Reference gene selection for head and neck squamous cell carcinoma gene expression studies

被引:45
作者
Lallemant, Benjamin [1 ,2 ,3 ,4 ]
Evrard, Alexandre [2 ,3 ,4 ,5 ]
Combescure, Christophe [6 ,7 ]
Chapuis, Heliette [8 ]
Chambon, Guillaume [1 ]
Raynal, Caroline [2 ,3 ,4 ,5 ]
Reynaud, Christophe [1 ]
Sabra, Omar [1 ]
Joubert, Dominique [2 ,3 ,4 ]
Hollande, Frederic [2 ,3 ,4 ]
Lallemant, Jean-Gabriel [1 ]
Lumbroso, Serge [2 ,3 ]
Brouillet, Jean-Paul [2 ,3 ]
机构
[1] Ctr Hosp Univ Nimes, Serv ORL & Chirurg Maxillofaciale, F-30029 Nimes 9, France
[2] CNRS, UMR 5203, Inst Genom Fonctionnelle, F-34094 Montpellier, France
[3] INSERM, U661, F-34094 Montpellier, France
[4] Univ Montpellier I, F-34094 Montpellier, France
[5] Ctr Hosp Univ Nimes, Biochim Lab, F-30029 Nimes 9, France
[6] Ctr Hosp Univ Nimes, Serv Informat Med & Biostat, F-30029 Nimes 9, France
[7] Univ Hosp Geneva, CRC Serv Epidemiol Clin, CH-1211 Geneva 14, Switzerland
[8] Ctr Hosp Univ Nimes, Serv Anat & Cytopathol, F-30029 Nimes 9, France
关键词
TRANSCRIPTION-PCR DATA; HOUSEKEEPING GENES; RT-PCR; NORMALIZATION; QUANTIFICATION; IDENTIFICATION; VALIDATION; STRATEGIES; BLADDER;
D O I
10.1186/1471-2199-10-78
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Background: It is no longer adequate to choose reference genes blindly. We present the first study that defines the suitability of 12 reference genes commonly used in cancer studies (ACT, ALAS, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPL27, RPS29, SHAD and TBP) for the normalization of quantitative expression data in the field of head and neck squamous cell carcinoma (HNSCC). Results: Raw expression levels were measured by RT-qPCR in HNSCC and normal matched mucosa of 46 patients. We analyzed the expression stability using geNorm and NormFinder and compared the expression levels between subgroups. In HNSCC and/or normal mucosa, the four best normalization genes were ALAS, GAPDH, RPS18 and SHAD and the most stable combination of two genes was GAPDH-SHAD. We recommend using KALPHA-TBP for the study of T1T2 tumors, RPL27-SHAD for T3T4 tumors, KALPHA-SHAD for N0 tumors, and ALAS-TBP for N+ tumors. ACT, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPS29, SHAD and TBP were slightly misregulated (<1.7-fold) between tumor and normal mucosa but can be used for normalization, depending on the resolution required for the assay. Conclusion: In the field of HNSCC, this study will guide researchers in selecting the most appropriate reference genes from among 12 potentially suitable reference genes, depending on the specific setting of their experiments.
引用
收藏
页数:10
相关论文
共 25 条
[1]   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 [J].
Andersen, CL ;
Jensen, JL ;
Orntoft, TF .
CANCER RESEARCH, 2004, 64 (15) :5245-5250
[2]   Introduction to the bootstrap world [J].
Boos, DD .
STATISTICAL SCIENCE, 2003, 18 (02) :168-174
[3]   Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems [J].
Bustin, SA .
JOURNAL OF MOLECULAR ENDOCRINOLOGY, 2002, 29 (01) :23-39
[4]   The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization [J].
Dheda, K ;
Huggett, JF ;
Chang, JS ;
Kim, LU ;
Bustin, SA ;
Johnson, MA ;
Rook, GAW ;
Zumla, A .
ANALYTICAL BIOCHEMISTRY, 2005, 344 (01) :141-143
[5]   Normalization strategies for mRNA expression data in cartilage research [J].
Fundel, K. ;
Haag, J. ;
Gebhard, P. M. ;
Zimmer, R. ;
Aigner, T. .
OSTEOARTHRITIS AND CARTILAGE, 2008, 16 (08) :947-955
[6]   Evaluation of reference genes for studies of gene expression in human adipose tissue [J].
Gabrielsson, BG ;
Olofsson, LE ;
Sjögren, A ;
Jernås, M ;
Elander, A ;
Lönn, M ;
Rudemo, M ;
Carlsson, LMS .
OBESITY RESEARCH, 2005, 13 (04) :649-652
[7]   Comparative study of different standardization concepts in quantitative competitive reverse transcription-PCR assays [J].
Haberhausen, G ;
Pinsl, J ;
Kuhn, CC ;
Markert-Hahn, C .
JOURNAL OF CLINICAL MICROBIOLOGY, 1998, 36 (03) :628-633
[8]   Equivalence test in quantitative reverse transcription polymerase chain reaction:: confirmation of reference genes suitable for normalization [J].
Haller, F ;
Kulle, B ;
Schwager, S ;
Gunawan, B ;
von Heydebreck, A ;
Sültmann, H ;
Füzesi, L .
ANALYTICAL BIOCHEMISTRY, 2004, 335 (01) :1-9
[9]   qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data [J].
Hellemans, Jan ;
Mortier, Geert ;
De Paepe, Anne ;
Speleman, Frank ;
Vandesompele, Jo .
GENOME BIOLOGY, 2007, 8 (02)
[10]   Real-time RT-PCR normalisation; strategies and considerations [J].
Huggett, J ;
Dheda, K ;
Bustin, S ;
Zumla, A .
GENES AND IMMUNITY, 2005, 6 (04) :279-284