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Journal of animal science and technology2015; 57; 18; doi: 10.1186/s40781-015-0050-8

GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells.

Abstract: Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, β-actin and β2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Methods: Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. Results: The expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p < 0.05). Differences in expression level of β-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, β-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. Conclusions: This study demonstrated that GAPDH and especially β-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.
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Publication Date: 2015-05-07 PubMed ID: 26290738PubMed Central: PMC4540241DOI: 10.1186/s40781-015-0050-8Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research article addresses the reliability of GAPDH, β-actin, and β2-microglobulin reference genes in gene expression studies on equine (horse) adipose- and marrow-derived mesenchymal stem cells (MSCs). The article concludes that these reference genes differ considerably in their expression across the two types of MSCs, making them unreliable for comparative gene expression analysis.

Study Purpose and Methodology

In molecular biology, quantitative real-time reverse transcription PCR (qRT-PCR) is a significant technique for analyzing gene expression. An essential step in this process is the choice of an appropriate internal control gene for data normalization. Traditionally, three common reference genes—GAPDH, β-actin, and β2-microglobulin—are used for data normalization. However, the researchers sought to understand if the source of MSCs—in this case, equine bone marrow and adipose tissue—impacts the expression levels of these reference genes.

  • Adipose tissue and bone marrow samples were collected from three mares, and MSCs were then isolated and cultured.
  • Upon reaching the 3rd culture passage (P3), the total RNA from these cells was extracted for cDNA synthesis.
  • Quantitative real-time PCR was then used to analyze these cDNAs.
  • A melting curve analysis was carried out to confirm the specificity of the PCR amplification.

Findings of the Study

The study’s results revealed a significant difference in the expression levels of the examined reference genes between adipose tissue-derived and bone marrow-derived MSCs.

  • Specifically, the levels of GAPDH were significantly different in the two types of MSCs (p < 0.05).
  • The expression levels of β-actin and B2M were substantially higher in bone marrow-derived MSCs than in adipose tissue-derived MSCs (P < 0.001 for β-actin and P < 0.006 for B2M).
  • The fold changes in expression levels of GAPDH, β-actin, and B2M in adipose tissue-derived MSCs compared to bone marrow-derived MSCs were 2.38, 6.76, and 7.76, respectively.

Conclusions

The researchers concluded that GAPDH, β-actin, and B2M are not reliable reference genes for comparative gene expression analysis conducted on MSCs derived from equine bone marrow and adipose tissue. This stems from the significant differences in the levels of these genes’ expression in the two types of MSCs, undermining their typical use as consistent reference points in gene expression studies.

Cite This Article

APA
Nazari F, Parham A, Maleki AF. (2015). GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells. J Anim Sci Technol, 57, 18. https://doi.org/10.1186/s40781-015-0050-8

Publication

Journal of animal science and technology
ISSN: 2055-0391
NlmUniqueID: 101661694
Country: Korea (South)
Language: English
Volume: 57
Pages: 18
PII: 18

Researcher Affiliations

Nazari, Fatemeh
  • Division of Physiology, Department of Basic Sciences, Veterinary Faculty, Ferdowsi University of Mashhad, Mashhad, Iran.
Parham, Abbas
  • Division of Physiology, Department of Basic Sciences, Veterinary Faculty, Ferdowsi University of Mashhad, Mashhad, Iran ; Embryonic and Stem Cell Biology and Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.
Maleki, Adham Fani
  • Division of Physiology, Department of Basic Sciences, Veterinary Faculty, Ferdowsi University of Mashhad, Mashhad, Iran.

References

This article includes 47 references
  1. Alves AG, Stewart AA, Dudhia J, Kasashima Y, Goodship AE, Smith RK. Cell-based therapies for tendon and ligament injuries.. Vet Clin North Am Equine Pract 2011 Aug;27(2):315-33.
    doi: 10.1016/j.cveq.2011.06.001pubmed: 21872761google scholar: lookup
  2. Li Y, Yu X, Lin S, Li X, Zhang S, Song YH. Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells.. Biochem Biophys Res Commun 2007 May 11;356(3):780-4.
    doi: 10.1016/j.bbrc.2007.03.049pubmed: 17382293google scholar: lookup
  3. Smith RK, Korda M, Blunn GW, Goodship AE. Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment.. Equine Vet J 2003 Jan;35(1):99-102.
    doi: 10.2746/042516403775467388pubmed: 12553472google scholar: lookup
  4. Nixon AJ, Dahlgren LA, Haupt JL, Yeager AE, Ward DL. Effect of adipose-derived nucleated cell fractions on tendon repair in horses with collagenase-induced tendinitis.. Am J Vet Res 2008 Jul;69(7):928-37.
    doi: 10.2460/ajvr.69.7.928pubmed: 18593247google scholar: lookup
  5. Radtke CL, Nino-Fong R, Esparza Gonzalez BP, Stryhn H, McD￮ LA. Characterization and osteogenic potential of equine muscle tissue- and periosteal tissue-derived mesenchymal stem cells in comparison with bone marrow- and adipose tissue-derived mesenchymal stem cells.. Am J Vet Res 2013 May;74(5):790-800.
    doi: 10.2460/ajvr.74.5.790pubmed: 23627394google scholar: lookup
  6. Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.. Stem Cells 2006 May;24(5):1294-301.
    doi: 10.1634/stemcells.2005-0342pubmed: 16410387google scholar: lookup
  7. Koch TG, Berg LC, Betts DH. Concepts for the clinical use of stem cells in equine medicine.. Can Vet J 2008 Oct;49(10):1009-17.
    pmc: PMC2553494pubmed: 19119371
  8. Strioga M, Viswanathan S, Darinskas A, Slaby O, Michalek J. Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells.. Stem Cells Dev 2012 Sep 20;21(14):2724-52.
    doi: 10.1089/scd.2011.0722pubmed: 22468918google scholar: lookup
  9. Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, Bae YC, Jung JS. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.. Cell Physiol Biochem 2004;14(4-6):311-24.
    doi: 10.1159/000080341pubmed: 15319535google scholar: lookup
  10. Burk J, Ribitsch I, Gittel C, Juelke H, Kasper C, Staszyk C, Brehm W. Growth and differentiation characteristics of equine mesenchymal stromal cells derived from different sources.. Vet J 2013 Jan;195(1):98-106.
    doi: 10.1016/j.tvjl.2012.06.004pubmed: 22841420google scholar: lookup
  11. Al-Nbaheen M, Vishnubalaji R, Ali D, Bouslimi A, Al-Jassir F, Megges M, Prigione A, Adjaye J, Kassem M, Aldahmash A. Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential.. Stem Cell Rev Rep 2013 Feb;9(1):32-43.
    doi: 10.1007/s12015-012-9365-8pmc: PMC3563956pubmed: 22529014google scholar: lookup
  12. Campioni D, Lanza F, Moretti S, Ferrari L, Cuneo A. Loss of Thy-1 (CD90) antigen expression on mesenchymal stromal cells from hematologic malignancies is induced by in vitro angiogenic stimuli and is associated with peculiar functional and phenotypic characteristics.. Cytotherapy 2008;10(1):69-82.
    doi: 10.1080/14653240701762364pubmed: 18202976google scholar: lookup
  13. Radcliffe CH, Flaminio MJ, Fortier LA. Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations.. Stem Cells Dev 2010 Feb;19(2):269-82.
    doi: 10.1089/scd.2009.0091pmc: PMC3138180pubmed: 19604071google scholar: lookup
  14. De Schauwer C, Meyer E, Van de Walle GR, Van Soom A. Markers of stemness in equine mesenchymal stem cells: a plea for uniformity.. Theriogenology 2011 May;75(8):1431-43.
    doi: 10.1016/j.theriogenology.2010.11.008pubmed: 21196039google scholar: lookup
  15. Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays.. J Mol Endocrinol 2000 Oct;25(2):169-93.
    doi: 10.1677/jme.0.0250169pubmed: 11013345google scholar: lookup
  16. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.. Clin Chem 2009 Apr;55(4):611-22.
    doi: 10.1373/clinchem.2008.112797pubmed: 19246619google scholar: lookup
  17. Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations.. Genes Immun 2005 Jun;6(4):279-84.
    doi: 10.1038/sj.gene.6364190pubmed: 15815687google scholar: lookup
  18. 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 Jun 18;3(7):RESEARCH0034.
    doi: 10.1186/gb-2002-3-7-research0034pmc: PMC126239pubmed: 12184808google scholar: lookup
  19. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR.. Nucleic Acids Res 2001 May 1;29(9):e45.
    doi: 10.1093/nar/29.9.e45pmc: PMC55695pubmed: 11328886google scholar: lookup
  20. 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 Oct 8;75(2-3):291-5.
    doi: 10.1016/S0168-1656(99)00163-7pubmed: 10617337google scholar: lookup
  21. Lee PD, Sladek R, Greenwood CM, Hudson TJ. Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies.. Genome Res 2002 Feb;12(2):292-7.
    doi: 10.1101/gr.217802pmc: PMC155273pubmed: 11827948google scholar: lookup
  22. Ranera B, Lyahyai J, Romero A, Vázquez FJ, Remacha AR, Bernal ML, Zaragoza P, Rodellar C, Martín-Burriel I. Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue.. Vet Immunol Immunopathol 2011 Nov 15;144(1-2):147-54.
    doi: 10.1016/j.vetimm.2011.06.033pubmed: 21782255google scholar: lookup
  23. Ranera B, Ordovás L, Lyahyai J, Bernal ML, Fernandes F, Remacha AR, Romero A, Vázquez FJ, Osta R, Cons C, Varona L, Zaragoza P, Martín-Burriel I, Rodellar C. Comparative study of equine bone marrow and adipose tissue-derived mesenchymal stromal cells.. Equine Vet J 2012 Jan;44(1):33-42.
    doi: 10.1111/j.2042-3306.2010.00353.xpubmed: 21668489google scholar: lookup
  24. Zhang YW, Davis EG, Bai J. Determination of internal control for gene expression studies in equine tissues and cell culture using quantitative RT-PCR.. Vet Immunol Immunopathol 2009 Jul 15;130(1-2):114-9.
    doi: 10.1016/j.vetimm.2009.01.012pubmed: 19269038google scholar: lookup
  25. Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues.. Physiol Genomics 2005 May 11;21(3):389-95.
    doi: 10.1152/physiolgenomics.00025.2005pubmed: 15769908google scholar: lookup
  26. Josson S, Nomura T, Lin JT, Huang WC, Wu D, Zhau HE, Zayzafoon M, Weizmann MN, Gururajan M, Chung LW. β2-microglobulin induces epithelial to mesenchymal transition and confers cancer lethality and bone metastasis in human cancer cells.. Cancer Res 2011 Apr 1;71(7):2600-10.
    doi: 10.1158/0008-5472.CAN-10-3382pmc: PMC3182156pubmed: 21427356google scholar: lookup
  27. Nomura T, Huang WC, Zhau HE, Josson S, Mimata H, Chung LW. β2-Microglobulin-mediated signaling as a target for cancer therapy.. Anticancer Agents Med Chem 2014 Mar;14(3):343-52.
    doi: 10.2174/18715206113139990092pmc: PMC3931390pubmed: 23848204google scholar: lookup
  28. Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR.. J Biochem Biophys Methods 2000 Nov 20;46(1-2):69-81.
    doi: 10.1016/S0165-022X(00)00129-9pubmed: 11086195google scholar: lookup
  29. Dorak MT. Real-Time PCR. New York: Taylor & Francis Group; 2006.
  30. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method.. Nat Protoc 2008;3(6):1101-8.
    doi: 10.1038/nprot.2008.73pubmed: 18546601google scholar: lookup
  31. Alipour F, Parham A, Kazemi Mehrjerdi H, Dehghani H. Equine adipose-derived mesenchymal stem cells: phenotype and growth characteristics, gene expression profile and differentiation potentials.. Cell J 2015 Winter;16(4):456-65.
    pmc: PMC4297484pubmed: 25685736doi: 10.22074/cellj.2015.491google scholar: lookup
  32. Zahedi M, Abavisani A, Dehghani H, Kazemi Mehrjerdi H. Isolation and characterization of horse bone marrow mesenchymal stem cells for treatment of joint injuries: an animal model for human studies. Artif Organs 2013;37(7):A50.
  33. Russell KC, Lacey MR, Gilliam JK, Tucker HA, Phinney DG, O'Connor KC. Clonal analysis of the proliferation potential of human bone marrow mesenchymal stem cells as a function of potency.. Biotechnol Bioeng 2011 Nov;108(11):2716-26.
    doi: 10.1002/bit.23193pmc: PMC3178709pubmed: 21538337google scholar: lookup
  34. Ragni E, Viganò M, Rebulla P, Giordano R, Lazzari L. What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes.. J Cell Mol Med 2013 Jan;17(1):168-80.
    doi: 10.1111/j.1582-4934.2012.01660.xpmc: PMC3823147pubmed: 23305553google scholar: lookup
  35. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR.. Nat Protoc 2006;1(3):1559-82.
    doi: 10.1038/nprot.2006.236pubmed: 17406449google scholar: lookup
  36. Lupberger J, Kreuzer KA, Baskaynak G, Peters UR, le Coutre P, Schmidt CA. Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.. Mol Cell Probes 2002 Feb;16(1):25-30.
    doi: 10.1006/mcpr.2001.0392pubmed: 12005444google scholar: lookup
  37. Amable PR, Teixeira MV, Carias RB, Granjeiro JM, Borojevic R. Identification of appropriate reference genes for human mesenchymal cells during expansion and differentiation.. PLoS One 2013;8(9):e73792.
    doi: 10.1371/journal.pone.0073792pmc: PMC3759474pubmed: 24023904google scholar: lookup
  38. Hjertner B, Olofsson KM, Lindberg R, Fuxler L, Fossum C. Expression of reference genes and T helper 17 associated cytokine genes in the equine intestinal tract.. Vet J 2013 Sep;197(3):817-23.
    doi: 10.1016/j.tvjl.2013.05.020pubmed: 23810185google scholar: lookup
  39. Sánchez-Matamoros A, Kukielka D, De las Heras AI, Sánchez-Vizcaíno JM. Development and evaluation of a SYBR Green real-time RT-PCR assay for evaluation of cytokine gene expression in horse.. Cytokine 2013 Jan;61(1):50-3.
    doi: 10.1016/j.cyto.2012.10.004pubmed: 23103121google scholar: lookup
  40. Bustin S, Penning LC. Improving the analysis of quantitative PCR data in veterinary research.. Vet J 2012 Mar;191(3):279-81.
    doi: 10.1016/j.tvjl.2011.06.044pubmed: 22005167google scholar: lookup
  41. Chooi WH, Zhou R, Yeo SS, Zhang F, Wang DA. Determination and validation of reference gene stability for qPCR analysis in polysaccharide hydrogel-based 3D chondrocytes and mesenchymal stem cell cultural models.. Mol Biotechnol 2013 Jun;54(2):623-33.
    doi: 10.1007/s12033-012-9604-xpubmed: 23054629google scholar: lookup
  42. Glare EM, Divjak M, Bailey MJ, Walters EH. beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels.. Thorax 2002 Sep;57(9):765-70.
    doi: 10.1136/thorax.57.9.765pmc: PMC1746418pubmed: 12200519google scholar: lookup
  43. Lin J, Redies C. Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression.. Dev Genes Evol 2012 Nov;222(6):369-76.
    doi: 10.1007/s00427-012-0420-xpubmed: 23099774google scholar: lookup
  44. Bursten SL, Stevenson F, Torrano F, Lovett DH. Mesangial cell activation by bacterial endotoxin. Induction of rapid cytoskeletal reorganization and gene expression.. Am J Pathol 1991 Aug;139(2):371-82.
    pmc: PMC1886077pubmed: 1867323
  45. Sirover MA. New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase.. Biochim Biophys Acta 1999 Jul 13;1432(2):159-84.
    doi: 10.1016/S0167-4838(99)00119-3pubmed: 10407139google scholar: lookup
  46. Zhu Y, Su Y, Cheng T, Chung LW, Shi C. Beta2-microglobulin as a potential factor for the expansion of mesenchymal stem cells.. Biotechnol Lett 2009 Sep;31(9):1361-5.
    doi: 10.1007/s10529-009-0027-0pmc: PMC2984555pubmed: 19466557google scholar: lookup
  47. Kim JW, Kim SJ, Han SM, Paik SY, Hur SY, Kim YW, Lee JM, Namkoong SE. Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in human cervical cancers.. Gynecol Oncol 1998 Nov;71(2):266-9.
    doi: 10.1006/gyno.1998.5195pubmed: 9826470google scholar: lookup

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  1. Nevone A, Lattarulo F, Russo M, Panno G, Milani P, Basset M, Avanzini MA, Merlini G, Palladini G, Nuvolone M. A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets. Biomedicines 2023 Apr 3;11(4).
    doi: 10.3390/biomedicines11041079pubmed: 37189697google scholar: lookup
  2. Xue M, Wen H, Xu P, Chen J, Wang Q, Tang Y, Ma X, Lv G, Li H, Song C. Validation and Functional Analysis of Reference and Tissue-Specific Genes in Adipose Tissue of Freshwater Drum, Aplodinotus grunniens, under Starvation and Hypothermia Stress. Cells 2023 May 6;12(9).
    doi: 10.3390/cells12091328pubmed: 37174728google scholar: lookup
  3. Riveroll AL, Skyba-Lewin S, Lynn KD, Mubyeyi G, Abd-El-Aziz A, Kibenge FST, Kibenge MJT, Cohen AM, Esparza-Gonsalez B, McD￮ L, Montelpare WJ. Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR. Genes (Basel) 2023 Mar 8;14(3).
    doi: 10.3390/genes14030673pubmed: 36980946google scholar: lookup
  4. Zhao L, Yang H, Li X, Zhou Y, Liu T, Zhao Y. Transcriptome-based selection and validation of optimal reference genes in perirenal adipose developing of goat (Capra hircus). Front Vet Sci 2022;9:1055866.
    doi: 10.3389/fvets.2022.1055866pubmed: 36467654google scholar: lookup
  5. Huang Q, Xu J, Ge Y, Shi Y, Wang F, Zhu M. NR4A1 inhibits the epithelial-mesenchymal transition of hepatic stellate cells: Involvement of TGF-β-Smad2/3/4-ZEB signaling. Open Life Sci 2022;17(1):447-454.
    doi: 10.1515/biol-2022-0047pubmed: 35600274google scholar: lookup
  6. Rashid M, Shah SG, Natu A, Verma T, Rauniyar S, Gera PB, Gupta S. RPS13, a potential universal reference gene for normalisation of gene expression in multiple human normal and cancer tissue samples. Mol Biol Rep 2021 Dec;48(12):7967-7974.
    doi: 10.1007/s11033-021-06828-6pubmed: 34657252google scholar: lookup
  7. Muñoz JJ, Anauate AC, Amaral AG, Ferreira FM, Watanabe EH, Meca R, Ormanji MS, Boim MA, Onuchic LF, Heilberg IP. Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models. Sci Rep 2021 Oct 5;11(1):19798.
    doi: 10.1038/s41598-021-99366-xpubmed: 34611276google scholar: lookup
  8. Johnson R, Rafuse M, Selvakumar PP, Tan W. Effects of recipient age, heparin release and allogeneic bone marrow-derived stromal cells on vascular graft remodeling. Acta Biomater 2021 Apr 15;125:172-182.
    doi: 10.1016/j.actbio.2021.02.028pubmed: 33639311google scholar: lookup
  9. Vermani L, Kumar R, Senthil Kumar N. GAPDH and PUM1: Optimal Housekeeping Genes for Quantitative Polymerase Chain Reaction-Based Analysis of Cancer Stem Cells and Epithelial-Mesenchymal Transition Gene Expression in Rectal Tumors. Cureus 2020 Dec 10;12(12):e12020.
    doi: 10.7759/cureus.12020pubmed: 33457124google scholar: lookup
  10. Wang L, Chen X, Song T, Zhang X, Zhan S, Cao J, Zhong T, Guo J, Li L, Zhang H, Wang Y. Using RNA-Seq to Identify Reference Genes of the Transition from Brown to White Adipose Tissue in Goats. Animals (Basel) 2020 Sep 10;10(9).
    doi: 10.3390/ani10091626pubmed: 32927876google scholar: lookup
  11. Lee GKC, Tessier L, Bienzle D. Salivary Scavenger and Agglutinin (SALSA) Is Expressed in Mucosal Epithelial Cells and Decreased in Bronchial Epithelium of Asthmatic Horses. Front Vet Sci 2019;6:418.
    doi: 10.3389/fvets.2019.00418pubmed: 31850379google scholar: lookup
  12. Borkowska P, Zielińska A, Paul-Samojedny M, Stojko R, Kowalski J. Evaluation of reference genes for quantitative real-time PCR in Wharton's Jelly-derived mesenchymal stem cells after lentiviral transduction and differentiation. Mol Biol Rep 2020 Feb;47(2):1107-1115.
