Skip to main content
SpringerLink
Log in

Introduction to the Gene Expression Analysis

  • Protocol
  • First Online:
Molecular Genetics of Asthma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1434))

Abstract

In 1941, Beadle and Tatum published experiments that would explain the basis of the central dogma of molecular biology, whereby the DNA through an intermediate molecule, called RNA, results proteins that perform the functions in cells. Currently, biomedical research attempts to explain the mechanisms by which develops a particular disease, for this reason, gene expression studies have proven to be a great resource. Strictly, the term “gene expression” comprises from the gene activation until the mature protein is located in its corresponding compartment to perform its function and contribute to the expression of the phenotype of cell.

The expression studies are directed to detect and quantify messenger RNA (mRNA) levels of a specific gene. The development of the RNA-based gene expression studies began with the Northern Blot by Alwine et al. in 1977. In 1969, Gall and Pardue and John et al. independently developed the in situ hybridization, but this technique was not employed to detect mRNA until 1986 by Coghlan. Today, many of the techniques for quantification of RNA are deprecated because other new techniques provide more information. Currently the most widely used techniques are qPCR, expression microarrays, and RNAseq for the transcriptome analysis. In this chapter, these techniques will be reviewed.

An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-4939-3652-6_19

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Saiki RK, Scharf S, Faloona F et al (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354

    Article  CAS  PubMed  Google Scholar 

  2. Mallona I (2008) Selección de genes de normalización para RT-PCR cuantitativa en Petunia hybrida. (Normalization gene selection for quantitative RT-PCR in Petunia hybrida). Available via http://repositorio.bib.upct.es/dspace/handle/10317/723. Accessed 25 Nov 2014

  3. Higuchi R, Dollinger G, Walsh PS et al (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10:413–417

    Article  CAS  PubMed  Google Scholar 

  4. Higuchi R, Fockler C, Dollinger G et al (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030

    Article  CAS  PubMed  Google Scholar 

  5. Clewley JP (1994) The polymerase chain reaction (PCR) for human viral diagnosis. CRC Press, Boca Ratón

    Google Scholar 

  6. Taylor S, Wakem M, Dijkman G et al (2010) A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods 50:S1–S5

    Article  CAS  PubMed  Google Scholar 

  7. Huggett J, Bustin S (2011) Standardization and reporting for nucleic acid quantification. Accred Qual Assur 16:399–405

    Article  CAS  Google Scholar 

  8. Kozera B, Rapacz M (2013) Reference genes in real-time PCR. J Appl Genet 54:391–406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zipper H, Brunner H, Bernhagen J et al (2004) Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res 32(12):e103

    Article  PubMed  PubMed Central  Google Scholar 

  10. Sigma-Aldrich (2008) qPCR Technical Guide. Available via http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/qpcr_technical_guide.pdf. Accessed 22 Nov 2014

  11. http://www.gene-quantification.de/hrm-dyes.html

  12. VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44:619–626

    Article  CAS  PubMed  Google Scholar 

  13. Qiagen (2006) Critical factors for successful real time PCR. Integrated solutions-real time PCR applications. Available via http://jornades.uab.cat/workshopmrama/sites/jornades.uab.cat.workshopmrama/files/Critical_factors_successful_real_time_PCR.pdf. Accessed 28 Nov 2014

  14. Leonard DGB (2007) Molecular pathology in clinical practice. Springer Science & Business Media, Berlin

    Book  Google Scholar 

  15. Bustin SA, Kessler HH (2010) Amplification and detection methods. In: Kessler HH (ed) Molecular diagnostics of infectious diseases. De Gruyter, Berlín

    Google Scholar 

  16. Louw TM, Booth CS, Pienaar E et al (2011) Experimental validation of a fundamental model for PCR efficiency. Chem Eng Sci 66:1783–1789

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Diez GO (2006) Técnicas de Genética Molecular II (Molecular Genetic Techniques II). In: Lasa A (ed) PCR cuantitativa (quantitative PCR). SEQC, Barcelona

    Google Scholar 

  18. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  19. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007

    Article  Google Scholar 

  20. Pfaffl MW (2004) Quantification strategies in real-time PCR. In: Bustin SA (ed) A-Z of quantitative PCR. International University Line, La Jolla

    Google Scholar 

  21. Bohla L, Dusanic D, Narat M et al (2012) Comparison of methods for relative quantification of gene expression using real-time PCR. Acta Agric Slov 100:97–106

    Google Scholar 

  22. Mallona I, Lischewski S, Weiss J et al (2010) Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol 10:4

    Article  PubMed  PubMed Central  Google Scholar 

  23. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  PubMed  Google Scholar 

  24. Valente V, Teixeira SA, Neder L et al (2009) Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR. BMC Mol Biol 10:17

    Article  PubMed  PubMed Central  Google Scholar 

  25. Podevin N, Krauss A, Henry I et al (2012) Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa. Mol Breed 30:1237–1252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Fu W, Xie W, Zhang Z et al (2013) Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella. Int J Biol Sci 9:792–802

    Article  PubMed  PubMed Central  Google Scholar 

  27. Gantasala NP, Papolu PK, Thakur PK et al (2013) Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L). BMC Res Notes 6:312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Paim RM, Pereira MH, Di Ponzio R et al (2012) Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodniusprolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR. BMC Res Notes 5:128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Tunbridge EM, Eastwood SL, Harrison PJ (2011) Changed relative to what? Housekeeping genes and normalization strategies in human brain gene expression studies. Biol Psychiatry 69:173–179

    Article  CAS  PubMed  Google Scholar 

  30. Southern E, Mir K, Schepinov M (1999) Molecular interactions on microarrays. Nat Genet 21:5–9

    Article  CAS  PubMed  Google Scholar 

  31. Eberwine JH, Valentino KL, Barchas JD (1994) In situ hybridization in neurobiology: advances in methodology. Oxford University Press, Oxford

    Google Scholar 

  32. McLachlan G, Do K, Ambroise C (2005) Analyzing microarray gene expression data. Wiley, Hoboken

    Google Scholar 

  33. Faiz A, Burgess JK (2012) How can microarrays unlock asthma? J Allergy 2012:241314

    Article  Google Scholar 

  34. Affymetrix (2002) Affymetrix, Stanford University and incyte resolve patent oppositions and interferences. Available via http://investor.affymetrix.com/phoenix.zhtml?c=116408&p=irol-newsArticle_pf&ID=362094. Accessed 21 Dec 2014

  35. Times Higher Education (2006) Background memo on the winners of the European inventor of the year 2006 awards. Available via http://www.timeshighereducation.co.uk/news/background-memo-on-the-winners-of-the-european-inventor-of-the-year-2006-awards/203002.article. Accessed 22 Dec 2014

  36. Shalon D, Smith SJ, Brown PO (1996) A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res 6:639–645

    Article  CAS  PubMed  Google Scholar 

  37. Lopez M, Mallorquín P, Vega M (2002) Microarrays y biochips de DNA, Informe de vigilancia tecnológica (DNA microarrays and biochips, technological surveillance report).Genoma España/CIBT-FGUAM

    Google Scholar 

  38. Daudén E (2007) Farmacogenética II. Métodos moleculares de estudio, bioinformática y aspectos éticos (Molecular study methods, bioinformatics and ethical aspects). Actas Dermosifiliogr 98:3–13

    Article  PubMed  Google Scholar 

  39. Alba R, Fei Z, Payton P et al (2004) ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development. Plant J 39:697–714

    Article  CAS  PubMed  Google Scholar 

  40. Lin SM, Johnson KF (2002) Methods of microarray data analysis II. Springer Science & Business Media, Berlin

    Book  Google Scholar 

  41. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Malone JH, Oliver B (2011) Microarray, deep sequencing and the true measure of the transcriptome. BMC Biol 9:34

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Fernández AI, Óvilo C, Fernández A et al (2008) Luces y sombras del análisis de expresión génica utilizando microarrays. Un ejemplo en cerdo ibérico (Lights and shadows of gene expression analysis using microarrays. An example Iberian pig.) ITEA 104:99–105

    Google Scholar 

  44. Liu L, Li Y, Li S et al (2012) Comparison of next-generation sequencing systems. J Biomed Biotechnol 2012:251364

    PubMed  PubMed Central  Google Scholar 

  45. Costa V, Angelini C, De Feis I et al (2010) Uncovering the complexity of transcriptomes with RNA-Seq. J Biomed Biotechnol 2010:853916

    Article  PubMed  PubMed Central  Google Scholar 

  46. Santos CA, Blanck DV, de Freitas PD (2014) RNA-seq as a powerful tool for penaeid shrimp genetic progress. Front Genet 5:298

    Article  PubMed  PubMed Central  Google Scholar 

  47. Oshlack A, Robinson MD, Young MD (2010) From RNA-seq reads to differential expression results. Genome Biol 11:220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by grants of the Junta de Castilla y León ref. GRS1047/A/14, GRS1189/A/15, and BIO/SA73/15; and by the project “Efecto del Ácido Retinóico en la enfermedad alérgica. Estudio transcripcional y su traslación a la clínica,” PI13/00564, integrated into the “Plan Estatal de I + D + I 2013–2016” and cofunded by the “ISCIII-Subdirección General de Evaluación y Fomento de la investigación” and the European Regional Development Fund (FEDER).

Author information

Authors and Affiliations

  1. Department of Clinical Biochemistry, University Hospital of Salamanca, Paseo de San Vicente 58, 37007, Salamanca, Spain

    Ignacio San Segundo-Val

  2. Salamanca Institute for Biomedical Research (IBSAL), Salamanca, Spain

    Ignacio San Segundo-Val & Catalina S. Sanz-Lozano

  3. Department of Microbiology and Genetics, University of Salamanca, Salamanca, Spain

    Catalina S. Sanz-Lozano

Authors
  1. Ignacio San Segundo-Val
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Catalina S. Sanz-Lozano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ignacio San Segundo-Val .

Editor information

Editors and Affiliations

  1. Department of Clinical Biochemistry, University Hospital of Salamanca, Salamanca, Spain

    María Isidoro García

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

San Segundo-Val, I., Sanz-Lozano, C.S. (2016). Introduction to the Gene Expression Analysis. In: Isidoro García, M. (eds) Molecular Genetics of Asthma. Methods in Molecular Biology, vol 1434. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3652-6_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3652-6_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3650-2

  • Online ISBN: 978-1-4939-3652-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation