Selection of reference genes for gene expression studies in porcine hepatic tissue using quantitative real-time polymerase chain reaction

• Autorzy: Pierzchala M. , Pareek C. S. , Urbanski P. , Goluch D. , Kamyczek M. , Rozycki M. , Kuryl J.
• Języki publikacji: EN

Abstrakty

EN

Quantitative real-time polymerase chain reaction (RT-qPCR) has become an indispensable technique for accurate determination of gene expression in variety of samples. Accurate and reliable quantification, however, depends on a proper normalization strategy. Normalization with multiple uniformly expressed reference genes is becoming the standard, although the most suitable reference genes dependent on the used experimental factors as well as the tissue or cell type studied. In this study, the stability of various reference genes was investigated in porcine hepatic tissue. The study was conducted on Polish Large White, Polish Landrace, Pietrain, Pulawska and Duroc pigs slaghtered at different ages. Nine reference genes (ACTB, B2M, GAPDH, HPRT1, RPL13A, SDHA, TBP, TOP2B and YWHAZ) were investigated on 180 mRNA samples of porcine hepatic tissue. Based on geNorm and NormFinder analysis, three most stable (HPRT1, TOP2B and TBP) and three moderately (GAPDH, ACTB and SDHA) stable reference genes were identified. The study provides a new panel of reference genes for normalization of the expression of a gene of interest in porcine liver tissue. It is concluded that the use of a single gene for normalization may lead to relatively large errors, so it is important to use multiple control genes based on a survey of potential reference genes applied to gene expression profiling studies of candidate genes for economic traits in pigs.

Słowa kluczowe

EN

selection; reference gene; gene expression; hepatic tissue; quantitative real-time polymerase chain reaction; pig; liver

• Wydawca: -
• Czasopismo: Animal Science Papers and Reports
• Rocznik: 2011
• Tom: 29
• Numer: 1
• Opis fizyczny: p.53-63,fig.,ref.

Twórcy

autor

Pierzchala M.

• Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzebiec, 05-552 Wolka Kosowska, Poland

autor

Pareek C. S.

autor

Urbanski P.

autor

Goluch D.

autor

Kamyczek M.

autor

Rozycki M.

autor

Kuryl J.

Bibliografia

• Andersen C.L., Jensen J.L., Orntoft T.F., 2004 – 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 Research 64, 5245-5250.
• Bustin S.A., Beaulieu J.F., Huggett J., Jaggi R., Kibenge F.S., Olsvik P.A., Penning L.C., Toegel S., 2010 – MIQE précis, Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Molecular Biology 11, 74.
• Bustin S.A., 2000 – Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25, 169-193.
• Bemeur C., Ste -Marie L., Desjardins P., Hazell A.S., Vachon L., Butterworth R., Montgomery J., 2004 – Decreased beta-actin mRNA expression in hyperglycemic focal cerebral ischemia in the rat. Neuroscience Letters 357, 211-214.
• Chomczynski P., Sacchi N., 1987 – Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156-159.
• DUVIGNEAU J.C., HART R.T., GROISS S., GEMEINER M., 2005 – Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. Journal of Immunological Methods 306, 1-2.
• Erkens T., Van Poucke M., Vandesompele J., Goossens K., Van Zeveren A.,Peelman L.J., 2006 – Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A. BMC Biotechnology 6, 41.
• Lee J.H., Fitzgerald J.B., Dimicco M.A., Grodzinsky A.J., 2005 – Mechanical injury of cartilage explants causes specific time-dependent changes in chondrocyte gene expression. Arthritis and Rheumatism 52, 2386-2395.
• Lee S., Jo M., Lee J., Koh S.S., Kim S., 2007 – Identification of novel universal housekeeping genes by statistical analysis of microarray data. The Journal of Biochemistry and Molecular Biology 40, 226-231.
• Gabrielsson B.G., Olofsson L.E., Sjögren A., Jernĺs M., Elander A., Lönn M., Rudemo M., Carlsson L.M., 2005 – Evaluation of reference genes for studies of gene expression in human adipose tissue. Obesity Research 13, 649-652.
• Gorodkin J., Cirera S., Hedegaard J., Gilchrist M.J., Panitz F., Jřrgensen C.,Scheibye -Knudsen K., Arvin T., Lumholdt S., Sawera M., Green T., Nielsen B.J.,Havgaard J.H., Rosenkilde C., Wang J., Li H., Li R., Liu B., Hu S., Dong W., Li W., Yu J., Wang J., Staefeldt H.H., Wernersson R., Madsen L.B., Thomsen B., Hornshřj H., Bujie Z., Wang X., Wang X, Bolund L., Brunak S., Yang H., Bendixen C.,Fredholm M., 2007 – Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags. Genome Biology 8, R45.
• Hellemans J., Mortier G., De Paepe A., Speleman F., Vandesompele J., 2007 -qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biology 8, R19.
• Jiwakanon J., Berg M., Fossum C., Persson E., Dalin A.M., 2009 – Influence of seminal plasma on TGFβ1 mRNA expression in the pig oviduct. Veterinary Immunology and Immunopathology 128, 1-3.
• Kadegowda A.K.G., Bionaz M., Thering B., Piperova L.S., Erdman R.A., Loor J.J.,2007 – Identification of internal control genes for quantitative polymerase chain reaction in mammary tissue of lactating cows receiving lipid supplements. Journal of Dairy Science 92, 2007-2019.
• Kuijk E.W., Puy L.D., van Tol H.T.A., Haagsman H.P., Colenbrander B., Roelen B.A.J., 2007 – Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos. BMC Developmental Biology 7, 58.
• Monaco E., Bionaz M., de Lima A.S., Hurley W.L., Loor J.J., Wheeler M.B.,2010 - Selection and reliability of internal reference genes for quantitative PCR verification of transcriptomics during the differentiation process of porcine adult mesenchymal stem cells. Stem Cell Research and Therapy 1, 7.
• Muráni E., Murániová M., Ponsuksili S., Schellander K., Wimmers K., 2007 – Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity. BMC Developmental Biology 1, 109.
• Nygard A.B., JOrgensen C.B., Cirera S., Fredholm .M., 2007 – Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Molecular Biology 8, 67.
• Ohl F., Jung M., Xu C., Stephan C., Rabien A., Burkhardt M., Nitsche A., Kristiansen G., Loening S.A., Radonic A., Jung K., 2005 – Gene expression studies in prostate cancer tissue, which reference gene should be selected for normalization? Journal of Molecular Medicine 83, 1014-1024.
• Qi S., Chen J., Guo R., Yu B., Chen D., 2009 – β-defensins gene expression in tissues of the crossbred and Tibetan pigs. Livestock Science 123, 161-168.
• Radonic A., Thulke S., Mackay I.M., Landt O., Siegert W., Nitsche A., 2004 –Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysics Research Communications 313, 856-862.
• Sieczkowska H., Zybert A., Krzęcio E., Antosik K., Koćwin -Podsiadła M.,Pierzchała M., Urbański P., 2010 – The expression of genes PKM2 and CAST in the muscle tissue of pigs differentiated by glycolytic potential and drip loss, with reference to the genetic group.Meat Science 84, 180-185.
• Suzuki T., Higgins P.J., Crawford D.R., 2000 – Control selection for RNA quantitation.Biotechniques 29, 332-337.
• Svobodová K., Bilek K., Knoll A., 2008 – Verification of reference genes for relative quantification of gene expression by real-time reverse transcription PCR in the pig. Journal of Applied Genetics 49, 263-265.
• Tramontana S., Bionaz M., Sharma A., Graugnard D.E., Cutler E.A., Ajmone -Marsan P., Hurley W.L., Loor J.J., 2008 – Internal controls for quantitative polymerase chain reaction of swine mammary glands during pregnancy and lactation. Journal of Dairy Science 91,3057-3066.
• 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. Genome Biology 3, 34.
• Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A.,Speleman F., 2004 – GeNorm software manual, update 6 Sept. 2004. (http,//medgen.ugent.be/~jvdesomp/genorm).
• Zhang X., Ding L., Sandford A.J., 2005 – Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC Molecular Biology 6, 4.