ABSTRACT: We performed microRNA sequencing (miRNA-seq) according to Illumina sequencing protocols. Total RNA extracted from MCF-7 cells expressing or not human Erbeta in absence of estrogen stimuli was subjected to size selection labrary preparation ond retrotranscribed. Obtained DNA was sequenced with the Illumina/Solexa Genome Analyzer (GAIIX).
Project description:We performed microRNA sequencing (miRNA-seq) according to Illumina sequencing protocols. Total RNA extracted from MCF-7 cells expressing human ER-beta in absence of estrogen stimuli was subjected to size selection labrary preparation ond retrotranscribed. Obtained DNA was sequenced with the Illumina/Solexa Genome Analyzer (GAIIX).
Project description:We performed microRNA sequencing (miRNA-seq) according to Illumina sequencing protocols. Total RNA extracted from ZR75.1 cells expressing or not human ER-beta in absence of estrogen stimuli was subjected to size selection library preparation and retrotranscribed. Obtained DNA was sequenced with the Illumina HiSeq 1500 sequencer .
Project description:We performed microRNA sequencing (miRNA-seq) according to Illumina sequencing protocols. Platelet-rich plasma (PRP) has been obtained by centrifuging whole blood at 160 x g for 15 min at room temperature. Platelets have been activated with 3 different physiologic agonists, namely ADP (20uM), collagen (60ug/mL), or Thrombin Receptor Activating Peptide (TRAP 25uM) under continuous stirring. An aliquot of platelets has been obtained at 120 minutes following ADP, collagen and TRAP and then processed to determine miRNA expression profiles. Time 0, indicating samples before any agonist treatment, was used as baseline. Total RNA was extracted by using mirVana Paris kit (life technologies) and then was subjected to size selection library preparation and retrotranscribed. Obtained cDNA was sequenced with the Illumina HiSeq 1000 sequencer .
Project description:Endometrial biopsies from tumor (T), adjacent normal (N) and hyperplastic (H) tissue specimens (when available) were obtained by histeroscopy from 10 patients and 3 healty donor. RNA isolated from these samples was used to perform smallRNA-Seq by NGS and gene expression profiling by microarrays in order to identify deregulated Small Non Coding RNAs that could be used as a signaturein the endometrial carcinogenesis process and to evaluate in parallel the target transcriptome disregulation.
Project description:Two subtypes of the estrogen receptor, ERalpha and ERbeta, mediate the actions of estrogens, and the majority of human breast tumors contain both ERalpha and ERbeta. To examine the possible interactions and modulatory effects of ERbeta on ERalpha activity, we have used adenoviral gene delivery to produce human breast cancer (MCF-7) cells expressing ERbeta, along with their endogenous ERalpha. We have examined the effects of ERβ expression on genome-wide gene expression by Affymetrix GeneChip microarrays. We find that ERbeta modulated estrogen gene expression on nearly 24% of E2-stimulated genes but only 8% of E2-inhibited genes. We find that ERbeta modulation is gene-specific, enhancing or counteracting ERalpha regulation for distinct subsets of estrogen target genes. Introduction of ERbeta into ERalpha-containing cells induced up/down-regulation of many estrogen target in the absence of any added ligand. In addition, ERbeta presence elicited the expression of a unique set of genes that were not regulated by ERalpha alone. ERbeta modulated the expression of genes in many functional categories, but the greatest numbers were associated with transcription factor and signal transduction pathways. Regulation of multiple components in the TGF beta, SDF1, and semaphorin pathways, may contribute to the suppression of proliferation observed with ERbeta both in the presence and absence of estrogen. Hence, ERbeta modulates ERalpha gene regulation in diverse ways that may contribute to its growth-inhibiting beneficial effects in breast cancer Experiment Overall Design: MCF-7 cells expressing endogenous ERalpha were infected with adenovirus carrying either estrogen receptor beta (AdERb) or no insert (Ad) at multiplicity of infection (moi) of 5 or 50. Cells were infected with adenovirus for a period of 48hr before treatment with ligand (vehicle control or 10nM 17beta-estradiol) for a additional period of 24hr before harvest.
Project description:Small noncoding (snc) RNAs represent a growing family of transcripts that regulate key cellular processes, including mRNA degradation, translational repression and transcriptional gene silencing. Among these, the PIWI-interacting RNAs (piRNAs), a major class of sncRNAs initially identified in the germline of a variety of species, are now being found to be functionally active also in somatic cells. However, whether the Piwi/piRNA pathway is associated with fundamental biological processes, such as cell cycle progression, remains elusive. Here we investigated the possibility that piRNAs are expressed in liver and modulated during regenerative proliferation of this organ. To this aim, smallRNA-Seq was applied to identify and quantitate expression of these RNAs in rat liver before, during and after the wave of cell proliferation that follows partial hepatectomy (PH). Q-PCR analysis revealed the presence in rat liver of two PIWI (PIWI-Like) subfamily members (PIWIL2/HILI and, to a much lower level, PIWIL4/HIWI2) and other components of the piRNA biogenesis pathways, suggesting that this is present and functional in hepatocytes. Indeed, ~1400 piRNAs originally identified in rat and other mammalian germline cells are expressed in adult rat liver, including 72 that show timed changes in expression during cell cycle progression. Most piRNAs are up-regulated 24-48h after hepatectomy, a timing that corresponds to cell transition through the S phase, and return to basal levels by 168 h, when organ regeneration is completed and hepatocytes reach quiescence. These results indicate that the piRNA pathway is active in somatic cells and, more important, that it is subject to regulation during physiological processes, such as cell proliferation, when piRNAs may exert their regulatory functions on the cell genome and transcriptome. smallRNA-Seq was applied to identify and quantitate expression of RNAs in rat liver before and after partial hepatectomy (PH).
Project description:E12.5 wild-type embryos were dissected to collect ventral midbrain regions. Samples were crosslinked 10min with 1% formaldehyde and processed for chromatin extraction and Chromatin Immunoprecipitation (ChIP) following the Millipore upstate protocol. Libraries were prepared using the illumina ChIP-Seq DNA Sample Prep Kit. ChIP-Seq libraries were sequenced on the Illumina GAIIx.
Project description:E12.5 wild-type and NestinCre Foxa1/2 Flox/ Flox mutant embryos were quickly snap-frozen, then 10um-thick cryostat coronal sections of the midbrain were cut and mounted on membrane slides (Zeiss) and the floor plate was Laser Capture Microdissected. RNA was extracted using the Picopure RNA Isolation Kit RNA sequencing library was prepared using the Ovation RNA-Seq system (Nugen). RNA-Seq libraries were sequenced on the Illumina GAIIx.
Project description:We used microarrays to detail the global transcriptional response mediated by ERalpha or ERbeta to the phytoestrogen genistein in the MCF-7 human breast cancer cell model. Experiment Overall Design: MCF-7 human breast cancer cells expressing endogenouse Estrogen Receptor Alpha (ERalpha) were infected with adenovirus carrying either estrogen receptor beta (AdERb) or no insert (Ad) at multiplicity of infection (moi) of 20. Cells were then treated with either vehicle control (veh), 6nM 17beta-estradiol (E2), 6nM genistein (LG), 300nM genistein (HG), 300nM S-Equol (EQ), HG+3uM ICI182,780 (IG), EQ+3uM ICI 182,780(IE) for a additional periods of 4h or 24hr before RNA extraction and hybridization on Affymetrix microarrays. We sought to determine if genistein and S-Equol, phytoestrogens selective for the ERbeta can elicit transcriptional response distinctive from those mediated by the ERalpha.
Project description:A single replicate of exponentially growing DT40 CL18 chicken B lymphoma cells were harvested and extracted RNA was subjected to Illumina GAIIx paired-end sequencing to determine global gene expression. Single replicate RNA-seq expression analysis of DT40 cells.