Reaction to Evaluate Prostate Stem Cell Antigen as a Tumor Marker in Pancreatic Cancer Ann. Surg. Oncol., December 1, 2006; 13(12): 1645 - 1654. [Abstract] [Full Text] [PDF]

				M. T. Sorensen, J. V. Norgaard, P. K. Theil, M. Vestergaard, and K. Sejrsen Cell Turnover and Activity in Mammary Tissue During Lactation and the Dry Period in Dairy Cows J Dairy Sci, December 1, 2006; 89(12): 4632 - 4639. [Abstract] [Full Text] [PDF]

				H. Beyan, M. R. Goodier, N. S. Nawroly, M. I. Hawa, S. A. Bustin, W. B. Ogunkolade, M. Londei, N. Yousaf, and R. D. G. Leslie Altered Monocyte Cyclooxygenase Response to Lipopolysaccharide in Type 1 Diabetes Diabetes, December 1, 2006; 55(12): 3439 - 3445. [Abstract] [Full Text] [PDF]

				B. Etschmann, K. S. Heipertz, A. von der Schulenburg, and M. Schweigel A vH+-ATPase is present in cultured sheep ruminal epithelial cells Am J Physiol Gastrointest Liver Physiol, December 1, 2006; 291(6): G1171 - G1179. [Abstract] [Full Text] [PDF]

				A. N. Harman, J. Wilkinson, C. R. Bye, L. Bosnjak, J. L. Stern, M. Nicholle, J. Lai, and A. L. Cunningham HIV Induces Maturation of Monocyte-Derived Dendritic Cells and Langerhans Cells J. Immunol., November 15, 2006; 177(10): 7103 - 7113. [Abstract] [Full Text] [PDF]

				B. C. Trainor and H. A. Hofmann Somatostatin Regulates Aggressive Behavior in an African Cichlid Fish Endocrinology, November 1, 2006; 147(11): 5119 - 5125. [Abstract] [Full Text] [PDF]

				A. De Ketelaere, K. Goossens, L. Peelman, and C. Burvenich Technical note: validation of internal control genes for gene expression analysis in bovine polymorphonuclear leukocytes. J Dairy Sci, October 1, 2006; 89(10): 4066 - 4069. [Abstract] [Full Text] [PDF]

				G. M. Anderson, D. R. Grattan, W. van den Ancker, and R. S. Bridges Reproductive Experience Increases Prolactin Responsiveness in the Medial Preoptic Area and Arcuate Nucleus of Female Rats Endocrinology, October 1, 2006; 147(10): 4688 - 4694. [Abstract] [Full Text] [PDF]

				G. M. Anderson, P. Beijer, A. S. Bang, M. A. Fenwick, S. J. Bunn, and D. R. Grattan Suppression of Prolactin-Induced Signal Transducer and Activator of Transcription 5b Signaling and Induction of Suppressors of Cytokine Signaling Messenger Ribonucleic Acid in the Hypothalamic Arcuate Nucleus of the Rat during Late Pregnancy and Lactation Endocrinology, October 1, 2006; 147(10): 4996 - 5005. [Abstract] [Full Text] [PDF]

				N. A. Henderson, G. M Cooke, and B. Robaire Region-specific expression of androgen and growth factor pathway genes in the rat epididymis and the effects of dual 5{alpha}-reductase inhibition. J. Endocrinol., September 1, 2006; 190(3): 779 - 791. [Abstract] [Full Text] [PDF]

				R. Karunakaran, K. Ebert, S. Harvey, M. E. Leonard, V. Ramachandran, and P. S. Poole Thiamine Is Synthesized by a Salvage Pathway in Rhizobium leguminosarum bv. viciae Strain 3841. J. Bacteriol., September 1, 2006; 188(18): 6661 - 6668. [Abstract] [Full Text] [PDF]

				A. Rodriguez, C. Diez, S. Ikeda, L.J. Royo, J.N. Caamano, C. Alonso-Montes, F. Goyache, I. Alvarez, N. Facal, and E. Gomez Retinoids during the in vitro transition from bovine morula to blastocyst Hum. Reprod., August 1, 2006; 21(8): 2149 - 2157. [Abstract] [Full Text] [PDF]

				S. Schulz, T. Hyslop, J. Haaf, C. Bonaccorso, K. Nielsen, M. E. Witek, R. Birbe, J. Palazzo, D. Weinberg, and S. A. Waldman A Validated Quantitative Assay to Detect Occult Micrometastases by Reverse Transcriptase-Polymerase Chain Reaction of Guanylyl Cyclase C in Patients with Colorectal Cancer Clin. Cancer Res., August 1, 2006; 12(15): 4545 - 4552. [Abstract] [Full Text] [PDF]

				W. B. Ogunkolade, B. J. Boucher, S. A. Bustin, J. M. Burrin, K. Noonan, N. Mannan, and G. A. Hitman Vitamin D Metabolism in Peripheral Blood Mononuclear Cells Is Influenced by Chewing "Betel Nut" (Areca catechu) and Vitamin D Status J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2612 - 2617. [Abstract] [Full Text] [PDF]

				M Kusakabe, I Nakamura, J Evans, P Swanson, and G Young Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes. J. Endocrinol., June 1, 2006; 189(3): 541 - 554. [Abstract] [Full Text] [PDF]

				S. M. Mense, A. Sengupta, M. Zhou, C. Lan, G. Bentsman, D. J. Volsky, and L. Zhang Gene expression profiling reveals the profound upregulation of hypoxia-responsive genes in primary human astrocytes Physiol Genomics, May 16, 2006; 25(3): 435 - 449. [Abstract] [Full Text] [PDF]

				R. Singh, S. Ellard, A. Hattersley, and L. W. Harries Rapid and Sensitive Real-Time Polymerase Chain Reaction Method for Detection and Quantification of 3243A>G Mitochondrial Point Mutation J. Mol. Diagn., May 1, 2006; 8(2): 225 - 230. [Abstract] [Full Text] [PDF]

				K. P. Choe, J. Havird, R. Rose, K. Hyndman, P. Piermarini, and D. H. Evans COX2 in a euryhaline teleost, Fundulus heteroclitus: primary sequence, distribution, localization, and potential function in gills during salinity acclimation J. Exp. Biol., May 1, 2006; 209(9): 1696 - 1708. [Abstract] [Full Text] [PDF]

				P. Bosi, M. Mazzoni, S. De Filippi, P. Trevisi, L. Casini, G. Petrosino, and G. Lalatta-Costerbosa A Continuous Dietary Supply of Free Calcium Formate Negatively Affects the Parietal Cell Population and Gastric RNA Expression for H+/K+-ATPase in Weaning Pigs J. Nutr., May 1, 2006; 136(5): 1229 - 1235. [Abstract] [Full Text] [PDF]

				A. B. Dydensborg, E. Herring, J. Auclair, E. Tremblay, and J.-F. Beaulieu Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon Am J Physiol Gastrointest Liver Physiol, May 1, 2006; 290(5): G1067 - G1074. [Abstract] [Full Text] [PDF]

				G. Mazon, S. Campoy, I. Erill, and J. Barbe Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria lexA gene. Microbiology, April 1, 2006; 152(Pt 4): 1109 - 1118. [Abstract] [Full Text] [PDF]

				N. P. Harrington, O. P. Surujballi, and J. F. Prescott CERVINE (CERVUS ELAPHUS) CYTOKINE mRNA QUANTIFICATION BY REAL-TIME POLYMERASE CHAIN REACTION. J. Wildl. Dis., April 1, 2006; 42(2): 219 - 233. [Abstract] [Full Text] [PDF]

				J.-P. Pichon, B. Bonnaud, P. Cleuziat, and F. Mallet Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling Nucleic Acids Res., March 22, 2006; 34(6): e46 - e46. [Abstract] [Full Text] [PDF]

				W. J. Lubbe, Z. Y. Zhou, W. Fu, D. Zuzga, S. Schulz, R. Fridman, R. J. Muschel, S. A. Waldman, and G. M. Pitari Tumor epithelial cell matrix metalloproteinase 9 is a target for antimetastatic therapy in colorectal cancer. Clin. Cancer Res., March 15, 2006; 12(6): 1876 - 1882. [Abstract] [Full Text] [PDF]

				S. M Dougherty, W. Mazhawidza, A. R Bohn, K. A Robinson, K. A Mattingly, K. A Blankenship, M. O Huff, W. G McGregor, and C. M Klinge Gender difference in the activity but not expression of estrogen receptors {alpha} and {beta} in human lung adenocarcinoma cells. Endocr. Relat. Cancer, March 1, 2006; 13(1): 113 - 134. [Abstract] [Full Text] [PDF]

				B. E. Stahli, H. Greutert, S. Mei, P. Graf, K. Frischknecht, M. Stalder, L. Englberger, A. Kunzli, L. Scharer, T. F. Luscher, et al. Absence of histamine-induced nitric oxide release in the human radial artery: implications for vasospasm of coronary artery bypass vessels Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1182 - H1189. [Abstract] [Full Text] [PDF]

				G. E. Byfield, H. Xue, and R. G. Upchurch Two Genes from Soybean Encoding Soluble {Delta}9 Stearoyl-ACP Desaturases Crop Sci., February 24, 2006; 46(2): 840 - 846. [Abstract] [Full Text] [PDF]

				T. Ishii, A. M. Wallace, X. Zhang, J. Gosselink, R. T. Abboud, J. C. English, P. D. Pare, and A. J. Sandford Stability of housekeeping genes in alveolar macrophages from COPD patients Eur. Respir. J., February 1, 2006; 27(2): 300 - 306. [Abstract] [Full Text] [PDF]

				S. Dames, L. K. Bromley, M. Herrmann, M. Elgort, M. Erali, R. Smith, and K. V. Voelkerding A Single-Tube Nucleic Acid Extraction, Amplification, and Detection Method Using Aluminum Oxide J. Mol. Diagn., February 1, 2006; 8(1): 16 - 21. [Abstract] [Full Text] [PDF]

				R. P. Radcliff, B. L. McCormack, D. H. Keisler, B. A. Crooker, and M. C. Lucy Partial Feed Restriction Decreases Growth Hormone Receptor 1A mRNA Expression in Postpartum Dairy Cows J Dairy Sci, February 1, 2006; 89(2): 611 - 619. [Abstract] [Full Text] [PDF]

				O. V. Nainan, G. Xia, G. Vaughan, and H. S. Margolis Diagnosis of Hepatitis A Virus Infection: a Molecular Approach Clin. Microbiol. Rev., January 1, 2006; 19(1): 63 - 79. [Abstract] [Full Text] [PDF]

				J. Chen, D. A. Rider, and R. Ruan Identification of Valid Housekeeping Genes and Antioxidant Enzyme Gene Expression Change in the Aging Rat Liver J. Gerontol. A Biol. Sci. Med. Sci., January 1, 2006; 61(1): 20 - 27. [Abstract] [Full Text] [PDF]

				M. E. Witek, K. Nielsen, R. Walters, T. Hyslop, J. Palazzo, S. Schulz, and S. A. Waldman The Putative Tumor Suppressor Cdx2 Is Overexpressed by Human Colorectal Adenocarcinomas Clin. Cancer Res., December 15, 2005; 11(24): 8549 - 8556. [Abstract] [Full Text] [PDF]

				K. Tiedemann, B. Olander, E. Eklund, L. Todorova, M. Bengtsson, M. Maccarana, G. Westergren-Thorsson, and A. Malmstrom Regulation of the chondroitin/dermatan fine structure by transforming growth factor-{beta}1 through effects on polymer-modifying enzymes Glycobiology, December 1, 2005; 15(12): 1277 - 1285. [Abstract] [Full Text] [PDF]

				F. Fiorani, A. L. Umbach, and J. N. Siedow The Alternative Oxidase of Plant Mitochondria Is Involved in the Acclimation of Shoot Growth at Low Temperature. A Study of Arabidopsis AOX1a Transgenic Plants Plant Physiology, December 1, 2005; 139(4): 1795 - 1805. [Abstract] [Full Text] [PDF]

				M. A. Cheatham, J. Zheng, K. H. Huynh, G. G. Du, J. Gao, J. Zuo, E. Navarrete, and P. Dallos Cochlear function in mice with only one copy of the prestin gene J. Physiol., November 15, 2005; 569(1): 229 - 241. [Abstract] [Full Text] [PDF]

				M. Stewart, M. Desport, N. Hartaningsih, and G. Wilcox TaqMan Real-Time Reverse Transcription-PCR and JDVp26 Antigen Capture Enzyme-Linked Immunosorbent Assay To Quantify Jembrana Disease Virus Load during the Acute Phase of In Vivo Infection J. Clin. Microbiol., November 1, 2005; 43(11): 5574 - 5580. [Abstract] [Full Text] [PDF]

				P. Luciani, S. Gelmini, E. Ferrante, A. Lania, S. Benvenuti, S. Baglioni, G. Mantovani, I. Cellai, F. Ammannati, A. Spada, et al. Expression of the Antiapoptotic Gene Seladin-1 and Octreotide-Induced Apoptosis in Growth Hormone-Secreting and Nonfunctioning Pituitary Adenomas J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6156 - 6161. [Abstract] [Full Text] [PDF]

				K. P. Choe, A. Kato, S. Hirose, C. Plata, A. Sindic, M. F. Romero, J. B. Claiborne, and D. H. Evans NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2005; 289(5): R1520 - R1534. [Abstract] [Full Text] [PDF]

				C. M. Luquet, D. Weihrauch, M. Senek, and D. W. Towle Induction of branchial ion transporter mRNA expression during acclimation to salinity change in the euryhaline crab Chasmagnathus granulatus J. Exp. Biol., October 1, 2005; 208(19): 3627 - 3636. [Abstract] [Full Text] [PDF]