Skip to main content

Advertisement

SpringerLink
Log in

Selection of reference genes for quantitative real-time PCR normalization in European quail tissues

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Coturniculture has been standing out as an industrial poultry activity in several countries around the world because of the several adaptive advantages of quails. Research that considers the analysis of gene expression can enhance this activity. This study aimed to analyze the stability of reference genes (RGs) in different tissues of quails (both males and females) for the recommendation of use in gene expression studies by the quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression stability of ten RGs (ACTA1, ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, MRPS27, MRPS30, and RPL5) was analyzed in four tissues (breast muscle, abdominal fat, liver, and intestine), and assessed using the statistical tools geNorm, NormFinder, comparative ΔCq method, and BestKeeper. The HPRT1 gene was the most stable in all quail tissues tested, followed by MRPS27 and MRPS30 in breast muscle, B2M and RPL5 in abdominal fat, HMBS and B2M in the liver, and RPL5 and HMBS in the intestine. These results may help studies using RT-qPCR assays to assess quail tissues from both sexes because they provide data on the most stable genes, which should be tested as candidate RGs for other experimental conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Barrete TLS, Quirino BJS, Brito CD, Umigi RT, Araújo MS, Coimbra JRS, Rojas GEE, Freitas FFJ, Reis SR (2007) Metabolizable energy levels for Japanese quails in the initial laying phase. Rev Bras Zoot 36:79–85. https://doi.org/10.1590/S1516-35982007000100010

    Article  Google Scholar 

  2. Lukanov H (2019) Domestic quail (Coturnix japonica domestica), is there such farm animal? W Poul Sci J 75:547–558. https://doi.org/10.1017/S0043933919000631

    Article  Google Scholar 

  3. López-Salazar SE, Flota-Bañuelos C, Fraire-Cordero S (2020) Agroecological production of codorniz (Coturnix coturnix japonica) as strategy for food security in Campeche, Mexico. Agroproductividad 13:3–7. https://doi.org/10.32854/agrop.vi0.1513

    Article  Google Scholar 

  4. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Vandesompele J (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. C Chem 55:611–622. https://doi.org/10.1373/clinchem.2008.112797

    Article  CAS  Google Scholar 

  5. Shukla P, Reddy RA, Ponnuvel KM, Rohela GK, Shabnam AA, Ghosh MK, Mishra RK (2019) Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses. Mol Biol Rep 46:1809–1817. https://doi.org/10.1007/s11033-019-04631-y

    Article  CAS  PubMed  Google Scholar 

  6. Wang B, Lirong WANG, Duan H, Chong P, Su S, Shan L, Li Y (2020) Selection and validation of reference genes for quantitative real-time PCR analysis of Nitraria tangutorum. https://doi.org/10.21203/rs.2.21923/v1

  7. Pfaffl MW, Tichopad A, Prgomet C (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515. https://doi.org/10.1023/B:BILE.0000019559.84305.47

    Article  CAS  PubMed  Google Scholar 

  8. Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalization; strategies and considerations. G Immun 6:279–284. https://doi.org/10.1038/sj.gene.6364190

    Article  CAS  Google Scholar 

  9. Sanders R, Mason DJ, Foy CA, Huggett JF (2014) Considerations for accurate gene expression measurement by reverse transcription quantitative PCR when analysing clinical samples. Anal Bioanal Chem 406:6471–6483. https://doi.org/10.1007/s00216-014-7857-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCRÐA perspective. J Mol Endoc 34:597–601. https://doi.org/10.1677/jme.1.01755

    Article  CAS  Google Scholar 

  11. Tang B, Dai W, Zhang C (2019) Selection of reference genes for quantitative real-time polymerase chain reaction normalization in Bradysia odoriphaga (Diptera: Sciaridae). Entom Sci 22:422–436. https://doi.org/10.1111/ens.12383

    Article  Google Scholar 

  12. Lee PD, Sladek R, Greenwood CM, Hudson TJ (2002) Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genon Res 12:292–297. https://doi.org/10.1101/gr.217802

    Article  CAS  Google Scholar 

  13. Nascimento CS, Barbosa LT, Brito C, Fernandes RP, Mann RS, Pinto APG, Oliveira HC, Dodson MV, Guimarães SEF, Duarte MS (2015) Identification of suitable reference genes for real time quantitative polymerase chain reaction assays on pectoralis major muscle in chicken (Gallus gallus). PLoS One 10:e0127935. https://doi.org/10.1371/journal.pone.0127935

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Jemiolo B, Trappe S (2004) Single muscle fiber gene expression in human skeletal muscle: validation of internal control with exercise. Bioch Biophys Res Commun 320:1043–1050. https://doi.org/10.1016/j.bbrc.2004.05.223

    Article  CAS  Google Scholar 

  15. Axtner J, Sommer S (2009) Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis. BMC Res Note 2:1–7. https://doi.org/10.1186/1756-0500-2-264

    Article  CAS  Google Scholar 

  16. Weyrich A, Axtner J, Sommer S (2010) Selection and validation of reference genes for realtime RT-PCR studies in the non-model species Delomys sublineatus, an endemic Brazilian rodent. Bioch Biophys Res Commun 392:145–149. https://doi.org/10.1016/j.bbrc.2009.12.173

    Article  CAS  Google Scholar 

  17. Zhang Y, Han X, Chen S, Zheng L, He X, Liu M, Zhuo R (2017) Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses. Sci Report 7:1–11. https://doi.org/10.1038/srep40290

    Article  CAS  Google Scholar 

  18. Li Z, Lu H, He Z, Wang C, Wang Y, Ji X (2019) Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions. PLoS One 14:e0225926. https://doi.org/10.1371/journal.pone.0225926

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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. Gen Biol 7:1–12. https://doi.org/10.1186/gb-2002-3-7-research0034

    Article  Google Scholar 

  20. 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. Can Res 64:5245–5250. https://doi.org/10.1158/0008-5472.CAN-04-0496

    Article  CAS  Google Scholar 

  21. Silver N, Best S, Jiang J (2006) Selection of housekeeping genes for genes expression studies in human reticulocytes using real-time PCR. BMC Mol Bio 7:1–7. https://doi.org/10.1186/1471-2199-7-33

    Article  CAS  Google Scholar 

  22. Lisowski P, Pierzchała M, Gościk J, Pareek CS, Zwierzchowski L (2008) Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid. J Appl Gen 49:367–372 https://doi.org/10.100/BF03195635

    Article  Google Scholar 

  23. Bonnet M, Bernard L, Bes S, Leroux C (2013) Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue, muscle, liver and mammary gland from ruminants. Animal 7:1344–1353. https://doi.org/10.1017/S1751731113000475

    Article  CAS  PubMed  Google Scholar 

  24. Cheng L, Yu J, Hu X, Xiang M, Xia Y, Tao B, Chen H (2020) Identification of reliable reference genes for expression studies in maternal reproductive tissues and fetal tissues of pregnant cows. R Dom Anim. https://doi.org/10.1111/rda.13808

  25. Martínez-Giner M, Noguera JL, Balcells I, Fernández-Rodríguez A, Pena RN (2013) Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy. PLoS One 8:e66023. https://doi.org/10.1371/journal.pone.0066023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zang R, Bai J, Xu H, Zhang L, Yang J, Yang L, Lu J, Wu J (2011) Selection of suitable reference genes for real-time quantitative PCR studies in Lanzhou fat-tailed sheep (Ovis ares). Asi J A Vet Advan Y 6:789–804. https://doi.org/10.3923/ajava.2011.789.804

    Article  CAS  Google Scholar 

  27. Najafpanah MJ, Sadeghi M, Bakhtiarizadeh MR (2013) Reference genes selection for quantitative real-time PCR using RankAggreg method in different tissues of Capra hircus. PLoS One 8:e83041. https://doi.org/10.1371/journal.pone.0083041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Katarzyńska-Banasik D, Grzesiak M, Sechman A (2017) Selection of reference genes for quantitative real-time PCR analysis in chicken ovary following silver nanoparticle treatment. Environ Toxicol Pharmacol 56:186–190. https://doi.org/10.1016/j.etap.2017.09.011

    Article  CAS  PubMed  Google Scholar 

  29. Yang CG, Wang XL, Tian J, Liu W, Wu F, Jiang M, Wen H (2013) Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus). Gene 527:183–192. https://doi.org/10.1016/j.gene.2013.06.013

    Article  CAS  PubMed  Google Scholar 

  30. Ahn K, Bae JH, Nam KH, Lee CE, Park KD, Lee HK, Cho BW, Kim HS (2011) Identification of reference genes for normalization of gene expression in thoroughbred and Jeju native horse (Jeju pony) tissues. G Gen 33:245–250. https://doi.org/10.1007/s13258-010-0114-6

    Article  CAS  Google Scholar 

  31. Ashish S, Bhure SK, Harikrishna P, Ramteke SS, Kutty VM, Shruthi N, Mihir S (2017) Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis). Theriogenology 92:6–13. https://doi.org/10.1016/j.theriogenology.2017.01.006

    Article  CAS  PubMed  Google Scholar 

  32. Kaur R, Sodhi M, Sharma A, Sharma VL, Verma P, Swami SK, Mukesh M (2018) Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis). PLoS One 13:e0191558. https://doi.org/10.1371/journal.pone.0191558

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wu Y, Zhang Y, Hou Z, Fan G, Pi J, Sun S, Hu G (2018) Population genomic data reveal genes related to important traits of quail. GigaScience 7:1–16. https://doi.org/10.1093/gigascience/giy049

    Article  CAS  PubMed  Google Scholar 

  34. Carvalho A, Couroussé N, Crochet S, Coustham V (2019) Identification of reference genes for quantitative gene expression studies in three tissues of Japanese Quail. Genes 10:1–12. https://doi.org/10.3390/genes10030197

    Article  CAS  Google Scholar 

  35. Olias P, Adam I, Meyer A, Scharff C, Gruber AD (2014) Reference genes for quantitative gene expression studies in multiple avian species. PLoS One 9:e99678. https://doi.org/10.1371/journal.pone.0099678

    Article  PubMed  PubMed Central  Google Scholar 

  36. Oliveira H, Garcia AA, Gromboni JG, Farias Filho R, Nascimento C, Wenceslau AA (2017) Influence of heat stress, sex and genetic groups on reference genes stability in muscle tissue of chicken. PLoS One 12:e0176402. https://doi.org/10.1371/journal.pone.0176402

    Article  CAS  Google Scholar 

  37. Hassanpour H, Aghajani Z, Bahadoran S, Farhadi N, Nazari H, Kaewduangta W (2019) Identification of reliable reference genes for quantitative real-time PCR in ovary and uterus of laying hens under heat stress. Stress 22:387–394. https://doi.org/10.1080/10253890.2019.1574294

    Article  CAS  PubMed  Google Scholar 

  38. Pfaffl MWA (2001) A new mathematical model for relative quantification in real-time RT- PCR. Nucl A Res 29:e45. https://doi.org/10.1093/nar/29.9.e45

    Article  CAS  Google Scholar 

  39. Livak JK, Schmittgen DT (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262

    Article  CAS  Google Scholar 

  40. Chapman JR, Helin AS, Wille M, Atterby C, Järhul JD, Fridlund JS, Waldenström J (2016) A panel of stably expressed reference genes for real-time qPCR gene expression studies of mallards (Anas platyrhynchos). PLoS One 11:e0149454. https://doi.org/10.1371/journal.pone.0149454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nygard AB, Jørgensen CB, Cirera S, Fredholm M (2007) Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol Bio 8:1–6. https://doi.org/10.1186/1471-2199-8-67

