Suitability of endogenous reference genes for gene expression studies with human intraocular endothelial cells

BMC Research Notes

Winfried M Amoaku¹  Elizabeth Anne Stewart¹  Ruoxin Wei¹ 

[1] University of Nottingham, Division of Ophthalmology and Visual Sciences, B Floor, Eye and ENT Building, Queen’s Medical Centre, Nottingham, NG7 2UH, UK

关键词: Polymerase chain reaction;    Endogenous control;    Housekeeping gene;    Endothelial cells;    Retina;    Choroid;   

Others  :  1144808
DOI  :  10.1186/1756-0500-6-46

 received in 2012-10-08, accepted in 2013-01-30,  发布年份 2013

【 摘 要 】

Background

The use of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has become widely applied as a method to measure transcript abundance. In order to be reflective of biological processes during health and disease this method is dependent on normalisation of data against stable endogenous controls. However, these genes can vary in their stability in different cell types. The importance of reference gene validation for a particular cell type is now well recognised and is an important step in any gene expression study.

Results

Cultured primary human choroidal and retinal endothelial cells were treated with the immunostimulant polyinosinic: polycytidylic acid or untreated. qRT-PCR was used to quantify the expression levels of 10 commonly used endogenous control genes, TBP, HPRT1, GAPDH, GUSB, PPIA, RPLP0, B2M, 18S rRNA, PGK1 and ACTB. Three different mathematical algorithms, GeNorm, NormFinder, and BestKeeper were used to analyse gene stability to give the most representative validation. In choroidal endothelial cells the most stable genes were ranked as HPRT1 and GUSB by GeNorm and NormFinder and HPRT1 and PPIA by BestKeeper. In retinal endothelial cells the most stable genes ranked were TBP and PGK1 by GeNorm and NormFinder and HPRT1 by BestKeeper. The least stable gene for both cell types was 18S with all 3 algorithms.

Conclusions

We have identified the most stable endogenous control genes in intraocular endothelial cells. It is suggested future qRT-PCR studies using these cells would benefit from adopting the genes identified in this study as the most appropriate endogenous control genes.

【 授权许可】

   
2013 Wei et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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【 参考文献 】

• [1]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, 7:95-96.
• [2]Suzuki T, Higgins PJ, Crawford DR: Control selection for RNA quantitation. Biotechniques 2000, 29:332-337.
• [3]Greer S, Honeywell R, Geletu M, Arulanandam R, Raptis L: Housekeeping genes; expression levels may change with density of cultured cells. J Immunol Methods 2010, 355:76-79.
• [4]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:RESEARCH0034.
• [5]Bustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000, 25:169-193.
• [6]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, 75:291-295.
• [7]Warrington JA, Nair A, Mahadevappa M, Tsyganskaya M: Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics 2000, 2:143-147.
• [8]Gebhardt FM, Scott HA, Dodd PR: Housekeepers for accurate transcript expression analysis in Alzheimer's disease autopsy brain tissue. Alzheimers Dement 2010, 6:465-474.
• [9]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, 37:112-114. 116, 118–119
• [10]Akira S, Uematsu S, Takeuchi O: Pathogen recognition and innate immunity. Cell 2006, 124:783-801.
• [11]Kluwe J, Mencin A, Schwabe RF: Toll-like receptors, wound healing, and carcinogenesis. J Mol Med (Berl) 2009, 87:125-138.
• [12]Akira S: TLR signaling. Curr Top Microbiol Immunol 2006, 311:1-16.
• [13]Lee MT, Hooper LC, Kump L, Hayashi K, Nussenblatt R, Hooks JJ, Detrick B: Interferon-beta and adhesion molecules (E-selectin and s-intracellular adhesion molecule-1) are detected in sera from patients with retinal vasculitis and are induced in retinal vascular endothelial cells by Toll-like receptor 3 signalling. Clin Exp Immunol 2007, 147:71-80.
• [14]Kumar MV, Nagineni CN, Chin MS, Hooks JJ, Detrick B: Innate immunity in the retina: Toll-like receptor (TLR) signaling in human retinal pigment epithelial cells. J Neuroimmunol 2004, 153:7-15.
• [15]Dasu MR, Devaraj S, Zhao L, Hwang DH, Jialal I: High glucose induces toll-like receptor expression in human monocytes: mechanism of activation. Diabetes 2008, 57:3090-3098.
• [16]Kleinman ME, Yamada K, Takeda A, Chandrasekaran V, Nozaki M, Baffi JZ, Albuquerque RJ, Yamasaki S, Itaya M, Pan Y, et al.: Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 2008, 452:591-597.
• [17]Yang Z, Stratton C, Francis PJ, Kleinman ME, Tan PL, Gibbs D, Tong Z, Chen H, Constantine R, Yang X, et al.: Toll-like receptor 3 and geographic atrophy in age-related macular degeneration. N Engl J Med 2008, 359:1456-1463.
• [18]University of Kentucky Research Foundation: Toll Like Receptor (TLR) Stimulation for Ocular Angiogenesis and Macular Degeneration. 2009. US Patent No. us 0238793
• [19]Park DW, Baek K, Lee JG, Park YK, Kim JH, Kim JR, Baek SH: Activation of toll-like receptor 4 modulates vascular endothelial growth factor synthesis through prostacyclin-IP signaling. Biochem Biophys Res Commun 2007, 362:1090-1095.
• [20]van Beijnum JR, Buurman WA, Griffioen AW: Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1). Angiogenesis 2008, 11:91-99.
• [21]Paone A, Galli R, Gabellini C, Lukashev D, Starace D, Gorlach A, De Cesaris P, Ziparo E, Del Bufalo D, Sitkovsky MV, et al.: Toll-like receptor 3 regulates angiogenesis and apoptosis in prostate cancer cell lines through hypoxia-inducible factor 1 alpha. Neoplasia 2010, 12:539-549.
• [22]Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 2004, 26:509-515.
• [23]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, 64:5245-5250.
• [24]Foldager CB, Munir S, Ulrik-Vinther M, Soballe K, Bunger C, Lind M: Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes. BMC Mol Biol 2009, 10:94. BioMed Central Full Text
• [25]Robinson TL, Sutherland IA, Sutherland J: Validation of candidate bovine reference genes for use with real-time PCR. Vet Immunol Immunopathol 2007, 115:160-165.
• [26]de Kok JB, Roelofs RW, Giesendorf BA, Pennings JL, Waas ET, Feuth T, Swinkels DW, Span PN: Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest 2005, 85:154-159.
• [27]Stewart EA, Samaranayake GJ, Browning AC, Hopkinson A, Amoaku WM: Comparison of choroidal and retinal endothelial cells: characteristics and response to VEGF isoforms and anti-VEGF treatments. Exp Eye Res 2011, 93:761-766.