Normalizing RT-qPCR Data: Are We Getting the Right Answers? An Appraisal of Normalization Approaches and Internal Reference Genes from a Case Study in the Finfish Lates calcarifer

Marine Biotechnology

Volumn 13, Issue 2, 2011, Pages 170-180

  de Santis, Christian ^a   Smith Keune, Carolyn ^a   Jerry, Dean R ^a  

^a JAMES COOK UNIVERSITY   (Australia)

Author keywords

Fasting; Lates calcarifer; Quantitative real time RT PCR; Reference genes; RT qPCR normalization methods

Indexed keywords

FASTING; LATES CALCARIFER; QUANTITATIVE REAL-TIME RT-PCR; REFERENCE GENES; RT-QPCR NORMALIZATION METHODS;

GROWTH KINETICS; NUCLEIC ACIDS; POLYMERASE CHAIN REACTION; TRANSCRIPTION;

GENE EXPRESSION REGULATION;

FISH PROTEIN; MESSENGER RNA;

COST-BENEFIT ANALYSIS; FASTING; GENE EXPRESSION; GENETIC ANALYSIS; GROWTH REGULATOR; PERCIFORM; POLYMERASE CHAIN REACTION; REAL TIME;

ANIMAL; ARTICLE; DIET RESTRICTION; EVALUATION; FOOD DEPRIVATION; GENE EXPRESSION; GENETICS; METABOLISM; METHODOLOGY; PERCIFORMES; PHYSIOLOGY; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; STANDARD;

ANIMALS; FASTING; FISH PROTEINS; FOOD DEPRIVATION; GENE EXPRESSION; PERCIFORMES; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION; RNA, MESSENGER;

LATES CALCARIFER;

EID: 79955531827     PISSN: 14362228     EISSN: None     Source Type: Journal    
DOI: 10.1007/s10126-010-9277-z     Document Type: Article

References (37)

1. Ali M, Nicieza A, Wootton RJ (2003) Compensatory growth in fishes: a response to growth depression. Fish Fish 4: 147-190.
2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403-410.
3. Amali AA, Lin CJF, Chen YH, Wang WL, Gomg HY, Lee CY, Ko YL, Lu JK, Her GM, Chen TT, Wu JL (2004) Up-regulation of muscle-specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knock-down of myostatin-1. Dev Dynam 229: 847-856.
4. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611-622.
5. Chauvigne F, Gabillard JC, Weil C, Rescan PY (2003) Effect of refeeding on IGFI, IGFII, IGF receptors, FGF2, FGF6, and myostatin mRNA expression in rainbow trout myotomal muscle. Gen Comp Endocr 132: 209-215.
6. De Santis C, Jerry DR (2007) Candidate growth genes in finfish-where should we be looking? Aquaculture 272: 22-38.
7. De Santis C, Evans BS, Smith-Keune C, Jerry DR (2008) Molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer) myostatin gene. BMC Genomics 9: 82.
8. Filby AL, Tyler CR (2007) Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish. BMC Mol Biol 8: 10.
9. Gaylord TG, Mackenzie DS, Gatlin DM (2001) Growth performance, body composition and plasma thyroid hormone status of channel catfish (Ictalurus punctatus) in response to short-term feed deprivation and refeeding. Fish Physiol Biochem 24: 73-79.
10. GenBank. http://www. ncbi. nlm. nih. gov/Genbank.
11. Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, van Wuytswinkel O (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Pl Biotech J 6: 609-618.
12. Hagen O, Fernandes JMO, Solberg C, Johnston IA (2009) Expression of growth-related genes in muscle during fasting and refeeding of juvenile Atlantic halibut, Hippoglossus hippoglossus L. Comp Biochem Physiol B 152: 47-53.
13. Hayward RS, Noltie DB, Wang N (1997) Use of compensatory growth to double hybrid sunfish growth rates. T Am Fish Soc 126: 316-322.
14. Infante C, Matsuoka MP, Asensio E, Canavate JP, Reith M, Manchado M (2008) Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR. BMC Mol Biol 9: 28.
15. Johansen KA, Overturf K (2006) Alterations in expression of genes associated with muscle metabolism and growth during nutritional restriction and refeeding in rainbow trout. Comp Biochem Phys B 144: 119-127.
16. Jorgensen SM, Kleveland EJ, Grimholt U, Gjoen T (2006) Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon. Mar Biotech 8: 398-408.
17. Kumar RS, Ijiri S, Trant JM (2000) Changes in the expression of genes encoding steroidogenic enzymes in the channel catfish (Ictalurus punctatus) ovary throughout a reproductive cycle. Biol Reprod 63: 1676-1682.
18. Lee SJ (2004) Regulation of muscle mass by myostatin. Annu Rev Cell Dev Biol 20: 61-86.
19. Lee SJ, McPherron AC (2001) Regulation of myostatin activity and muscle growth. PNAS 98: 9306-9311.
20. Ma K, Mallidis C, Bhasin S, Mahabadi V, Artaza J, Gonzalez-Cadavid N, Arias J, Salehian B (2003) Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression. Am J Physiol-Endoc M 285: E363-E371.
21. Marshall O (2004) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20(15): 2471-2472.
22. Murray A, Lawrence GP (1993) How should the repeatability of clinical measurements be analyzed-an assessment of analysis techniques with data from cardiovascular autonomic function-tests. Q J Med 86: 831-836.
23. Nikki J, Pirhonen J, Jobling M, Karjalainen J (2004) Compensatory growth in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), held individually. Aquaculture 235: 285-296.
24. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: 2002-2007.
25. 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, pp 21-34.
26. Resuehr D, Spiess AN (2003) A real-time polymerase chain reaction-based evaluation of cDNA synthesis priming methods. Anal Biochem 322: 287-291.
27. Rodgers BD, Weber GM, Kelley KM, Levine MA (2003) Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates. Am J Physiol Reg-I 284: 1277-1286.
28. Ruan WJ, Lai MD (2007) Actin, a reliable marker of internal control? Clin Chim Acta 385: 1-5.
29. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press Vol 1: 7. 10.
30. Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, Griffiths LR (2001) Beta-actin-an unsuitable internal control for RT-PCR. Mol Cell Probes 15: 307-311.
31. Small BC, Murdock CA, Bilodeau-Bourgeois AL, Peterson BC, Waldbieser GC (2008) Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions. Comp Bioch Phys B 151: 296-304.
32. SPSS Inc. (2006) SPSS Base 16. 0 for Windows User's Guide. Chicago IL.
33. Terova G, Bernardini G, Binelli G, Gornati R, Saroglia M (2006) cDNA encoding sequences for myostatin and FGF6 in sea bass (Dicentrarchus labrax, L.) and the effect of fasting and refeeding on their abundance levels. Dom Anim Endocr 30: 304-319.
34. Vandesompele J, De Preter K, Pattyn F, Poppe B, van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Gen Biol 3(7): 3, RESEARCH0034.
35. Weber TE, Bosworth BG (2005) Effects of 28 day exposure to cold temperature or feed restriction on growth, body composition, and expression of genes related to muscle growth and metabolism in channel catfish. Aquaculture 246: 483-492.
36. Xu YX, Zhu ZY, Lo LC, Wang CM, Lin G, Feng F, Yue GH (2006) Characterization of two parvalbumin genes and their association with growth traits in Asian seabass (Lates calcarifer). Anim Genet 37: 266-268.
37. Zhong QW, Zhang QQ, Wang ZG, Qi J, Chen YJ, Li S, Sun YY, Li CM, Lan X (2008) Expression profiling and validation of potential reference genes during Paralichthys olivaceus embryogenesis. Mar Biotechnol 10: 310-318.