Project description:RNA pull-down assay. For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified. RNA purification and amplification: RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual. Microarray analysis: The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.

Project description:P. syringae pv. phaseolicola, the causal agent of halo blight disease in bean, produces a toxin known as phaseolotoxin, whose synthesis involves the product of some of the genes found within the Pht region. This region, considered a pathogenicity island, comprises 23 genes arranged in five transcriptional units; two single-gene units (argK, phtL) and three arranged as operons (phtA, phtD, phtM), most with unknown function. In P. syringae pv. phaseolicola, maximal expression of most of the genes encoded in the Pht region and the synthesis of phaseolotoxin require the product of the phtL gene, which has been proposed to have a regulatory function. In order to evaluate the role of phtL gene in P. syringae pv. phaseolicola, we performed a comparative transcriptional analysis with the wild type and a phtL- mutant strains using microarrays. The microarray data analysis showed that PhtL regulates the expression not only of genes within the Pht region, but also alters the expression of genomic genes related with the iron-acquisition system, pathogenicity, oxidative stress and virulence. This study suggests the possible relation of the PhtL protein with the iron response genes and with the pathogenicity and or virulence of this bacterium. The microarrays used in this study correspond to an assembled DNA microarray of P. syringae pv. phaseolicola NPS3121 previously reported (GEO accession number GPL7115) (HernM-CM-!ndez-Morales et al., 2009). This microarray consist of 1911 probes, with an average length 2.4 Kbp, representing 1X the genome of P. syringae pv. phaseolicola NPS3121, as well as several PCR products corresponding to various known genes, which were printed as controls. Each microarray experiment was repeated six times (two technical replicates with the same RNA samples and three biological replicates using RNA isolated from a different culture). cDNA synthesis, labeling, hybridization, washing, and chip scanning were performed at the Microarray Core Facility at Cinvestav-Langebio. Hybridized microarray slides were scanned (GenePix 4000, Axon Instruments, Inc) at a 10 um resolution adjusting the laser and gain parameters to obtain similar levels of fluorescence intensity in both channels. The spot intensities were quantified using Axon GenePrix Pro 6.0 image analysis software. First, an automatic spot finding and quantification option of the software was used. Subsequently, all spots were inspected individually and in some cases, the spot diameters were corrected or the spots were removed from the analysis. The mean of the signals and the median of backgrounds were used for further analysis. Raw data were imported into the R.2.2.1 software. Background signals were subtracted using the Robust Multichip Analysis (RMA) whereas the normalization of the signal intensities within slides was carried out using M-bM-^@M-^\print-tip loessM-bM-^@M-^] method and the LIMMA package. Normalized data were log2 transformed and then fitted into mixed model ANOVAs using the Mixed procedure. The p-values of the phtL effects were adjusted for by the False Discovery Rate Method M-bM-^@M-^\FDRM-bM-^@M-^]. Changes in signal intensity of M-BM-11.5-fold or higher/lower between phtL- mutant and wild type were highly significant (FDR, p-value M-bM-^IM-$0.05), however we focused only in differential expressed genes that fell within the more traditional criteria, which is the cut-off threshold for up-regulated (M-bM-^IM-%1.8) and down-regulated genes (M-bM-^IM-$0.50).

Project description:Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker-positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD34- and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD34- cells (p = 0.03), whereas mRNA of the mesenchymal marker PdgfrM-NM-1 (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p = 0.04). FACS analysis confirmed the existence of a PdgfrM-NM-1 positive subpopulation within Epcam+/Sca-1+/CD34- epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD34- cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,- cells in the biology of the murine lung. Single cells of 10 Sca-1+/CD34+/CD31-/CD45-, 7 Sca-1+/CD34-/CD31-/CD45-, and 12 Sca-1-/CD34-/CD31-/CD45- were analyzed. Although the raw data are two channel only Cy5 signal of each file as analyzed. The Cy5 channel for each gene is normalized to the average Cy5 intensity of the gene across all samples.

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Long-term exposure of Atlantic Cod (Gadus morhua) to components of produced water

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