Skip to main content
SpringerLink
Log in
Book cover

Parasite Genomics Protocols pp 207–219Cite as

RNA-Seq Approaches for Determining mRNA Abundance in Leishmania

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1201))

Abstract

High-throughput sequencing of cDNA copies of mRNA (RNA-seq) provides a digital read-out of mRNA levels over several orders of magnitude, as well as mapping the transcripts to the nucleotide level. Here we describe an RNA-seq approach that exploits the 39-nucleotide mini-exon or spliced leader (SL) sequence found at the 5′ end of all Leishmania (and other trypanosomatid) mRNAs.

Key words

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

Protocol
EUR   44.95
Price includes VAT (France)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
EUR   85.59
Price includes VAT (France)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
EUR   116.04
Price includes VAT (France)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
EUR   105.49
Price includes VAT (France)
  • 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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Ivens AC, Peacock CS, Worthey EA et al (2005) The genome of the kinetoplastid parasite, Leishmania major Science 309:436–442

    Article  PubMed Central  PubMed  Google Scholar 

  2. Berriman M, Ghedin E, Hertz-Fowler C et al (2005) The genome of the African trypanosome, Trypanosoma brucei Science 309:416–422

    Article  CAS  PubMed  Google Scholar 

  3. El-Sayed NM, Myler PJ, Bartholomeu DC et al (2005) The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 309:409–415

    Article  CAS  PubMed  Google Scholar 

  4. El-Sayed NM, Myler PJ, Blandin G et al (2005) Comparative genomics of trypanosomatid parasitic protozoa. Science 309:404–409

    Article  CAS  PubMed  Google Scholar 

  5. Saxena A, Lahav T, Holland N et al (2007) Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in gene expression during differentiation. Mol Biochem Parasitol 152:53–65

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Lahav T, Sivam D, Volpin H et al (2011) Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania. FASEB J 25:515–525

    Article  CAS  PubMed  Google Scholar 

  7. Duncan R (2004) DNA microarray analysis of protozoan parasite gene expression: outcomes correlate with mechanisms of regulation. Trends Parasitol 20:211–215

    Article  CAS  PubMed  Google Scholar 

  8. Duncan RC, Salotra P, Goyal N et al (2004) The application of gene expression microarray technology to kinetoplastid research. Curr Mol Med 4:611–621

    Article  CAS  PubMed  Google Scholar 

  9. Guimond C, Trudel N, Brochu C et al (2003) Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays. Nucleic Acids Res 31:5886–5896

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. McNicoll F, Drummelsmith J, Muller M et al (2006) A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum. Proteomics 6:3567–3581

    Article  CAS  PubMed  Google Scholar 

  11. Rochette A, Raymond F, Ubeda JM et al (2008) Genome-wide gene expression profiling analysis of Leishmania major and Leishmania infantum developmental stages reveals substantial differences between the two species. BMC Genomics 9:255

    Article  PubMed Central  PubMed  Google Scholar 

  12. Almeida R, Gilmartin BJ, McCann SH et al (2004) Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome. Mol Biochem Parasitol 136:87–100

    Article  CAS  PubMed  Google Scholar 

  13. Holzer TR, McMaster WR, Forney JD (2006) Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana Mol. Biochem Parasitol 146:198–218

    Article  CAS  Google Scholar 

  14. Leifso K, Cohen-Freue G, Dogra N et al (2007) Genomic and proteomic expression analysis of Leishmania promastigote and amastigote life stages: The Leishmania genome is constitutively expressed. Mol Biochem Parasitol 152:35–46

    Article  CAS  PubMed  Google Scholar 

  15. Cohen-Freue G, Holzer TR, Forney JD, McMaster WR (2007) Global gene expression in Leishmania. Int J Parasitol 37:1077–1086

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Clayton CE (2002) Life without transcriptional control? From fly to man and back again. EMBO J 21:1881–1888

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Nilsson D, Gunasekera K, Mani J et al (2010) Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei. PLoS Pathog 6:e1001037

    Article  PubMed Central  PubMed  Google Scholar 

  19. Mittra B, Cortez M, Haydock A et al (2013) Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels. J Exp Med 210:401–416

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. van Luenen H, Farris C, Jan S et al (2012) Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania. Cell 150:909–921

    Article  PubMed Central  PubMed  Google Scholar 

  21. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11:R25

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Seattle BioMed, 307 Westlake Ave N., Suite 500, Seattle, WA, 98109-5219, USA

    Andrew Haydock, Aarthi Sekar, Gowthaman Ramasamy, Loren Baugh & Peter J. Myler

  2. Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, Ribeirão Preto, SP, Brazil

    Monica Terrao

Authors
  1. Andrew Haydock
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Monica Terrao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aarthi Sekar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gowthaman Ramasamy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Loren Baugh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peter J. Myler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter J. Myler .

Editor information

Editors and Affiliations

  1. University of Western Australia, Nedlands, West Australia, Australia

    Christopher Peacock

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Haydock, A., Terrao, M., Sekar, A., Ramasamy, G., Baugh, L., Myler, P.J. (2015). RNA-Seq Approaches for Determining mRNA Abundance in Leishmania . In: Peacock, C. (eds) Parasite Genomics Protocols. Methods in Molecular Biology, vol 1201. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1438-8_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1438-8_12

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1437-1

  • Online ISBN: 978-1-4939-1438-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation