Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Springer Science and Business Media LLC - 2010
Guangmao Shen1, Hong‐Bo Jiang1, Xiaona Wang1, Jinjun Wang1
1Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing, 400715, P. R. China

Tóm tắt

Abstract Background

Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, Bactrocera dorsalis (Hendel).

 Results

Two different programs, geNorm and Normfinder, were used to analyze the data. According to geNorm, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by Normfinder are quite the same as the results with geNorm; α-TUB is always one of the most stable genes in each sample validated by the two programs.

 Conclusions

In this study, we validated the suitable reference genes for gene expression profiling in different tissues of B. dorsalis. Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in B. dorsalis, but also will serve as a resource to screen reference genes for gene expression studies in any other insects.

Từ khóa


Tài liệu tham khảo

Hong SY, Seo PJ, Yang MS, Xiang F, Park CM: Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol. 2008, 8: 112- 10.1186/1471-2229-8-112

Wong ML, Medrano JF: Real-time PCR for mRNA quantization. BioTechniques. 2005, 39 (1): 75-85. 10.2144/05391RV01

Huggett J, Dheda K, Bustin S, Zumla A: Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005, 6 (4): 279-284. 10.1038/sj.gene.6364190

Strube C, Buschbaum S, Wolken S, Schnieder T: Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus. Gene. 2008, 425 (1-2): 36-43. 10.1016/j.gene.2008.08.001

Bustin SA, Benes V, Nolan T, Pfaffl MW: Quantitative real-time RT-PCR - a perspective. J Mol Endocrinol. 2005, 34 (3): 597-601. 10.1677/jme.1.01755

Folkersen L, Kurtovic S, Razuvaev A, Agardh HE, Gabrielsen A, Paulsson-Berne G: Endogenous control genes in complex vascular tissue samples. BMC Genomics. 2009, 10: 516- 10.1186/1471-2164-10-516

Thorrez L, Van Deun K, Tranchevent LC, Van Lommel L, Engelen K, Marchal K, Moreau Y, Van Mechelen I, Schuit F: Using ribosomal protein genes as reference: a tale of caution. PLoS One. 2008, 3 (3): e1854- 10.1371/journal.pone.0001854

Butte AJ, Dzau VJ, Glueck SB: Further defining housekeeping, or "maintenance, " genes Focus on "A compendium of gene expression in normal human tissues". Physiol Genomics. 2001, 7 (2): 95-96.

Infante C, Matsuoka MP, Asensio E, Canavate JP, Reith M, Manchado M: Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR. BMC Mol Biol. 2008, 9: 28- 10.1186/1471-2199-9-28

Ruan WJ, Lai MD: Actin, a reliable marker of internal control?. Clin Chim Acta. 2007, 385 (1-2): 1-5. 10.1016/j.cca.2007.07.003

Jain M, Nijhawan A, Tyagi AK, Khurana JP: Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun. 2006, 345 (2): 646-651. 10.1016/j.bbrc.2006.04.140

Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A: Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques. 2004, 37 (1): 112-119.

Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, Griffiths LR: Beta-actin-an unsuitable internal control for RT-PCR. Mol Cell Probes. 2001, 15 (5): 307-311. 10.1006/mcpr.2001.0376

Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E: Housekeeping genes as internal standards: use and limits. J Biotechnol. 1999, 75 (2-3): 291-295. 10.1016/S0168-1656(99)00163-7

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3 (7): research0034.0031-0034.0011. 10.1186/gb-2002-3-7-research0034. 10.1186/gb-2002-3-7-research0034

Clarke AR, Armstrong KF, Carmichael AE, Milne JR, Raghu S, Roderick GK, Yeates DK: Invasive phytophagous pests arising through a recent tropical evolutionary radiation: The Bactrocera dorsalis complex of fruit flies. Annu Rev Entomol. 2005, 50: 293-319. 10.1146/annurev.ento.50.071803.130428

Yu DJ, Xu L, Nardi F, Li JG, Zhang RJ: The complete nucleotide sequence of the mitochondrial genome of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae). Gene. 2007, 396 (1): 66-74. 10.1016/j.gene.2007.02.023

Zimowska GJ, Nirmala X, Handler AM: The beta 2-tubulin gene from three tephritid fruit fly species and use of its promoter for sperm marking. Insect Biochem Mol Biol. 2009, 39 (8): 508-515. 10.1016/j.ibmb.2009.05.004

Chen S, Dai S, Lu K, Chang C: Female-specific double sex dsRNA interrupts yolk protein gene expression and reproductive ability in oriental fruit fly, Bactrocera dorsalis (Hendel). Insect Biochem Mol Biol. 2008, 38 (2): 155-165. 10.1016/j.ibmb.2007.10.003

Chen P, Ye H: Relationship among five populations of Bactrocera dorsalis based on mitochondrial DNA sequences in western Yunnan, China. J Appl Entomol. 2008, 132 (7): 530-537. 10.1111/j.1439-0418.2008.01302.x. 10.1111/j.1439-0418.2008.01302.x

Yu DJ, Chen ZL, Zhang RJ, Yin WY: Real-time qualitative PCR for the inspection and identification of Bactrocera philippinensis and Bactrocera occipitalis (Diptera: Tephritidae) using SYBR Green assay. Raffles Bull Zool. 2005, 53 (1): 73-78.

Dai SM, Lin CC, Chang C: Polymorphic microsatellite DNA markers from the Oriental Fruit Fly Bactrocera dorsalis (Hendel). Mol Ecol Notes. 2004, 4 (4): 629-631. 10.1111/j.1471-8286.2004.00732.x. 10.1111/j.1471-8286.2004.00732.x

Chou M, Haymer DS, Feng H, Mau RFL, Hsu J: Potential for insecticide resistance in populations of Bactrocera dorsalis in Hawaii: spinosad susceptibility and molecular characterization of a gene associated with organophosphate resistance. Entomol Exp Appl. 2010, 134 (3): 296-303. 10.1111/j.1570-7458.2009.00962.x. 10.1111/j.1570-7458.2009.00962.x

Hsu J, Wu W, Haymer DS, Liao H, Feng H: Alterations of the acetylcholinesterase enzyme in the oriental fruit fly Bactrocera dorsalis are correlated with resistance to the organophosphate insecticide fenitrothion. Insect Biochem Mol Biol. 2008, 38 (2): 146-154. 10.1016/j.ibmb.2007.10.002

Hsu J, Haymer DS, Wu W, Feng H: Mutations in the acetylcholinesterase gene of Bactrocera dorsalis associated with resistance to organophosphorus insecticides. Insect Biochem Mol Biol. 2006, 36 (5): 396-402. 10.1016/j.ibmb.2006.02.002

Chung H, Sztal T, Pasricha S, Sridhar M, Batterham P, Daborn PJ: Characterization of Drosophila melanogaster cytochrome P450 genes. Proc Natl Acad Sci. 2009, 106 (14): 5731-5736. 10.1073/pnas.0812141106

Yang J, McCart C, Woods DJ, Terhzaz S, Greenwood KG, ffrench-Constant RH, Dow JAT: A Drosophila systems approach to xenobiotic metabolism. Physiol Genomics. 2007, 30 (3): 223-231. 10.1152/physiolgenomics.00018.2007

Dow J, Davies S: The Malpighian tubule: rapid insights from post-genomic biology. J Insect Physiol. 2006, 52 (4): 365-378. 10.1016/j.jinsphys.2005.10.007

Petersen RA, Zangerl AR, Berenbaum MR, Schuler MA: Expression of CYP6B1 and CYP6B3 cytochrome P450 monooxygenases and furanocoumarin metabolism in different tissues of Papilio polyxenes (Lepidoptera: Papilionidae). Insect Biochem Mol Biol. 2001, 31 (6-7): 679-690. 10.1016/S0965-1748(00)00174-0

Arbeitman MN, Fleming AA, Siegal ML, Null BH, Baker BS: A genomic analysis of Drosophila somatic sexual differentiation and its regulation. Development. 2004, 131 (9): 2007-2021. 10.1242/dev.01077

McGraw LA, Gibson G, Clark AG, Wolfner MF: Genes Regulated by Mating, Sperm, or Seminal Proteins in Mated Female Drosophila melanogaster. Curr Biol. 2004, 14 (16): 1509-1514. 10.1016/j.cub.2004.08.028

Jiang H, Liu Y, Tang P, Zhou A, Wang J: Validation of endogenous reference genes for insecticide-induced and developmental expression profiling of Liposcelis bostrychophila (Psocoptera: Liposcelididae). Mol Biol Rep. 2009, 37 (2): 1019-1029. 10.1007/s11033-009-9803-0. 10.1007/s11033-009-9803-0

Scharlaken B, de Graaf DC, Goossens K, Brunain M, Peelman LJ, Jacobs FJ: Reference gene selection for insect expression studies using quantitative real-time PCR: The head of the honeybee, Apis mellifera, after a bacterial challenge. J Insect Sci. 2008, 8: 33-10.1673/031.008.3301. 10.1673/031.008.3301

Exposito-Rodriguez M, Borges AA, Borges-Perez A, Perez JA: Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol. 2008, 8: 131- 10.1186/1471-2229-8-131

Bustin SA: Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol. 2002, 29 (1): 17-10.1677/jme.0.0290023. 10.1677/jme.0.0290023

Perez R, Tupac-Yupanqui I, Dunner S: Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue. BMC Mol Biol. 2008, 9: 79- 10.1186/1471-2199-9-79

Zhu J, He FH, Song SH, Wang J, Yu J: How many human genes can be defined as housekeeping with current expression data?. BMC Genomics. 2008, 9: 172- 10.1186/1471-2164-9-172

He JQ, Sandford AJ, Wang IM, Stepaniants S, Knight DA, Kicic A, Stick SM, Pare PD: Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells. Eur Respir J. 2008, 32 (3): 755-762. 10.1183/09031936.00129107

Coulson DTR, Brockbank S, Quinn JG, Murphy S, Ravid R, Irvine GB, Johnston JA: Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue. BMC Mol Biol. 2008, 9: 46- 10.1186/1471-2199-9-46

Cicinnati VR, Shen QL, Sotiropoulos GC, Radtke A, Gerken G, Beckebaum S: Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR. BMC Cancer. 2008, 8: 350- 10.1186/1471-2407-8-350

Nijhof A, Balk J, Postigo M, Jongejan F: Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86. BMC Mol Biol. 2009, 10: 112- 10.1186/1471-2199-10-112

Boda E, Pini A, Hoxha E, Parolisi R, Tempia F: Selection of Reference Genes for Quantitative Real-time RT-PCR Studies in Mouse Brain. J Mol Neurosci. 2009, 37 (3): 238-253. 10.1007/s12031-008-9128-9

Fernandes JMO, Mommens M, Hagen O, Babiak I, Solberg C: Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development. Comp Biochem Physiol B-Biochem Mol Biol. 2008, 150 (1): 23-32. 10.1016/j.cbpb.2008.01.003

Ahn K, Huh JW, Park SJ, Kim DS, Ha HS, Kim YJ, Lee JR, Chang KT, Kim HS: Selection of internal reference genes for SYBR green qRT-PCR studies of rhesus monkey (Macaca mulatta) tissues. BMC Mol Biol. 2008, 9: 78- 10.1186/1471-2199-9-78

Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M: Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol. 2009, 10: 11- 10.1186/1471-2199-10-11

Jian B, Liu B, Bi YR, Hou WS, Wu CX, Han TF: Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol. 2008, 9: 59- 10.1186/1471-2199-9-59

Brunner AM, Yakovlev IA, Strauss SH: Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol. 2004, 4: 14- 10.1186/1471-2229-4-14

Van Hiel M, Van Wielendaele P, Temmerman L, Van Soest S, Vuerinckx K, Huybrechts R, Broeck J, Simonet G: Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions. BMC Mol Biol. 2009, 10: 56- 10.1186/1471-2199-10-56

Nygard AB, Jorgensen CB, Cirera S, Fredholm M: Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol Biol. 2007, 8: 67- 10.1186/1471-2199-8-67

Zhang XZ, Ding L, Sandford AJ: Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Mol Biol. 2005, 6: 4- 10.1186/1471-2199-6-4

Andersen CL, Jensen JL, Orntoft TF: Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets. Cancer Res. 2004, 64 (15): 5245-5250. 10.1158/0008-5472.CAN-04-0496

Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J: qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biology. 2007, 8 (2): 14-10.1186/gb-2007-8-2-r19. 10.1186/gb-2007-8-2-r19

Olsvik PA, Lie KK, Jordal AEO, Nilsen TO, Hordvik I: Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol Biol. 2005, 6: 21- 10.1186/1471-2199-6-21

Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, Bustin SA, Orlando C: Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem. 2002, 309: 293-300. 10.1016/S0003-2697(02)00311-1

Tang RY, Dodd A, Lai D, McNabb WC, Love DR: Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin. 2007, 39 (5): 384-390. 10.1111/j.1745-7270.2007.00283.x

Robinson TL, Sutherland IA, Sutherland J: Validation of candidate bovine reference genes for use with real-time PCR. Vet Immunol Immunopathol. 2007, 115 (1-2): 160-165. 10.1016/j.vetimm.2006.09.012

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT: The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin Chem. 2009, 55 (4): 611-622. 10.1373/clinchem.2008.112797

Thông tin
Thông tin xuất bản

Springer Science and Business Media LLC

Thông tin tác giả