    Article  CAS  Google Scholar 

  42. Hildyard JC, Wells DJ (2014) Identification and validation of quantitative PCR reference genes suitable for normalizing expression in normal and dystrophic cell culture models of myogenesis. PLoS One 11:e0149454. https://doi.org/10.1371/currents.md.faafdde4bea8df4aa7d06cd5553119a6

    Article  Google Scholar 

  43. Feng X, Xiong Y, Qian H, Lei M, Xu D, Ren Z (2010) Selection of reference genes for gene expression studies in porcine skeletal muscle using SYBR green qPCR. J Biotec 150:288–293. https://doi.org/10.1016/j.jbiotec.2010.09.949

    Article  CAS  Google Scholar 

  44. Chapman JR, Waldenström J (2015) With reference to reference genes: a systematic review of endogenous controls in gene expression studies. PLoS One 10:e0141853. https://doi.org/10.1371/journal.pone.0141853

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, Griffiths LR (2001) β-Actin an unsuitable internal control for RT-PCR. Mol Cell Probes 15:307–311. https://doi.org/10.1006/mcpr.2001.0376

    Article  CAS  PubMed  Google Scholar 

  46. Li X, Huang K, Chen F, Li W, Sun S, Shi XE, Yang G (2016) Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells. Animal 10:947–952. https://doi.org/10.1017/S1751731115002748

    Article  CAS  PubMed  Google Scholar 

  47. Cedraz de Oliveira H, Pinto Garcia AA, Gonzaga Gromboni JG, Vasconcelos Farias Filho R, Souza do Nascimento C, Arias Wenceslau A (2017) Influence of heat stress, sex and genetic groups on reference genes stability in muscle tissue of chicken. PLoS One 12:e0176402. https://doi.org/10.1371/journal.pone.0176402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

We thank the Fundação CAPES (Finance code 001) and Fundação de Amparo à Pesquisa do Estado do Piauí (FAPEPI) for the financial support.

Author information

Authors and Affiliations

  1. Department of Animal Science, Universidade Federal do Piauí, Campus Profª Cinobelina Elvas, Bom Jesus, PI, 64900-000, Brazil

    Fabiana Cristina Belchior de Sousa

  2. Department of Animal Science, Universidade Federal de Sergipe, São Cristóvão, SE, 49100-000, Brazil

    Carlos Souza do Nascimento, Maíse dos Santos Macário & Leandro Teixeira Barbosa

  3. Department of Entomology, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil

    Renan dos Santos Araújo

  4. Department of Animal Science, Instituto Federal de Educação, Ciência e Tecnologia do Maranhão, São Luís, MA, 65095-460, Brazil

    Geraldo Fábio Viana Bayão

  5. Department of Oceanography and Limnology, Universidade Federal do Maranhão, São Luís, MA, 65080-805, Brazil

    Katiene Régia Silva Sousa

Authors
  1. Fabiana Cristina Belchior de Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carlos Souza do Nascimento
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maíse dos Santos Macário
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Renan dos Santos Araújo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Leandro Teixeira Barbosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Geraldo Fábio Viana Bayão
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Katiene Régia Silva Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

FCBS, CSN, MSM, LTB, GFVB, KRSS: conceived and the study; FCBS, CSN, MSM, LTB: conducted the experiments; FCBS, CSN, RSA, GFVB, KRSS: analyzed the data; FCBS, RSA, KRSS: wrote the manuscript. All authors read and approved the final version.

Corresponding author

Correspondence to Renan dos Santos Araújo.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures applied in this study were approved by the Animal Ethics and Protection Committee of the Federal University of Sergipe (Universidade Federal de Sergipe), Sergipe, Brazil.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

de Sousa, F.C.B., do Nascimento, C.S., Macário, M.d.S. et al. Selection of reference genes for quantitative real-time PCR normalization in European quail tissues. Mol Biol Rep 48, 67–76 (2021). https://doi.org/10.1007/s11033-020-06134-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-020-06134-7

Keywords

Search

Navigation