RNA immunoprecipitation of Trypanosoma cruzi to study messenger ribonucleoproteins (mRNPs) containing TcDhh1.
ABSTRACT: To study the composition of mRNPs containing TcDhh1, we carried out immunoprecipitation assays with anti-TcDhh1 and epimastigotes lysates. Pre-immune serum was used as control. We also carried out a ribonomic approach to identify the mRNAs present within the TcDhh1 immunoprecipitated complexes. For this purpose, competitive microarray hibridizations were performed against negative controls, the non-precipitated fraction.
Project description:Exploration of transcriptome expression in 5 control and 4 familial dysautonomia (FD) human olfactory ecto-mesenchymal stem cells (hOE-MSCs) at very early (P1 and P2) and later (P5 and P9) cell passages.
Project description:Synechococcus elongatus PCC7942 wild type, the IdiB-free S.elongatus mutant K10 (Michel et al. 1999; Microbiology, 145: page 1473-1484) and the IdiC-merodiploid mutant MuD (Pietsch et al. 2007, Photosynth. Res. 94: page 91-108) were cultivated in BG11 medium, continuously bubbled with 2%CO2-enriched air and illuminated with fluorescent bulbs with a light intensity of 100 ﾵmol photons m-s s-1. After an inoculation of a cell density of OD750nm 0.3 the cells were cultivated with iron-sufficient or iron-deficient BG11 medium. The growth time for wild type was 24h and 72h. The two mutants were cultivated for 72h only. Possible differences in the RNA amount after the cultivation with or without iron were examined using the microarray technique. .
Project description:Four independent pools of zebrafish embryo were injected with prp2 morphants and after 24 hours post fertilization, gene expression profiles were compared to their respective controls, using microarray. A dye swap design experiment using four microarray slides were conducted.
Project description:Population dynamics of methanogenic genera was investigated in pilot anaerobic digesters. Cattle manure and two-phase olive mill wastes were codigested at a 3:1 ratio in two reactors operated at 37 ﾰC and 55 ﾰC. Other two reactors were run with either residue at 37 ﾰC. Sludge DNA extracted from samples taken from all four reactors on days 4, 14 and 28 of digestion was used for hybridisation with the AnaeroChip, an oligonucleotide microarray targeting those groups of methanogenic archaea that are commonly found under mesophilic and thermophilic conditions (Franke-Whittle et al. 2009, in press, doi:10.1016/j.mimet.2009.09.017).
Project description:RNA pull-down assay.<br>For the recombinant protein pull-down assays, 50 µg of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4°C. 100 µg of total RNA from epimastigotes were incubated with the bound protein in 500 µl EMSA buffer at 4°C 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 supernatant fractions were purified.<br><br>RNA purification and amplification:<br><br>RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpII aRNA Amplification kit (Ambion), according to the manufacturers manual.<br><br>Microarray analysis:<br>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 µM 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. <br>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:This study newly identified Tripelennamine (TA) as an inhibitor of yeast meiosis and sporulation. To examine if and how exposure of sporulating yeast cells to TA changes the meiotic transcriptional program cells were sporulated for 0, 4, and 8 hours in the presence or absence of 100 uM TA.