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Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures

Overview of attention for article published in BMC Genomics, October 2017
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Title
Trypanosoma cruzi specific mRNA amplification by in vitro transcription improves parasite transcriptomics in host-parasite RNA mixtures
Published in
BMC Genomics, October 2017
DOI 10.1186/s12864-017-4163-y
Pubmed ID
Authors

Rafael Luis Kessler, Daniela Parada Pavoni, Marco Aurelio Krieger, Christian Macagnan Probst

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. 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. 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). 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.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 21 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 21 100%

Demographic breakdown

Readers by professional status Count As %
Researcher 4 19%
Student > Master 4 19%
Student > Bachelor 3 14%
Student > Doctoral Student 2 10%
Professor > Associate Professor 2 10%
Other 1 5%
Unknown 5 24%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 4 19%
Agricultural and Biological Sciences 4 19%
Medicine and Dentistry 3 14%
Computer Science 1 5%
Chemistry 1 5%
Other 0 0%
Unknown 8 38%

Attention Score in Context

This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 19 May 2018.
All research outputs
#7,475,536
of 12,963,687 outputs
Outputs from BMC Genomics
#4,025
of 7,614 outputs
Outputs of similar age
#152,598
of 311,334 outputs
Outputs of similar age from BMC Genomics
#419
of 821 outputs
Altmetric has tracked 12,963,687 research outputs across all sources so far. This one is in the 40th percentile – i.e., 40% of other outputs scored the same or lower than it.
So far Altmetric has tracked 7,614 research outputs from this source. They receive a mean Attention Score of 4.3. This one is in the 42nd percentile – i.e., 42% of its peers scored the same or lower than it.
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We're also able to compare this research output to 821 others from the same source and published within six weeks on either side of this one. This one is in the 45th percentile – i.e., 45% of its contemporaries scored the same or lower than it.