Project description:We evaluated whether targeted next-generation sequencing (NGS) using the Ion Torrent Personal Genome Sequencer of cfDNA could identify prognostic or predictive factors for overall survival (OS) or progression free survival (PFS) within a large cohort of patients with advanced lung adenocarcinoma enrolled in the GALAXY-1 trial.
Project description:Purpose: The goals of this study are to determine the effect of microRNA-17 overexpression on 20,803 human genes in RASFs using Ion ProtonTM System platform. Human RASFs from two RA patients were transfected with pre-miR-17 or NC-pre-miR for 48 h and total RNA was prepared using miRNeasy kit (Qiagen). Total RNA integrity was checked using an Agilent Technologies 2100 Bio analyzer (Santa Clara, CA). 10 ng of high quality RNA was used to make cDNA for amplification with the Ion AmpliSeq Transcriptome Human Gene Expression kit (ThermoFisher Scientific). The cDNA was subjected to 12 cycles of amplification with panel primers and barcoded with adapters as recommended. Resulting sequencing libraries were quantified by qPCR using SYBR FAST master mix from KapaBiosystems (Wilmington, MA). Sets of eight libraries were balanced, pooled and sequencing beads produced on an Ion Chef. Sequencing was performed on an Ion P1 semi-conductor sequencing chip using an Ion Proton™ System (ThermoFisher Scientific, Grand Island, NY). Data was collected and primary analysis performed using Torrent Suite software version 5.0.3. Reads were mapped to the panel and expression values determined. R Software version R-3.2.3 was used to generate heatmap. Among the panel of 20,803 genes, the expression of 15,067 genes as shown in the representative heat map was observed in pre-miR-17 and NC-pre-miR transfected RASFs. A total of 664 significantly modulated genes (301 upregulated and 363 downregulated) using Student ‘t’ test were further utilized for the IPA analysis. The result of IPA predicted the protein ubiquitin pathway as a major canonical pathway affected by the differentially regulated genes. Interestingly, IPA analysis generated an interactome that showed connectivity among various ubiquitin ligases, NF-ԟB family, AP-1/cJun, 20S and 26S proteasome system. Conclusion: Our results clearly shows the major pathways affected by miR-17 overexpression in RASFs were Protein ubiquitination related. mRNA profiles of pre-miR-17 and NC-pre-miR transfected RASFs were generated by AmpliSeq, in duplicate, using Ion Proton™ System.
Project description:Estrogens receptor a (ERα) is essential for breast tumors,since about seventy percent of breast cancers are detected as ERα positive.Recent studies suggest that ERα is related with the epithelial cell morphology. Recently, it has demonstrated that the suppression of ERα induced epithelial-mesenchymal transition (EMT) in the MCF-7 breast cacner cells. Interestingly, the loss of ERa resulted in strong differences on the gene expression profile of a variety of genes. Therefore, the aim of the RNA-seq is to elucidate the effect of the silencing of ERα on the mRNA levels of a larger variety of genes, thus revealing possible target genes which may be implicated on the aggressive phenotype and behavior of the ERα-suppresed MCF-7/SP10+ breast cancer cells. For this reason total RNA from both MCF-7/SP10+ cells and of their internal control MCF-7/C cells was extracted in 3 biological replicates and 3 technical replicates.
Project description:In this study, we have investigated the physiological consequences of PHB (poly(3-hydroxybutyrate)) synthesis in H. seropedicae by characterising the trancriptional changes in a mutant strain lacking phaC1 gene which codes for the PHA synthase enzyme essential for the last step in the synthesis of PHB. To do this experiment both wild type (SmR1) and phaC1 mutant were cultured on Nfb-Malate-HP media suplemented with 20 mM of ammonium chloride under 30 Celsius degrees and 120 rpm shaking rate until the late log-phase (O.D600 = 1.0), when the peak of PHB production is observed. The cultures obtained as above were harvested for RNA extraction and transcriptome analysis.
Project description:Background: Repair of DNA damage requires chromatin remodeling to permit removal of the lesions. How nucleosomes are remodelled to initiate repair of DNA damage remains largely unknown. Here, we describe how chromatin is altered during repair of UV-induced DNA damage at the level of the linear organisation of nucleosomes. Results: Using MNase-seq, we identified a subset of nucleosomes in the genome that are remodelled in UV-damaged wild-type yeast cells. We mapped the genomic location of these nucleosomes, showing that they contain the histone variant H2A.Z. The remodelling observed is consistent with histone exchange or eviction at these positions. This depends on the yeast SWI/SNF global genome nucleotide excision repair (GG-NER) chromatin-remodelling complex. Remarkably, we found that in the absence of DNA damage, the GG-NER complex occupies chromatin at nucleosome free regions separating adjacent nucleosomes. This establishes the nucleosome structure at these genomic locations, which we refer to as GG-NER complex binding sites (GCBS’s). We observed that these sites are frequently located precisely at certain boundary regions that delineate chromasomally interacting domains (CIDs). These boundaries define chromosomal domains of higher-order nucleosome-nucleosome interaction. We demonstrate that the GG-NER complex redistributes following remodelling of these nucleosomes after DNA damage taking up genomic positions located within the CIDs. This permits the efficient removal of DNA damage at these sites. Conclusions: We argue that organising DNA repair in the genome as described may define origins of DNA repair that greatly reduces the genomic search space for DNA damage recognition, thus ensuring the efficient repair of damage in chromatin.
Project description:Genetic heterogeneity can provide tumors with opportunities for therapy evasion, however the degree of genetic heterogeneity within metastatic melanomas has not been thoroughly investigated. We therefore isolated DNA from different regions of formalin fixed paraffin embedded metastatic melanoma tissue samples and subjected them to amplicon sequencing-based profiling of mutations in a panel of well known cancer genes using the Ion Ampliseq Cancer Panel.
Project description:microRNAs (miRNAs) accomplish a remarkable variety of biological functions. Their expression is tightly controlled, and the final production of a miRNA is dependent on the cooperation of multiple mechanisms and their net effect. Here we show that miR-124-1 is transcriptionally activated during erythroid differentiation by GATA-1, however its post-transcriptional processing is attenuated. We found that QKI5 binds to a distal QKI response element (QRE) embedded in the primary transcript of miR-124-1 (pri-124-1) and modulates Microprocessor function by direct association with DGCR8. Strikingly, Microprocessor recruitment to pri-124-1 is disrupted upon RNAi-mediated depletion of QKI5, consistent with the decrease in mature miR-124. Moreover, addition of QKI5 increases the conversion efficiency of pri-124-1 in cell-free extracts. For erythropoiesis, the decreased QKI5 leads to attenuated Microprocessor-mediated processing of pri-124-1, which confers the exquisite miRNA abundance necessary for development. This regulation also gives rise to a unique miRNA signature required for normal erythropoiesis. Thus, this QKI5-regulated miRNA processing may represent a common paradigm for erythroid development, and specifically, it may serve as a post-transcriptional fault security to prevent misexpression of certain miRNAs, that is essential for the establishment of particular gene expression patterns during development. Two samples are analyzed: K562 cells transduced with GFP lentivirus; and K562 cells transduced with QKI5-overexpressing lentivirus.