Gene expression between high and low altitude Mus musculus domesticus
ABSTRACT: Analysis of gene expression profiles is an attractive method for discovering how animals respond to environmental challenges in nature. Compared to low altitudes, high altitudes are characterized by reduced partial pressures of oxygen (hypoxia) and cooler ambient temperatures To better understand how mammals cope with high altitudes, we trapped wild house mice (Mus musculus domesticus) from 3 populations in La Paz, Bolivia (3000 - 3600 m) and 3 populations in Lima, Peru (0 – 200 m). Affymetrix GeneChip® Mouse Genome 430 2.0 Arrays were use to measure mRNA abundance in the livers of these mice. Eighteen male house mice were trapped from three different locations (3 mice per location)at high alttiude (La Paz, Bolivia, 3600 m) and from three locations at low altiditude (Lima, Peru, 100 m). Total mRNA was extracted from the livers and used for hybridization of Affymetrix GeneChip Mouse expression set 420.
Project description:Patients were recruited at the Hospital do Cancer AC Camargo (São Paulo/Brazil), Instituto de Enfermedades Neoplasicas - INEN (Lima, Peru), Hospital Araujo Jorge (Goiania, Brazil) and Hospital Heliopolis (São Paulo, Brazil).All patients signed a pre-informed consent and the study was approved by the internal ethics committee. Tissue samples were snap frozen in liquid nitrogen . Before RNA extraction histopathological diagnosis was re-confirmed, all samples were micro-dissected and only tissues with at least 70% of tumor cells and no visible infiltrating inflammatory cells were used as tumor. Morphologically disease-free tissue obtained from surgical margins was considered as normal. A total of 38 samples were analyzed: 8 normal tissues, 10 goiters, 10 adenomas and 10 papillary carcinomas. Keywords: other
Project description:In Coronary Artery Bypass Grafting (CABG), the combined use of Left or Right Internal Mammary Artery (LIMA or RIMA) -collectively known as Bilateral IMAs (BIMAs)- provides a survival advantage over the LIMA alone. Several studies analyzed the gene expression in LIMAs and other conduits, however they either used a candidate gene approach or analyzed a small number of samples. Additionally, RIMA has never been analyzed compared to LIMA. Here we report a genome-wide transcriptional analysis of BIMA to investigate the expression profile of these conduits in patients undergoing CABG. Marginal differences were reported between LIMA and RIMA (p <0.05) using a linear model for microarray data. Ingenuity Pathway Assist (IPA) analysis found no consistent set of over-represented pathways and no trends in patterns of gene expression. As expected, in comparing the BIMAs to the aorta, we found differences in pathways and processes associated with atherosclerosis, inflammation, and cell signaling. Although evidence in favor of the use of BIMA in CABG has been available for over a decade, their routine use in clinical practice remains very low accounting for only 4% of CABG procedures in the US. Despite differences in embryologic development, our genome-wide transcriptional analysis, show marginal differences between LIMA and RIMA. Taken together, clinical and genomic analyses provide evidences that could impact the independent or combined use of the BIMAs as a conduit in CABG. We selected 32 patients from whom we had frozen archival tissue from aorta, LIMA, and RIMA in the Cardiovascular Blood and Tissue Bank at the Valley Columbia Heart Center . The mammary and aortic tissues were harvested at time of CABG. The surgeon dissected the two most distal aspects of LIMA and RIMA segments and obtained a ≥1cm sample for the tissue bank. The tissue sample was placed in a cryomold and processed as a “frozen section” specimen using standard OCT gel. Total RNA was extracted using Qiagen RNeasy Mini Kit, RNA integrity was assessed using the Agilent 2100 BioAnalyzer and RNA quantities and purity were determined using a NanoDrop Spectrophotometer. RNA samples were amplified using the NuGen Ovation RNA Amplification System V2 The resulting cDNA was labeled and hybridized to the Affymetrix U133A 2.0 GeneChip. 73 arrays at the end.
Project description:Solanum lycopersicoides (LA2408), collected at higher altitudes (up to 3600 meters) than any of other solanum species, is a wild nightshade distant-allied to cultivated tomato. Many traits of Solanum lycopersicoides including cold tolerance, resistance to virus diseases and insect pests were previously confirmed. Thus, it is an ideal candidate plant for isolating cold-tolerance-related genes. In this study, we successfully cloned the full-length cDNA of the CBF1 gene from Solanum lycopersicoides which was designated as SsCBF1. In order to investigate the possible functions of SsCBF1 in plant growth and stress responses, we generated transgenic Arabidopsis overexpressing SsCBF1. We employed the RNA-seq approach to identify the differentially expressed genes between the two genotypes. Processing of RNA samples on the Illumina HiSeq 2000 system produced more than 20 million reads, each 100bp in length, encompassing 2.0 Gb of sequence data for each sample which was then mapped to the reference genome. The statistical analysis identified a total of 338 differentially expressed genes between Col-0 (WT) and transgenic Arabidopsis overexpressing SsCBF1 with the criteria of Q-value < 0.001 and fold change >2, among which 120 (35.5%) were up-regulated and 218 (64.5%) were down-regulated. RNA-sequencing was carried out using one transgenic line (35S:SsCBF1-11) and the Col-0 (WT) plants. Total RNA was isolated with Trizol reagent (Invitrogen, USA) from the aerial parts of 4-week-old seedlings grown in parallel under unstressed conditions. Materials from 20 plants of each genotype were pooled for RNA isolation.
Project description:To determine the effects of age and lipoic acid supplementation on hepatic gene expression, we fed young (3 months) and old (24 months) male Fischer 344 rats a diet with or without 0.2% (w/w) R-α-lipoic acid (LA) for two weeks. Total RNA isolated from liver tissue was analyzed by Affymetrix microarray to examine changes in transcriptional profile. Results showed an increase in pro-inflammatory gene expression in the aging liver, with increased immune cell function and tissue remodeling genes, representing 45% of the age-related transcriptome changes. Increased inflammation was corroborated by increases in soluble ICAM1 levels with age. There were also observed age-related increases in transcription of genes related to lipid and cholesterol synthesis including Acetyl CoA Carboxylase (Acacb) and Fatty acid Synthase (Fasn). Supplementation of old animals with LA did not reverse this necro-inflammatory phenotype, yet limited age-associated hepatic dyslipidemia. Dietary LA further affected a small but concerted number of hepatic genes regardless of age. These included declines in lipid and bile synthesis genes. Decline in lipid synthesis genes was further corroborated by a decrease in Fasn and Acc protein levels. Intriguingly, LA also altered the expression of genes governing circadian rhythm, most notably Bmal1, Npas2, and Per2, which changed in a coordinated manner with respect to their rhythmic transcription. Thus, advanced age is associated with a necro-inflammatory phenotype and increased lipid synthesis, while chronic LA supplementation influences hepatic genes associated with energy metabolism and circadian rhythm regardless of age. Young (3 months) and old (24 months) Fischer 344 male rats were fed an AIN-93M diet ± 0.2% (w/w) R-α-lipoic acid for two weeks prior to sacrifice. Total RNA was extracted from the median lobe of the liver and analyzed using Affymetrix Rat Genome 230 2.0 Genechips. There were hybridizations for 8 animals in each of the young control, young LA-supplemented, and old control groups, and 6 animals in the old LA-supplemented group. There are only 6 animals in the old LA group because two animals died during the two-week feeding period. This is the reason for nonsequential numbering of animals in this group.
Project description:Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics. Various Argonautes associated small RNA profiles were generated by deep sequencing the Agos-IP samples in HEK293 Cells transfected with corresponding Argonaute.