    doi: 10.1007/s11033-019-05207-6pubmed: 31781918google scholar: lookup
  13. Jin Y, Liu F, Huang W, Sun Q, Huang X. Identification of reliable reference genes for qRT-PCR in the ephemeral plant Arabidopsis pumila based on full-length transcriptome data. Sci Rep 2019 Jun 10;9(1):8408.
    doi: 10.1038/s41598-019-44849-1pubmed: 31182737google scholar: lookup
  14. Duan M, Wang J, Zhang X, Yang H, Wang H, Qiu Y, Song J, Guo Y, Li X. Identification of Optimal Reference Genes for Expression Analysis in Radish (Raphanus sativus L.) and Its Relatives Based on Expression Stability. Front Plant Sci 2017;8:1605.
    doi: 10.3389/fpls.2017.01605pubmed: 28966627google scholar: lookup
  15. Kim BS, Tilstam PV, Springenberg-Jung K, Boecker AH, Schmitz C, Heinrichs D, Hwang SS, Stromps JP, Ganse B, Kopp R, Knobe M, Bernhagen J, Pallua N, Bucala R. Characterization of adipose tissue macrophages and adipose-derived stem cells in critical wounds. PeerJ 2017;5:e2824.
    doi: 10.7717/peerj.2824pubmed: 28070458google scholar: lookup
  16. Zhang WX, Fan J, Ma J, Rao YS, Zhang L, Yan YE. Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Three Types of Rat Adipose Tissue. Int J Mol Sci 2016 Jun 22;17(6).
    doi: 10.3390/ijms17060968pubmed: 27338366google scholar: lookup
  17. Zhao SH, Yue Y, Yu RT, Gao Q, Zhao JQ, Zhang SP, Zhou N, Xu GL. Validation of Stable Reference Genes for RT-qPCR Normalization in Oxycetonia jucunda (Coleoptera: Scarabaeidae). Insects 2026 Jan 1;17(1).
    doi: 10.3390/insects17010057pubmed: 41598911google scholar: lookup
  18. Wang S, Feng L, Sun J. Evaluation of Suitable Reference Gene During the Development of Paired or Unpaired Female Schistosoma japonicum on the 18th and the 23rd Days Post Infection. Pathogens 2025 Oct 21;14(10).
    doi: 10.3390/pathogens14101066pubmed: 41156676google scholar: lookup
  19. Stoikos P, Giannakoulas A, Kouvata E, Stefani G, Vassilopoulos G, Giannakoulas N. Transcriptional analysis of BMP2 and BMP6 in patients with newly diagnosed multiple myeloma. Oncol Lett 2025 Oct;30(4):448.
    doi: 10.3892/ol.2025.15194pubmed: 40747375google scholar: lookup
  20. Ismail NZ, Khairuddean M, Alidmat MM, Abubakar S, Arsad H. Investigating the potential of mono-chalcone compounds in targeting breast cancer receptors through network pharmacology, molecular docking, molecular dynamics simulation, antiproliferative effects, and gene expressions. 3 Biotech 2024 Jun;14(6):151.
    doi: 10.1007/s13205-024-03991-ypubmed: 38737798google scholar: lookup
  21. Ivanova Z, Petrova V, Grigorova N, Vachkova E. Identification of the Reference Genes for Relative qRT-PCR Assay in Two Experimental Models of Rabbit and Horse Subcutaneous ASCs. Int J Mol Sci 2024 Feb 14;25(4).
    doi: 10.3390/ijms25042292pubmed: 38396967google scholar: lookup
  22. Guo W, Yang Y, Ma B, Wang W, Hu Z, Leng P. Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing. Genes (Basel) 2024 Jan 21;15(1).
    doi: 10.3390/genes15010131pubmed: 38275612google scholar: lookup
  23. Zhou Y, Li X, Zhang X, Li M, Luo N, Zhao Y. Screening of Candidate Housekeeping Genes in Uterus Caruncle by RNA-Sequence and qPCR Analyses in Different Stages of Goat (Capra hircus). Animals (Basel) 2023 Jun 6;13(12).
    doi: 10.3390/ani13121897pubmed: 37370406google scholar: lookup
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