Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/28685
Title: Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures
Authors: Kessler, Rafael Luis
Pavoni, Daniela Parada
Krieger, Marco Aurélio
Probst, Christian Macagnan
Affilliation: Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Bioinformática e Biologia Computacional. Curitiba, PR, Brasil.
Abstract: Trypanosomatids are a group of protozoan parasites that includes the etiologic agents of important human illnesses as Chagas disease, sleeping sickness and leishmaniasis. These parasites have a significant distinction from other eukaryotes concerning mRNA structure, since all mature mRNAs have an identical species-specific sequence of 39 nucleotides at the 5′ extremity, named spliced leader (SL). Considering this peculiar aspect of trypanosomatid mRNA, the aim of the present work was to develop a Trypanosoma cruzi specific in vitro transcription (IVT) linear mRNA amplification method in order to improve parasite transcriptomics analyses.For this research, we designed an oligonucleotide complementary to the last 21 bases of T. cruzi SL sequence, bearing an upstream T7 promoter (T7SL primer), which was used to direct the synthesis of second-strand cDNA. Original mRNA was then amplified by IVT using T7 RNA polymerase. T7SL-amplified RNA from two distinct T. cruzi stages (epimastigotes and trypomastigotes) were deep sequenced in SOLiD platform. Usual poly(A) + RNA and and T7- oligo(dT) amplified RNA (Eberwine method) were also sequenced. RNA-Seq reads were aligned to our new and improved T. cruzi Dm28c genome assembly (PacBio technology) and resulting transcriptome pattern from these three RNA preparation methods were compared, mainly concerning the conservation of mRNA transcritional levels and DEGs detection between epimastigotes and trypomastigotes. As resulted, T7SL IVT method detected more potential differentially expressed genes in comparison to either poly(A) + RNA or T7dT IVT, and was also able to produce reliable quantifications of the parasite transcriptome down to 3 ng of total RNA. Furthermore, amplification of parasite mRNA in HeLa/epimastigote RNA mixtures showed that T7SL IVT generates transcriptome quantification with similar detection of differentially expressed genes when parasite RNA mass was only 0.1% of the total mixture (R = 0.78 when compared to poly(A) + RNA). This way, concluded that, the T7SL IVT amplification method presented here allows the detection of more potential parasite differentially expressed genes (in comparison to poly(A) + RNA) in host-parasite mixtures or samples with low amount of RNA. This method is especially useful for trypanosomatid transcriptomics because it produces less bias than PCR-based mRNA amplification. Additionally, by simply changing the complementary region of the T7SL primer, the present method can be applied to any trypanosomatid species.
Keywords: Sequence Analysis
Nucleic Acid Amplification Techniques
Keywords in spanish: ARN Mensajero
Análisis de Secuencia de ARN
Técnicas de Amplificación de Ácido Nucleico
keywords: Trypanosoma cruzi
DeCS: RNA Mensageiro
Trypanosomatina
Análise de Sequência de RNA
Técnicas de Amplificação de Ácido Nucleico
Issue Date: 2017
Publisher: Springer Nature
Citation: KESSLER, Rafael Luis et al. Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in hostparasite RNA mixtures. BMC Genomics, v.18, n. 793, 2017.
DOI: 10.1186/s12864-017-4163-y
ISSN: 1471-2164
Copyright: open access
Appears in Collections:PR - ICC - Artigos de Periódicos

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