Abstract
Real-time PCR is an indispensable technique for mRNA expression analysis but conclusions depend on appropriate reference gene selection. However, while reference gene selection has been a topic of publications, this issue is often disregarded when measuring target mRNA expression. Therefore, we (1) evaluated the frequency of appropriate reference gene selection, (2) suggest an easy-to-use tool for least variability reference gene selection, (3) demonstrate application of this tool, and (4) show effects on target gene expression profiles. All 2015 published articles in Naunyn-Schmiedeberg’s Archives of Pharmacology were screened for the use of quantitative real-time PCR analysis and selection of reference genes. Target gene expression (Vegfa, Grk2, Sirt4, and Timp3) in H9c2 cells was analyzed following various interventions (hypoxia, hyperglycemia, and/or isoflurane exposure with and without subsequent hypoxia) in relation to putative reference genes (Actb, Gapdh, B2m, Sdha, and Rplp1) using the least variability method vs. an arbitrarily selected but established reference gene. In the vast majority (18 of 21) of papers, no information was provided regarding selection of an appropriate reference gene. In only 1 of 21 papers, a method of appropriate reference gene selection was described and in 2 papers reference gene selection remains unclear. The method of reference gene selection had major impact on interpretation of target gene expression. With hypoxia, for instance, the least variability gene was Rplp1 and target gene expression (Vefga) heavily showed a 2-fold up-regulation (p = 0.022) but no change (p = 0.3) when arbitrarily using Gapdh. Frequency of appropriate reference gene selection in this journal is low, and we propose our strategy for reference gene selection as an easy tool for proper target gene expression.
Similar content being viewed by others
Reference
Adamzik M, Schafer S, Frey UH, Becker A, Kreuzer M, Winning S, et al. (2013) The NFKB1 promoter polymorphism (−94ins/delATTG) alters nuclear translocation of NF-kappaB1 in monocytes after lipopolysaccharide stimulation and is associated with increased mortality in sepsis. Anesthesiology 118(1):123–133
Andersen CL, Jensen JL, Orntoft TF (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 Res 64(15):5245–5250
Banai S, Shweiki D, Pinson A, Chandra M, Lazarovici G, Keshet E (1994) Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis. Cardiovasc Res 28(8):1176–1179
Bas A, Forsberg G, Hammarstrom S, Hammarstrom ML (2004) Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol 59(6):566–573
Berger MM, Huhn R, Oei GT, Heinen A, Winzer A, Bauer I, et al. (2010) Hypoxia induces late preconditioning in the rat heart in vivo. Anesthesiology 113(6):1351–1360
Bollmann F, Casper I, Henke J, Pautz A (2012) qRT-PCR: a method and its difficulties. Naunyn Schmiedeberg's Arch Pharmacol 385(10):949–951
Buldak L, Labuzek K, Buldak RJ, Machnik G, Boldys A, Okopien B (2015) Exenatide (a GLP-1 agonist) improves the antioxidative potential of in vitro cultured human monocytes/macrophages. Naunyn Schmiedeberg's Arch Pharmacol 388(9):905–919
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25(2):169–193
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622
Cabiati M, Raucci S, Caselli C, Guzzardi MA, D’Amico A, Prescimone T, et al. (2012) Tissue-specific selection of stable reference genes for real-time PCR normalization in an obese rat model. J Mol Endocrinol 48(3):251–260
Cacciola NA, Sepe R, Forzati F, Federico A, Pellecchia S, Malapelle U, et al. (2015) Restoration of CBX7 expression increases the susceptibility of human lung carcinoma cells to irinotecan treatment. Naunyn Schmiedeberg's Arch Pharmacol 388(11):1179–1186
Carotenuto M, de Antonellis P, Chiarolla CM, Attanasio C, Damiani V, Boffa I, et al. (2015) A therapeutic approach to treat prostate cancer by targeting Nm23-H1/h-Prune interaction. Naunyn Schmiedeberg's Arch Pharmacol 388(2):257–269
Cheng WC, Chang CW, Chen CR, Tsai ML, Shu WY, Li CY, et al. (2011) Identification of reference genes across physiological states for qRT-PCR through microarray meta-analysis. PLoS One 6(2):e17347
Cheng HB, Bo Y, Shen WX, Ren XG, Tan JN, Jia ZR, et al. (2015) Longikaurin E induces apoptosis of pancreatic cancer cells via modulation of the p38 and PI3K/AKT pathways by ROS. Naunyn Schmiedeberg's Arch Pharmacol 388(6):623–634
Choi SS, Park EK, Kwack MH, Sung YK (2015) Effects of dexamethasone, a synthetic glucocorticoid, on human periodontal ligament stem cells. Naunyn Schmiedeberg's Arch Pharmacol 388(9):991–995
de Barbi MM, Uppala R, Zhang Y, Van HB, Goetzman ES (2014) Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells. PLoS One 9(8):e106028
de Lucia C, Femminella GD, Gambino G, Pagano G, Allocca E, Rengo C, et al. (2014) Adrenal adrenoceptors in heart failure. Front Physiol 5:246
Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques 37(1):112–119
Elshal M, Abu-Elsaad N, El-Karef A, Ibrahim TM (2015) The multi-kinase inhibitor pazopanib targets hepatic stellate cell activation and apoptosis alleviating progression of liver fibrosis. Naunyn Schmiedeberg's Arch Pharmacol 388(12):1293–1304
Fiorentino L, Cavalera M, Menini S, Marchetti V, Mavilio M, Fabrizi M, et al. (2013) Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay. EMBO Mol Med 5(3):441–455
Foster SR, Porrello ER, Stefani M, Smith NJ, Molenaar P, CG d R, et al. (2015) Cardiac gene expression data and in silico analysis provide novel insights into human and mouse taste receptor gene regulation. Naunyn Schmiedeberg's Arch Pharmacol 388(10):1009–1027
Frey UH, Lieb W, Erdmann J, Savidou D, Heusch G, Leineweber K, et al. (2008a) Characterization of the GNAQ promoter and association of increased Gq expression with cardiac hypertrophy in humans. Eur Heart J 29(7):888–897
Frey UH, Bachmann HS, Peters J, Siffert W (2008b) PCR-amplification of GC-rich regions: 'slowdown PCR'. Nat Protoc 3(8):1312–1317
Gachon C, Mingam A, Charrier B, Real-time PCR (2004) What relevance to plant studies? J Exp Bot 55(402):1445–1454
Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60(2):487–493
Hakami NY, Wong H, Shah MH, Dusting GJ, Jiang F, Peshavariya HM (2015) Smad-independent pathway involved in transforming growth factor beta1-induced Nox4 expression and proliferation of endothelial cells. Naunyn Schmiedeberg's Arch Pharmacol 388(3):319–326
Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6(4):279–284
Imeri F, Blanchard O, Jenni A, Schwalm S, Wunsche C, Zivkovic A, et al. (2015) FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P(3) and phosphoinositide 3-kinase. Naunyn Schmiedeberg's Arch Pharmacol 388(12):1283–1292
Kim BW, Choi M, Kim YS, Park H, Lee HR, Yun CO, et al. (2008) Vascular endothelial growth factor (VEGF) signaling regulates hippocampal neurons by elevation of intracellular calcium and activation of calcium/calmodulin protein kinase II and mammalian target of rapamycin. Cell Signal 20(4):714–725
Kozera B, Rapacz M (2013) Reference genes in real-time PCR. J Appl Genet 54(4):391–406
Kuznetsov AV, Javadov S, Sickinger S, Frotschnig S, Grimm M (2015) H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation. Biochim Biophys Acta 1853(2):276–284
Lee SH, Wolf PL, Escudero R, Deutsch R, Jamieson SW, Thistlethwaite PA (2000) Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med 342(9):626–633
Li QF, Wang XR, Yang YW, DS S (2006) Up-regulation of hypoxia inducible factor 1alpha by isoflurane in Hep3B cells. Anesthesiology 105(6):1211–1219
Malfitano C, Alba Loureiro TC, Rodrigues B, Sirvente R, Salemi VM, Rabechi NB, et al. (2010) Hyperglycaemia protects the heart after myocardial infarction: aspects of programmed cell survival and cell death. Eur J Heart Fail 12(7):659–667
Malhotra R, Tyson DW, Rosevear HM, Brosius FC, III. Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells. BMC Cardiovasc Disord 2008;8:9.
Marfella R, D’Amico M, Di FC, Piegari E, Nappo F, Esposito K, et al. (2002) Myocardial infarction in diabetic rats: role of hyperglycaemia on infarct size and early expression of hypoxia-inducible factor 1. Diabetologia 45(8):1172–1181
McGinn S, Poronnik P, Gallery ED, Pollock CA (2004) The effects of high glucose and atorvastatin on endothelial cell matrix production. Diabet Med 21(10):1102–1107
Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh LVEGF (2006) Receptor signalling—in control of vascular function. Nat Rev Mol Cell Biol 7(5):359–371
Qian K, Xu H, Dai T, Shi K (2015) Effects of Tanshinone IIA on osteogenic differentiation of mouse bone marrow mesenchymal stem cells. Naunyn Schmiedeberg's Arch Pharmacol 388(11):1201–1209
Reuter T, Warta R, Theile D, Meid AD, Rigalli JP, Mogler C, et al. (2015) Role of NR1I2 (pregnane X receptor) polymorphisms in head and neck squamous cell carcinoma. Naunyn Schmiedeberg's Arch Pharmacol 388(11):1141–1150
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491
Samatiwat P, Prawan A, Senggunprai L, Kukongviriyapan V (2015) Repression of Nrf2 enhances antitumor effect of 5-fluorouracil and gemcitabine on cholangiocarcinoma cells. Naunyn Schmiedeberg's Arch Pharmacol 388(6):601–612
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108
Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46(1–2):69–81
Serafim KG, Navarro SA, Zarpelon AC, Pinho-Ribeiro FA, Fattori V, Cunha TM, et al. (2015) Bosentan, a mixed endothelin receptor antagonist, inhibits superoxide anion-induced pain and inflammation in mice. Naunyn Schmiedeberg's Arch Pharmacol 388(11):1211–1221
Sereni F, Dal Monte M, Filippi L, Bagnoli P (2015) Role of host beta1- and beta2-adrenergic receptors in a murine model of B16 melanoma: functional involvement of beta3-adrenergic receptors. Naunyn Schmiedeberg's Arch Pharmacol 388(12):1317–1331
Shin SH, Bak SS, Kim MK, Sung YK, Kim JC (2015) Baicalin, a flavonoid, affects the activity of human dermal papilla cells and promotes anagen induction in mice. Naunyn Schmiedeberg's Arch Pharmacol 388(5):583–586
Soltek S, Karakhanova S, Golovastova M, D’Haese JG, Serba S, Nachtigall I, et al. (2015) Anti-tumor properties of the cGMP/protein kinase G inhibitor DT3 in pancreatic adenocarcinoma. Naunyn Schmiedeberg's Arch Pharmacol 388(11):1121–1128
Stahlberg A, Hakansson J, Xian X, Semb H, Kubista M (2004) Properties of the reverse transcription reaction in mRNA quantification. Clin Chem 50(3):509–515
Suarez L, Bilal U, Bordallo J, Cantabrana B, Sanchez M (2015) Androgens block outward potassium currents and decrease spontaneous action potentials in GH3 cells. Naunyn Schmiedeberg's Arch Pharmacol 388(1):67–78
Tan SC, Carr CA, Yeoh KK, Schofield CJ, Davies KE, Clarke K (2012) Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors. Mol Biol Rep 39(4):4857–4867
Thellin O, Zorzi W, Lakaye B, De BB, Coumans B, Hennen G, et al. (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75(2–3):291–295
Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, et al. (2002) Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 309(2):293–300
Tsuzuki T, Okada H, Cho H, Tsuji S, Nishigaki A, Yasuda K, et al. (2012) Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1alpha and inhibits stromal cell-derived factor-1 in human endometrial stromal cells. Hum Reprod 27(2):523–530
Vandesompele J, De PK, Pattyn F, Poppe B, Van RN, De PA, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3(7):RESEARCH0034.
Weiss J, Theile D, Haefeli WE (2012) Rifampicin alters the expression of reference genes used to normalize real-time quantitative RT-PCR data. Naunyn Schmiedeberg's Arch Pharmacol 385(10):1025–1034
Werner K, Kalble S, Wolter S, Schneider EH, Buschauer A, Neumann D, et al. (2015) Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor. Naunyn Schmiedeberg's Arch Pharmacol 388(10):1039–1052
Woodall MC, Ciccarelli M, Woodall BP, Koch WJG (2014) Protein-coupled receptor kinase 2: a link between myocardial contractile function and cardiac metabolism. Circ Res 114(10):1661–1670
XY Y, Geng YJ, Liang JL, Lin QX, Lin SG, Zhang S, et al. (2010) High levels of glucose induce apoptosis in cardiomyocyte via epigenetic regulation of the insulin-like growth factor receptor. Exp Cell Res 316(17):2903–2909
Yao L, Chen X, Tian Y, Lu H, Zhang P, Shi Q, et al. (2012) Selection of housekeeping genes for normalization of RT-PCR in hypoxic neural stem cells of rat in vitro. Mol Biol Rep 39(1):569–576
Zaitone SA, Barakat BM, Bilasy SE, Fawzy MS, Abdelaziz EZ, Farag NE (2015) Protective effect of boswellic acids versus pioglitazone in a rat model of diet-induced non-alcoholic fatty liver disease: influence on insulin resistance and energy expenditure. Naunyn Schmiedeberg's Arch Pharmacol 388(6):587–600
Zhang HF, Li TB, Liu B, Lou Z, Zhang JJ, Peng JJ, et al. (2015) Inhibition of myosin light chain kinase reduces NADPH oxidase-mediated oxidative injury in rat brain following cerebral ischemia/reperfusion. Naunyn Schmiedeberg's Arch Pharmacol 388(9):953–963
Zhao P, Zuo Z (2004) Isoflurane preconditioning induces neuroprotection that is inducible nitric oxide synthase-dependent in neonatal rats. Anesthesiology 101(3):695–703
Zhu J, Zhang H, Zhu Z, Zhang Q, Ma X, Cui Z, et al. (2015) Effects and mechanism of flavonoids from Astragalus complanatus on breast cancer growth. Naunyn Schmiedeberg's Arch Pharmacol 388(9):965–972
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Klenke, S., Renckhoff, K., Engler, A. et al. Easy-to-use strategy for reference gene selection in quantitative real-time PCR experiments. Naunyn-Schmiedeberg's Arch Pharmacol 389, 1353–1366 (2016). https://doi.org/10.1007/s00210-016-1305-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00210-016-1305-8