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https://www.arca.fiocruz.br/handle/icict/56919
A SUITABLE RNA PREPARATION METHODOLOGY FOR WHOLE TRANSCRIPTOME SHOTGUN SEQUENCING HARVESTED FROM PLASMODIUM VIVAX‑INFECTED PATIENTS
Autor(es)
Afiliação
University of Campinas. Institute of Biology. Department of Genetics, Evolution, Microbiology and Immunology. Prof. Dr. Luiz Jacintho da Silva. Laboratory of Tropical Diseases. Campinas, SP, Brazil.
Fundação Oswaldo Cruz. Instituto Leônidas & Maria Deane. Manaus, AM, Brasil.
Fundação Oswaldo Cruz. Instituto Leônidas & Maria Deane. Manaus, AM, Brasil.
University of Campinas. Institute of Biology. Department of Genetics, Evolution, Microbiology and Immunology. Prof. Dr. Luiz Jacintho da Silva. Laboratory of Tropical Diseases. Campinas, SP, Brazil.
University of Campinas. Institute of Biology. Department of Genetics, Evolution, Microbiology and Immunology. Prof. Dr. Luiz Jacintho da Silva. Laboratory of Tropical Diseases. Campinas, SP, Brazil.
Fundação Oswaldo Cruz. Instituto Leônidas & Maria Deane. Manaus, AM, Brasil.
Fundação Oswaldo Cruz. Instituto Leônidas & Maria Deane. Manaus, AM, Brasil.
University of Campinas. Institute of Biology. Department of Genetics, Evolution, Microbiology and Immunology. Prof. Dr. Luiz Jacintho da Silva. Laboratory of Tropical Diseases. Campinas, SP, Brazil.
University of Campinas. Institute of Biology. Department of Genetics, Evolution, Microbiology and Immunology. Prof. Dr. Luiz Jacintho da Silva. Laboratory of Tropical Diseases. Campinas, SP, Brazil.
Resumo em Inglês
Plasmodium vivax is a world-threatening human malaria parasite, whose biology remains elusive. The unavailability of in vitro culture, and the difficulties in getting a high number of pure parasites makes RNA isolation in quantity and quality a challenge. Here, a methodological outline for RNA-seq from P. vivax isolates with low parasitemia is presented, combining parasite maturation and enrichment with efficient RNA extraction, yielding ~ 100 pg.µL−1 of RNA, suitable for SMART-Seq Ultra-Low Input RNA library and Illumina sequencing. Unbiased coding transcriptome of ~ 4 M reads was achieved for four patient isolates with ~ 51% of transcripts mapped to the P. vivax P01 reference genome, presenting heterogeneous profiles of expression among individual isolates. Amongst the most transcribed genes in all isolates, a parasite-staged mixed repertoire of conserved parasite metabolic, membrane and exported proteins was observed. Still, a quarter of transcribed genes remain functionally uncharacterized. In parallel, a P. falciparum Brazilian isolate was also analyzed and 57% of its transcripts mapped against IT genome. Comparison of transcriptomes of the two species revealed a common trophozoite-staged expression profile, with several homologous genes being expressed. Collectively, these results will positively impact vivax research improving knowledge of P. vivax biology.
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