Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptome analysis of the Trypanosoma cruzi life-cycle

ABSTRACT: To generate a high quality, annotated gene expression database of T. cruzi trypomastigotes(TRP), amastigotes (AMA), epimastigotes (EPI), and metacyclic trypomastigotes (MET). The global assessment of transcript abundances in each life-cycle stage of T. cruzi will identify stage-regulated genes and provide an essential element of an integrated database of T. cruzi genomic, proteomic, and transcriptomic data. RNAs from 3 biological replicates from a single time point in each stage will be subjected to DNA microarrays containing probes for the complete, annotated T. cruzi genome, as it is currently known. The arrays for this project will be provided by the Pathogen Functional Genomics Resource Center (PFGRC) at The Institute for Genomic Research (TIGR) sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), and will contain long oligonucleotides complementary to every annotated gene in the newly sequenced T. cruzi genome. The resulting data and analysis results will be deposited in TcruziDB (http://TcruziDB.org). Keywords: Gene expression comparisons between the four life-cycle stages of T. cruzi Six hybridizations were performed for each life-cycle stage. The hybridizations consisted of three dye-swap experiments from three independent samples (biological replicates). In each case, the experimental sample was from a single life-cycle stage and the control sample was an equal mixture of all four life-cycle stages. A total of 24 hybridizations were preformed.

ORGANISM(S): Trypanosoma cruzi

SUBMITTER: D Weatherly

PROVIDER: E-GEOD-14641 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The steady-state transcriptome of the four major life-cycle stages of Trypanosoma cruzi.

Minning Todd A TA Weatherly D Brent DB Atwood James J Orlando Ron R Tarleton Rick L RL

BMC genomics 20090807

<h4>Background</h4>Chronic chagasic cardiomyopathy is a debilitating and frequently fatal outcome of human infection with the protozoan parasite, Trypanosoma cruzi. Microarray analysis of gene expression during the T. cruzi life-cycle could be a valuable means of identifying drug and vaccine targets based on their appropriate expression patterns, but results from previous microarray studies in T. cruzi and related kinetoplastid parasites have suggested that the transcript abundances of most gene ...[more]

PMID: 19664227

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Project description:Ambient temperature affects organisms comprehensively, however cold responses are different among tissues. Here, we adopt a transcript screening approach to explore and compare the cold responses in zebrafish gills and brain. Zebrafish were exposed to cold and the oligonucleotide-based microarray was used to identify cold-induced genes. Principle component analysis (PCA) of the gene expression profiles indicated that gills develop different strategies for the increasing of exposure period while brain relatively remained stable. Combining statistic and clustering methods, we found that gills showed higher protein metabolism and cell activity while brain showed higher stress responses and detoxification during cold acclimation. According to the microarray data sets, we extended the study on ionocyte- and isotocin neuron-related genes in gills and brain, respectively, and found these genes were broadly stimulated by cold. These data suggest that cold activates specific physiological functions in different tissues. Taken together, our results provide molecular evidences to elucidate the cold acclimation in zebrafish gills and brain. Keywords: Time course, Tissue types After the low-temperature treatment, brain and gill tissues dissected from 10 individuals were pooled as a sample and then homogenized in 0.8 ml Trizol reagent (Invitrogen, Carlsbad, CA). 120 individuals (60 for low-temperature treatment and 60 for control) were sacrificed for each microarray hybridization experiment, and another 200 individuals were used for quantitative reverse-transcription polymerase chain reaction. After chloroform extraction, RNA precipitation and ethanol washing, the RNA samples were purified and treated with DNase1 to remove the genomic DNA by using RNeasy Mini Kit (Qiagen, Huntsville, Alabama). The quantity and quality of total RNA were assessed by spectrophotometry and Bioanalyzer (Aglient Technologies, Foster City, CA). The commercial zebrafish 14K oligonucleotides set (MWG Biotech AG, Ebersbach, Germany) were obtained and were printed on UltraGAPS Coated slide (Corning, New York, NY ) with use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI) according to the manufactureâ??s instructions. The 14,067 oligonucleotides represent 9666 genes (7009 singlet genes and 2657 redundant genes), and the redundancy of this chip is 31 %. The detailed description of the oligonucleotides information can be obtained on the Ocimun Biosolution website. cDNA probes were synthesized and purified by reverse transcription of 20 Î¼g total RNA using a SuperScript indirect cDNA labeling system (Invitrogen) and MinElute PCR purification kit (Qiagen) and were labeled with Alexa 647 dye (cold treatment groups) and Alexa 555 dye (control groups)(Invitrogen), respectively. The zebrafish 14K OciChip array chip was pretreated with 1% bovine serum albumin (BSA) (fraction V), 4x saline-sodium citrate (SSC), and 1% sodium dodecylsulfate (SDS) at 42 Â°C for 45 min, and then hybridized overnight in a cocktail containing 5x Denhardt's solution, 6x SSC, 0.5% SDS, 50% formamide, 50 mM sodium phosphate, and 2 Âµg/Âµl yeast tRNA. Slides were washed with 2x SSC and 0.1% SDS (5 min), 1x SSC and 0.1% SDS (5 min), 0.5x SSC (5 min), and twice with 0.1x SSC (2 min each). Scanning was performed with a Genepix scanner (Molecular Devices, Sunnyvale, CA). The acquired images were analyzed using Genepix and Genespring software (Aglient Technologies, Foster City, CA). The measurements of spots were filtered by flags, and the Lowess normalization was performed after subtraction of the median background. Each experiment contained 3 biological replicates (including 1 dye swap) with different samples. The differentially expressed genes were selected from those with at least 2 of 3 significant signals (ratio > 2 or < 0.5), and then the Significant Analysis of Microarray method (SAM 3.02) was used to determine statistical significances.

Project description:Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress. To evaluate the gamma radiation effect on T. cruzi epimastigote cells, two identical and independent experiments (biological replicates) were performed in triplicate. Each triplicate was composed of seven samples: cell culture growth control (not used in the microarray experiments), non-irradiated cells (used as reference sample in the microarray analysis), and five irradiated samples that had their RNA extracted immediately after irradiation (or i.a.i.), at 4, 24, 48, and 96 hours post-irradiation. Triplicates were pooled after the RNA extraction and before the purification/amplification steps. Each pool corresponding to one of the five time points was hybridized twice against the reference pool from each experiment, applying a reference time-course dye-swap design. Thus, four microarray slides for each time point were produced, two for each biological replicate, totaling 20 slides.

Dataset Information

Transcriptome analysis of the Trypanosoma cruzi life-cycle

Publications

The steady-state transcriptome of the four major life-cycle stages of Trypanosoma cruzi.

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