Skip to main content
SpringerLink
Log in

Radiolabeled Semi-quantitative RT-PCR Assay for the Analysis of Alternative Splicing of Interleukin Genes

  • Protocol
  • First Online:
Cytokine Bioassays

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1172))

Abstract

Alternative splicing evolved as a very efficient way to generate proteome diversity from a limited number of genes, while at the same time modulating posttranscriptional events of gene expression—such as stability, turnover, subcellular localization, binding properties, and general activity of both mRNAs and proteins. Since the vast majority of human genes undergo alternative splicing, it comes to no surprise that interleukin genes also show extensive alternative splicing. In fact, there is a growing body of evidence indicating that alternative splicing plays a central role in modulating the pleiotropic functions of cytokines, and aberrant expression of alternatively spliced interleukin mRNAs has been linked to disease. However, while several interleukin splice variants have been described, their function is still poorly understood. This is particularly relevant, since alternatively spliced cytokine isoforms can act both as disease biomarkers and as candidate entry points for therapeutic intervention. In this chapter we describe a protocol that uses radiolabeled semi-quantitative RT-PCR to efficiently detect, analyze, and quantify alternative splicing patterns of cytokine genes.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Wang ET, Sandberg R, Luo S et al (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Maniatis T, Tasic B (2002) Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature 418:236–243

    Article  CAS  PubMed  Google Scholar 

  3. Black DL (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72:291–336

    Article  CAS  PubMed  Google Scholar 

  4. Stamm S, Ben-Ari S, Rafalska I et al (2005) Function of alternative splicing. Gene 344:1–20

    Article  CAS  PubMed  Google Scholar 

  5. Braunschweig U, Gueroussov S, Plocik AM et al (2013) Dynamic integration of splicing within gene regulatory pathways. Cell 152:1252–1269

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Pan Q, Shai O, Lee LJ et al (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40:1413–1415

    Article  CAS  PubMed  Google Scholar 

  7. Singh RK, Cooper TA (2012) Pre-mRNA splicing in disease and therapeutics. Trends Mol Med 18:472–482

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Lopez-Bigas N, Audit B, Ouzounis C et al (2005) Are splicing mutations the most frequent cause of hereditary disease? FEBS Lett 579:1900–1903

    Article  CAS  PubMed  Google Scholar 

  9. Lynch KW (2004) Consequences of regulated pre-mRNA splicing in the immune system. Nat Rev Immunol 4:931–940

    Article  CAS  PubMed  Google Scholar 

  10. Martinez NM, Pan Q, Cole BS et al (2012) Alternative splicing networks regulated by signaling in human T cells. RNA 18:1029–1040

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Martinez NM, Lynch KW (2013) Control of alternative splicing in immune responses: many regulators, many predictions, much still to learn. Immunol Rev 253:216–236

    Article  PubMed Central  PubMed  Google Scholar 

  12. Sahoo A, Im SH (2010) Interleukin and interleukin receptor diversity: role of alternative splicing. Int Rev Immunol 29:77–109

    Article  CAS  PubMed  Google Scholar 

  13. Goodwin RG, Lupton S, Schmierer A et al (1989) Human interleukin 7: molecular cloning and growth factor activity on human and murine B-lineage cells. Proc Natl Acad Sci U S A 86:302–306

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Welch PA, Namen AE, Goodwin RG et al (1989) Human IL-7: a novel T cell growth factor. J Immunol 143:3562–3567

    CAS  PubMed  Google Scholar 

  15. Fry TJ, Mackall CL (2002) Interleukin-7: from bench to clinic. Blood 99:3892–3904

    Article  CAS  PubMed  Google Scholar 

  16. Krawczenko A, Kieda C, Dus D (2005) The biological role and potential therapeutic application of interleukin 7. Arch Immunol Ther Exp (Warsz) 53:518–525

    CAS  Google Scholar 

  17. Maeurer MJ, Walter W, Martin D et al (1997) Interleukin-7 (IL-7) in colorectal cancer: IL-7 is produced by tissues from colorectal cancer and promotes preferential expansion of tumour infiltrating lymphocytes. Scand J Immunol 45:182–192

    Article  CAS  PubMed  Google Scholar 

  18. Trinder P, Seitzer U, Gerdes J et al (1999) Constitutive and IFN-gamma regulated expression of IL-7 and IL-15 in human renal cell cancer. Int J Oncol 14:23–31

    CAS  PubMed  Google Scholar 

  19. Korte A, Moricke A, Beyermann B et al (1999) Extensive alternative splicing of interleukin-7 in malignant hematopoietic cells: implication of distinct isoforms in modulating IL-7 activity. J Interferon Cytokine Res 19:495–503

    Article  CAS  PubMed  Google Scholar 

  20. Cosenza L, Gorgun G, Urbano A et al (2002) Interleukin-7 receptor expression and activation in nonhaematopoietic neoplastic cell lines. Cell Signal 14:317–325

    Article  CAS  PubMed  Google Scholar 

  21. Hartgring SA, Bijlsma JW, Lafeber FP et al (2006) Interleukin-7 induced immunopathology in arthritis. Ann Rheum Dis 65 Suppl 3:iii69–iii74

    Google Scholar 

  22. Fry TJ, Connick E, Falloon J et al (2001) A potential role for interleukin-7 in T-cell homeostasis. Blood 97:2983–2990

    Article  CAS  PubMed  Google Scholar 

  23. Huang HW, Chiang YJ, Hung SI et al (2007) An IL-7 splicing-defect lymphopenia mouse model revealed by genome-wide mutagenesis. J Biomed Sci 14:169–181

    Article  CAS  PubMed  Google Scholar 

  24. Chung B, Dudl E, Toyama A et al (2008) Importance of interleukin-7 in the development of experimental graft-versus-host disease. Biol Blood Marrow Transplant 14:16–27

    Article  CAS  PubMed  Google Scholar 

  25. Pan D, Liu B, Jin X et al (2012) IL-7 splicing variant IL-7delta5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway. Biochem Biophys Res Commun 422:727–731

    Article  CAS  PubMed  Google Scholar 

  26. Kroemer RT, Kroncke R, Gerdes J et al (1998) Comparison of the 3D models of four different human IL-7 isoforms with human and murine IL-7. Protein Eng 11:31–40

    Article  CAS  PubMed  Google Scholar 

  27. Vudattu NK, Magalhaes I, Hoehn H et al (2009) Expression analysis and functional activity of interleukin-7 splice variants. Genes Immun 10:132–140

    Article  CAS  PubMed  Google Scholar 

  28. Rane L, Vudattu N, Bourcier K et al (2010) Alternative splicing of interleukin-7 (IL-7) and interleukin-7 receptor alpha (IL-7Ralpha) in peripheral blood from patients with multiple sclerosis (MS). J Neuroimmunol 222:82–86

    Article  CAS  PubMed  Google Scholar 

  29. Lundmark F, Duvefelt K, Iacobaeus E et al (2007) Variation in interleukin 7 receptor alpha chain (IL7R) influences risk of multiple sclerosis. Nat Genet 39:1108–1113

    Article  CAS  PubMed  Google Scholar 

  30. Moors M, Vudattu NK, Abel J et al (2010) Interleukin-7 (IL-7) and IL-7 splice variants affect differentiation of human neural progenitor cells. Genes Immun 11:11–20

    Article  CAS  PubMed  Google Scholar 

  31. Paonessa G, Graziani R, De Serio A et al (1995) Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling. EMBO J 14:1942–1951

    CAS  PubMed Central  PubMed  Google Scholar 

  32. Bihl MP, Heinimann K, Rudiger JJ et al (2002) Identification of a novel IL-6 isoform binding to the endogenous IL-6 receptor. Am J Respir Cell Mol Biol 27:48–56

    Article  CAS  PubMed  Google Scholar 

  33. Rincon M, Anguita J, Nakamura T et al (1997) Interleukin (IL)-6 directs the differentiation of IL-4-producing CD4+ T cells. J Exp Med 185:461–469

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Zhang XY, Zhang HJ, Zhang Y et al (2006) Identification and expression analysis of alternatively spliced isoforms of human interleukin-23 receptor gene in normal lymphoid cells and selected tumor cells. Immunogenetics 57:934–943

    Article  CAS  PubMed  Google Scholar 

  35. Guo R, Li Y, Ning J et al (2013) HnRNP A1/A2 and SF2/ASF regulate alternative splicing of interferon regulatory factor-3 and affect immunomodulatory functions in human non-small cell lung cancer cells. PLoS One 8:e62729

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Ruggiu M, Herbst R, Kim N et al (2009) Rescuing Z+ agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing. Proc Natl Acad Sci U S A 106:3513–3518

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. Biotechniques 39:75–85

    Article  CAS  PubMed  Google Scholar 

  38. Gibson UE, Heid CA, Williams PM (1996) A novel method for real time quantitative RT-PCR. Genome Res 6:995–1001

    Article  CAS  PubMed  Google Scholar 

  39. Horner RM (2006) Relative RT-PCR: determining the linear range of amplification and optimizing the primers:competimers ratio. CSH Protoc 2006

    Google Scholar 

  40. Bastien P, Procop GW, Reischl U (2008) Quantitative real-time PCR is not more sensitive than “conventional” PCR. J Clin Microbiol 46:1897–1900

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Rio DC, Ares MJ, Hannon GJ et al (2010) Polyacrylamide gel electrophoresis of RNA. Cold Spring Harb Protoc 2010:pdb.prot5444

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biological Sciences, St. John’s University, 8000 Utopia Parkway, Queens, NY, 11439, USA

    Felitsiya Shakola, Stephen Byrne, Kainaat Javed & Matteo Ruggiu

Authors
  1. Felitsiya Shakola
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen Byrne
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kainaat Javed
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matteo Ruggiu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matteo Ruggiu .

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, St. John's University, New York, New York, USA

    Ivana Vancurova

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Shakola, F., Byrne, S., Javed, K., Ruggiu, M. (2014). Radiolabeled Semi-quantitative RT-PCR Assay for the Analysis of Alternative Splicing of Interleukin Genes. In: Vancurova, I. (eds) Cytokine Bioassays. Methods in Molecular Biology, vol 1172. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0928-5_31

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0928-5_31

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0927-8

  • Online ISBN: 978-1-4939-0928-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation