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Leishmania pp 95-108 | Cite as

Quantitative RNA Analysis Using RNA-Seq

  • Peter J. MylerEmail author
  • Jacqueline A. McDonald
  • Pedro J. Alcolea
  • Aakash Sur
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

RNA-seq Transcriptome mRNA Differential gene expression 

References

  1. 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):436–442CrossRefGoogle Scholar
  2. 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):839–847CrossRefGoogle Scholar
  3. 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):2129–2142CrossRefGoogle Scholar
  4. 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):2143–2156CrossRefGoogle Scholar
  5. 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):1131–1147CrossRefGoogle Scholar
  6. 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):5886–5896CrossRefGoogle Scholar
  7. 7.
    Duncan R (2004) DNA microarray analysis of protozoan parasite gene expression: outcomes correlate with mechanisms of regulation. Trends Parasitol 20(5):211–215CrossRefGoogle Scholar
  8. 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):611–621CrossRefGoogle Scholar
  9. 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):3567–3581CrossRefGoogle Scholar
  10. 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):87–100CrossRefGoogle Scholar
  11. 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):198–218CrossRefGoogle Scholar
  12. 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):53–65CrossRefGoogle Scholar
  13. 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):35–46CrossRefGoogle Scholar
  14. 14.
    Cohen-Freue G, Holzer TR, Forney JD, McMaster WR (2007) Global gene expression in Leishmania. Int J Parasitol 37(10):1077–1086CrossRefGoogle Scholar
  15. 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 9:255CrossRefGoogle Scholar
  16. 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):515–525CrossRefGoogle Scholar
  17. 17.
    Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63CrossRefGoogle Scholar
  18. 18.
    Clayton CE (2002) Life without transcriptional control? From fly to man and back again. EMBO J 21(8):1881–1888CrossRefGoogle Scholar
  19. 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.1001037CrossRefPubMedPubMedCentralGoogle Scholar
  20. 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):909–921CrossRefGoogle Scholar
  21. 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):401–416CrossRefGoogle Scholar
  22. 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):e1003938CrossRefGoogle Scholar
  23. 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):e1005494CrossRefGoogle Scholar
  24. 24.
    Hunt AG (2015) A rapid, simple, and inexpensive method for the preparation of strand-specific RNA-Seq libraries. Methods Mol Biol 1255:195–207CrossRefGoogle Scholar
  25. 25.
    Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139–140CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Peter J. Myler
    • 1
    • 2
    • 3
    Email author
  • Jacqueline A. McDonald
    • 1
  • Pedro J. Alcolea
    • 1
    • 4
  • Aakash Sur
    • 1
    • 3
  1. 1.Center for Global Infectious Disease ResearchSeattle Childrens Research InstituteSeattleUSA
  2. 2.Department of Global HealthUniversity of WashingtonSeattleUSA
  3. 3.Department of Biomedical Informatics and Medical EducationUniversity of WashingtonSeattleUSA
  4. 4.Department of Cellular and Molecular BiologyCentro de Investigaciones Biológicas (CSIC)MadridSpain

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