Skip to main content
SpringerLink
Log in

Quantitative RNA Analysis Using RNA-Seq

  • Protocol
  • First Online:
Leishmania

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1971))

Abstract

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

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
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

Similar content being viewed by others

References

  1. Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M, Sisk E, Rajandream MA, Adlem E, Aert R, Anupama A, Apostolou Z, Attipoe P, Bason N, Bauser C, Beck A, Beverley SM, Bianchettin G, Borzym K, Bothe G, Bruschi CV, Collins M, Cadag E, Ciarloni L, Clayton C, Coulson RM, Cronin A, Cruz AK, Davies RM, De Gaudenzi J, Dobson DE, Duesterhoeft A, Fazelina G, Fosker N, Frasch AC, Fraser A, Fuchs M, Gabel C, Goble A, Goffeau A, Harris D, Hertz-Fowler C, Hilbert H, Horn D, Huang Y, Klages S, Knights A, Kube M, Larke N, Litvin L, Lord A, Louie T, Marra M, Masuy D, Matthews K, Michaeli S, Mottram JC, Muller-Auer S, Munden H, Nelson S, Norbertczak H, Oliver K, O'Neil S, Pentony M, Pohl TM, Price C, Purnelle B, Quail MA, Rabbinowitsch E, Reinhardt R, Rieger M, Rinta J, Robben J, Robertson L, Ruiz JC, Rutter S, Saunders D, Schafer M, Schein J, Schwartz DC, Seeger K, Seyler A, Sharp S, Shin H, Sivam D, Squares R, Squares S, Tosato V, Vogt C, Volckaert G, Wambutt R, Warren T, Wedler H, Woodward J, Zhou S, Zimmermann W, Smith DF, Blackwell JM, Stuart KD, Barrell B, Myler PJ (2005) The genome of the kinetoplastid parasite, Leishmania major. Science 309(5733):436–442

    Article  Google Scholar 

  2. Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, Quail MA, Peters N, Adlem E, Tivey A, Aslett M, Kerhornou A, Ivens A, Fraser A, Rajandream MA, Carver T, Norbertczak H, Chillingworth T, Hance Z, Jagels K, Moule S, Ormond D, Rutter S, Squares R, Whitehead S, Rabbinowitsch E, Arrowsmith C, White B, Thurston S, Bringaud F, Baldauf SL, Faulconbridge A, Jeffares D, Depledge DP, Oyola SO, Hilley JD, Brito LO, Tosi LR, Barrell B, Cruz AK, Mottram JC, Smith DF, Berriman M (2007) Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet 39(7):839–847

    Article  CAS  Google Scholar 

  3. Rogers MB, Hilley JD, Dickens NJ, Wilkes J, Bates PA, Depledge DP, Harris D, Her Y, Herzyk P, Imamura H, Otto TD, Sanders M, Seeger K, Dujardin JC, Berriman M, Smith DF, Hertz-Fowler C, Mottram JC (2011) Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania. Genome Res 21(12):2129–2142

    Article  CAS  Google Scholar 

  4. Downing T, Imamura H, Decuypere S, Clark TG, Coombs GH, Cotton JA, Hilley JD, de Doncker S, Maes I, Mottram JC, Quail MA, Rijal S, Sanders M, Schonian G, Stark O, Sundar S, Vanaerschot M, Hertz-Fowler C, Dujardin JC, Berriman M (2011) Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance. Genome Res 21(12):2143–2156

    Article  CAS  Google Scholar 

  5. Raymond F, Boisvert S, Roy G, Ritt JF, Legare D, Isnard A, Stanke M, Olivier M, Tremblay MJ, Papadopoulou B, Ouellette M, Corbeil J (2012) Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res 40(3):1131–1147

    Article  CAS  Google Scholar 

  6. Guimond C, Trudel N, Brochu C, Marquis N, El F, Peytavi R, Briand G, Richard D, Messier N, Papadopoulou B, Corbeil J, Bergeron MG, Legare D, Ouellette M (2003) Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays. Nucleic Acids Res 31(20):5886–5896

    Article  CAS  Google Scholar 

  7. Duncan R (2004) DNA microarray analysis of protozoan parasite gene expression: outcomes correlate with mechanisms of regulation. Trends Parasitol 20(5):211–215

    Article  CAS  Google Scholar 

  8. Duncan RC, Salotra P, Goyal N, Akopyants NS, Beverley SM, Nakhasi HL (2004) The application of gene expression microarray technology to kinetoplastid research. Curr Mol Med 4(6):611–621

    Article  CAS  Google Scholar 

  9. McNicoll F, Drummelsmith J, Muller M, Madore E, Boilard N, Ouellette M, Papadopoulou B (2006) A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum. Proteomics 6(12):3567–3581

    Article  CAS  Google Scholar 

  10. Almeida R, Gilmartin BJ, McCann SH, Norrish A, Ivens AC, Lawson D, Levick MP, Smith DF, Dyall SD, Vetrie D, Freeman T, Coulson RM, Sampaio I, Schneider H, Blackwell JM (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(1):87–100

    Article  CAS  Google Scholar 

  11. 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(2):198–218

    Article  CAS  Google Scholar 

  12. Saxena A, Lahav T, Holland N, Aggarwal G, Anupama A, Huang Y, Volpin H, Myler PJ, Zilberstein D (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(1):53–65

    Article  CAS  Google Scholar 

  13. Leifso K, Cohen-Freue G, Dogra N, Murray A, McMaster WR (2007) Genomic and proteomic expression analysis of Leishmania promastigote and amastigote life stages: The Leishmania genome is constitutively expressed. Mol Biochem Parasitol 152(1):35–46

    Article  CAS  Google Scholar 

  14. Cohen-Freue G, Holzer TR, Forney JD, McMaster WR (2007) Global gene expression in Leishmania. Int J Parasitol 37(10):1077–1086

    Article  CAS  Google Scholar 

  15. Rochette A, Raymond F, Ubeda JM, Smith M, Messier N, Boisvert S, Rigault P, Corbeil J, Ouellette M, Papadopoulou B (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  Google Scholar 

  16. Lahav T, Sivam D, Volpin H, Ronen M, Tsigankov P, Green A, Holland N, Kuzyk M, Borchers C, Zilberstein D, Myler PJ (2011) Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania. FASEB J 25(2):515–525

    Article  CAS  Google Scholar 

  17. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63

    Article  CAS  Google Scholar 

  18. Clayton CE (2002) Life without transcriptional control? From fly to man and back again. EMBO J 21(8):1881–1888

    Article  CAS  Google Scholar 

  19. Nilsson D, Gunasekera K, Mani J, Osteras M, Farinelli L, Baerlocher L, Roditi I, Ochsenreiter T (2010) Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei. PLoS Pathog 6(8):e1001037. https://doi.org/10.1371/journal.ppat.1001037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. van Luenen HG, Farris C, Jan S, Genest PA, Tripathi P, Velds A, Kerkhoven RM, Nieuwland M, Haydock A, Ramasamy G, Vainio S, Heidebrecht T, Perrakis A, Pagie L, van Steensel B, Myler PJ, Borst P (2012) Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania. Cell 150(5):909–921

    Article  Google Scholar 

  21. Mittra B, Cortez M, Haydock A, Ramasamy G, Myler PJ, NW A (2013) Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels. J Exp Med 210(2):401–416

    Article  CAS  Google Scholar 

  22. Martin JL, Yates PA, Soysa R, Alfaro JF, Yang F, Burnum-Johnson KE, Petyuk VA, Weitz KK, Camp DG 2nd, Smith RD, Wilmarth PA, David LL, Ramasamy G, Myler PJ, Carter NS (2014) Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani. PLoS Pathog 10(2):e1003938

    Article  Google Scholar 

  23. Goldman-Pinkovich A, Balno C, Strasser R, Zeituni-Molad M, Bendelak K, Rentsch D, Ephros M, Wiese M, Jardim A, Myler PJ, Zilberstein D (2016) An arginine deprivation response pathway is induced in Leishmania during macrophage invasion. PLoS Pathog 12(4):e1005494

    Article  Google Scholar 

  24. Hunt AG (2015) A rapid, simple, and inexpensive method for the preparation of strand-specific RNA-Seq libraries. Methods Mol Biol 1255:195–207

    Article  CAS  Google Scholar 

  25. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139–140

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Center for Global Infectious Disease Research, Seattle Childrens Research Institute, 307 Westlake Ave N, Suite 500, Seattle, 98109-5219, WA, USA

    Peter J. Myler, Jacqueline A. McDonald, Pedro J. Alcolea & Aakash Sur

  2. Department of Global Health, University of Washington, Seattle, 98195, WA, USA

    Peter J. Myler

  3. Department of Biomedical Informatics and Medical Education, University of Washington, Seattle, 98195, WA, USA

    Peter J. Myler & Aakash Sur

  4. Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas (CSIC), Madrid, 28040, Spain

    Pedro J. Alcolea

Authors
  1. Peter J. Myler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jacqueline A. McDonald
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pedro J. Alcolea
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aakash Sur
    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. Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

    Joachim Clos

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Myler, P.J., McDonald, J.A., Alcolea, P.J., Sur, A. (2019). Quantitative RNA Analysis Using RNA-Seq. In: Clos, J. (eds) Leishmania. Methods in Molecular Biology, vol 1971. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9210-2_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9210-2_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9209-6

  • Online ISBN: 978-1-4939-9210-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation