Selecting Suitable Reference Gene for RT-qPCR Normalization in FFPE Breast Cancer Tissues for Duhok/Iraqi Women
Keywords:
RT-qPCR , Reference gene , Breast cancer , FFPE tissue blocks, geNorm, Norm finder , Best Keeper , comparative ΔCt methodAbstract
Nowadays, with the continuously increasing advances in understanding cancer system biology. It has led to the discovery of valuable disease-specific biomarkers. By targeting these markers using molecular-based techniques a highly specific and sensitive measurements can be obtained. Molecular diagnosis through PCR-based methods and next-generation sequencing (NGS) can detect many diseases caused by DNA and RNA changes that cannot be detected through antibody-based techniques. RT-qPCR is a valuable tool in measuring biomarker’s transcriptional signature and will have a great potential in personalized medicine or molecular diagnostics. In related gene expression analysis, using a suitable reference gene is crucial for the accurate interpretation of the results. For this reason, ref. gene should be validated for particular tissue, cell and experimental conditions. This study was conducted to identify and validate the most suitable housekeeping gene (HKG) as a reference (ref.) gene to obtain accurate normalization for AXL receptor tyrosine kinase gene expression in Formalin-Fixed Paraffin-Embedded “FFPE” of breast cancer tissues. Based on the five different statistical algorithms used, ACTB was the least variable ref. gene and ꞵ-GLOBIN showed the highest variability. Norm finder, Best Keeper and the comparative ΔCt method suggested to use ACTB as a ref. gene in our experiment while geNorm suggested to use the combination of both ACTB and UBC since they had the same exact stability value. In conclusion, Reffinder suggested that ACTB had the least geomean of ranking values of the four used methods showing the least variance among the other genes and the three groups of our samples.
References
Christian Grätz ; Bui Maria L. U.; Thaqi Granit; Kirchner Benedikt; Loewe Robert P.and Pfaffl Michael W.; "Obtaining Reliable RT-qPCR Results in Molecular Diagnostics—MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery." Life, 12(3):386,2022.
Kabanova S.; P. Kleinbongard; J. Volkmer; B. Andrée; M. Kelmand T. W. Jax; "Gene expression analysis of human red blood cells." Int. J. Med. Sci., 6(4):156-9,2009.
de Kok Jacques B.; Rian W. Roelofs; Belinda A. Giesendorf; Jeroen L. Pennings; Erwin T. Waas; Ton Feuth; Dorine W. Swinkelsand Paul N. Span; "Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes." Lab. Invest., 85(1):154-159,2005.
Song J.; Z. Bai; W. Han; J. Zhang; H. Meng; J. Bi; X. Ma; S. Hanand Z. Zhang; "Identification of suitable reference genes for qPCR analysis of serum microRNA in gastric cancer patients." DDS, 57(4):897-904,2012.
Czechowski T.; M. Stitt; T. Altmann; M. K. Udvardiand W. R. Scheible; "Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis." Plant Physiol., 139(1):5-17,2005.
Drury S.; H. Andersonand M. Dowsett; "Selection of REFERENCE genes for normalization of qRT-PCR data derived from FFPE breast tumors." Diagn. Mol. Pathol., 18(2):103-7,2009.
García-Pérez Omar; Leticia Melgar-Vilaplana; Elizabeth Córdoba-Lanúsand Ricardo Fernández-de-Misa; "Gene Expression Studies in Formalin-Fixed Paraffin-Embedded Samples of Cutaneous Cancer: The Need for Reference Genes". Curr. Issues Mol. Biol., 43(3):2167-2176,2021.
Song J.; J. Cho; J. Parkand J. H. Hwang; "Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages." BMC Genomics, 23(1):585,2022.
Xie F.; J. Wangand B. Zhang; "RefFinder: a web-based tool for comprehensively analyzing and identifying reference genes." Funct. Integr. Genomics, 23(2):125,2023.
Fu J.; L. Bian; L. Zhao; Z. Dong; X. Gao; H. Luan; Y. Sunand H. Song; "Identification of genes for normalization of quantitative real-time PCR data in ovarian tissues." Acta Biochim Biophys Sin, 42(8):568-74,2010.
Tilli Tatiana M.; Cláudio da Silva Castro; Jack A. Tuszynskiand Nicolas Carels; "A strategy to identify housekeeping genes suitable for analysis in breast cancer diseases." BMC Genomics, 17(1):639,2016.
Nielsen Steffen; Niels Bassler; Leszek Grzanka; Jan Swakon; Pawel Olko; Christian Nicolaj Andreassen; Jan Alsnerand Brita Singers Sørensen; "Optimal reference genes for normalization of qPCR gene expression data from proton and photon irradiated dermal fibroblasts." Sci. Rep., 8(1):12688,2018.
Irie Nanami; Katsuhiko Warita; Jiro Tashiro; Yaxuan Zhou; Takuro Ishikawa; Zoltán N. Oltvaiand Tomoko Warita; "Expression of housekeeping genes varies depending on mevalonate pathway inhibition in cancer cells." Heliyon, 9(7):e18017,2023.
Mosaferi E.; N. Alizadeh Gharamaleki; L. Farzadi; J. Majidi; Z. Babaloo; T. Kazemi; M. Ramezani; M. Tabatabaei; H. Ahmadi; L. Aghebati Malekiand B. Baradaran; "The Study of HLA-G Gene and Protein Expression in Patients with Recurrent Miscarriage." Adv. Pharm. Bull., 9(1):70-75,2019.
Linardić M.and S. A. Braybrook; "Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses." PLoS One, 16(4):e0233249,2021.
Pfaffl M. W.; A. Tichopad; C. Prgometand T. P. Neuvians; "Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations." Biotechnol. Lett., 26(6):509-15,2004.
Formosa A.; E. Acton; A. Lee; P. Turgeon; S. Izhar; P. Plant; J. N. Tsoporis; S. Soussi; U. Trahtemberg; A. Bakerand C. C. Dos Santos; "Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness." PLoS One, 18(8):e0286871,2023.
Li R.; W. Xie; S. Wang; Q. Wu; N. Yang; X. Yang; H. Pan; X. Zhou; L. Bai; B. Xu; X. Zhouand Y. Zhang; "Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)." PLoS One, 8(1):e53006,2013.
Andersen C. L.; J. L. Jensenand T. F. Ørntoft; "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):5245-50,2004.
Christensen Julie N.; Henrik Schmidt; Torben Steinicheand Mette Madsen; "Identification of robust reference genes for studies of gene expression in FFPE melanoma samples and melanoma cell lines." Melanoma Res., 30(1),2020.
Xie Fuliang; Peng Xiao; Dongliang Chen; Lei Xuand Baohong Zhang; "miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs." Plant M.B., 80(1):75-84,2012.
Silveira G. O.; M. S. Amaral; H. S. Coelho; L. F. Maciel; A. S. A. Pereira; G. G. O. Olberg; P. A. Miyasato; E. Nakanoand S. Verjovski-Almeida; "Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR." Sci. Rep., 11(1):16816,2021.
Bustin S. A.; V. Benes; J. A. Garson; J. Hellemans; J. Huggett; M. Kubista; R. Mueller; T. Nolan; M. W. Pfaffl; G. L. Shipley; J. Vandesompeleand C. T. Wittwer; "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments." Clin. Chem., 55(4):611-22,2009.
Pennock Nathan D.; Sonali Jindal; Wesley Horton; Duanchen Sun; Jayasri Narasimhan; Lucia Carbone; Suzanne S. Fei; Robert Searles; Christina A. Harrington; Julja Burchard; Sheila Weinmann; Pepper Schedinand Zheng Xia; "RNA-seq from archival FFPE breast cancer samples: molecular pathway fidelity and novel discovery." BMC Med Genomics, 12(1):195,2019.
Evers D. L.; J. He; Y. H. Kim; J. T. Masonand T. J. O'Leary; "Paraffin embedding contributes to RNA aggregation, reduced RNA yield, and low RNA quality." J Mol Diagn. , 13(6):687-94,2011.
Macabelli C. H.; R. M. Ferreira; L. U. Gimenes; N. A. de Carvalho; J. G. Soares; H. Ayres; M. L. Ferraz; Y. F. Watanabe; O. Y. Watanabe; J. R. Sangalli; L. C. Smith; P. S. Baruselli; F. V. Meirellesand M. R. Chiaratti; "Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer." PLoS One, 9(3):e93287,2014.
Wang J.; X. Yu; X. Cao; L. Tan; B. Jia; R. Chenand J. Li; "GAPDH: A common housekeeping gene with an oncogenic role in pan-cancer." Comput Struct Biotechnol J. , 21:4056-4069,2023.
Downloads
Published
Issue
Section
License
Copyright (c) 2024 Sada S. Mohammed, Hanaa AL-Mahmoodi, Mayada I. Yalda
This work is licensed under a Creative Commons Attribution 4.0 International License.
.jpg)
