Project description:We used microarrays to analyze the global expression patterns for 22 commercially available pancreatic cancer cell lines Overall design: 22 pancreatic cancer cell lines were grown to 80% confluence in DMEM/FBS/PenStrep media and then harvested for total RNA
Project description:We used microarrays to analyze the global expression patterns for 22 commercially available pancreatic cancer cell lines 22 pancreatic cancer cell lines were grown to 80% confluence in DMEM/FBS/PenStrep media and then harvested for total RNA
Project description:Library preparation for whole genome bisulphite sequencing (WGBS) is challenging due to side effects of the bisulphite treatment, which leads to extensive DNA damage. Recently, a new generation of methods for bisulphite sequencing library preparation have been devised. They are based on initial bisulphite treatment of the DNA, followed by adaptor tagging of single stranded DNA fragments, and enable WGBS using low quantities of input DNA. In this study, we present a novel approach for quick and cost effective WGBS library preparation that is based on splinted adaptor tagging (SPLAT) of bisulphite-converted single-stranded DNA. Moreover, we validate SPLAT against three commercially available WGBS library preparation techniques, two of which are based on bisulphite treatment prior to adaptor tagging and one is a conventional WGBS method. Overall design: WGBS libraries were prepared with DNA from human lymphoblastoid cell line NA10860 and the ALL cell line REH
Project description:Dormancy in non-sporulating bacteria is an interesting and underexplored phenomenon with significant medical implications. In particular, latent tuberculosis may result from the maintenance of Mycobacterium tuberculosis bacilli in non-replicating states in infected individuals. Uniquely, growth of M. tuberculosis in aerobic conditions in potassium-deficient media resulted in the generation of bacilli that were non-culturable (NC) on solid media but detectable in liquid media. These bacilli were morphologically distinct and tolerant to cell-wall-targeting antimicrobials. Bacterial counts on solid media quickly recovered after washing and incubating bacilli in fresh resuscitation media containing potassium. This resuscitation of growth occurred too quickly to be attributed to M. tuberculosis replication. Transcriptomic and proteomic profiling through adaptation to, and resuscitation from, this NC state revealed a switch to anaerobic respiration and a shift to lipid and amino acid metabolism. High concordance with mRNA signatures derived from M. tuberculosis infection models suggests that analogous NC mycobacterial phenotypes may exist during disease and may represent unrecognized populations in vivo. Resuscitation of NC bacilli in potassium-sufficient media was characterized by time-dependent activation of metabolic pathways in a programmed series of processes that probably transit bacilli through challenging microenvironments during infection. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-151]
Project description:The success of shotgun proteomic analysis depends largely on how samples are prepared. Current approaches such as gel-, solution- or filter-based, although being extensively employed in the field, are time-consuming and less effective with respect to the repetitive sample processing, recovery, and overall yield. As an alternative, suspension trapping (S-Trap) filter is commercially available very recently in the format of single or 96-well filter plate. In contrast to conventional filter aided sample preparation (FASP) approach, which utilizes a molecular weight cutoff (MWCO) membrane as the filter and requires hours of processing before digestion-ready proteins can be obtained, S-Trap employs a three-dimensional porous material as filter media, and traps particulate protein suspension with subsequent depletion of interfering substances and in-filter digestion. Due to the large (sub-micron) pore size, each centrifugation cycle of S-Trap filter only takes around 1 minute, which significantly reduces the total processing time from approximately 3 hours by FASP to less than 15 minutes, suggesting an ultrafast sample preparation approach for shotgun proteomics. Here, for the first time, we comprehensively evaluate the performance of individual S-Trap filter and 96-well filter plate in the context of global protein identification and quantitation using whole cell lysate and clinically relevant sputum samples.
Project description:We generated hiPSCs from patients fibloblast with retinitis pigmentosa (RP) using retrovirus and Sendai virus vectors, which we differentiated into hiPSC derived retinal pigment epithelium using two different methods (SDIA and SFEB methods). We investigated whether these hiPSC-RPE colonies, which were differentiated from various cell lines and methods, showed similar gene expression patterns to those of native RPE. We classified hiPSC-RPE, hiPSCs, and fibroblasts from RP patients, hRPE (commercially available human fetal RPE, Lonza) , ARPE19 (a human RPE cell line), and other human tissues from 54,675 probe sets using microarray data.
Project description:The aim of the study was to characterize the molecular mechanism involved in TGF-ß mediated smooth muscle formation in vitro. We employed rat bone marrow derived Oct4 expressing clones of multipotent adult progenitor cells (rMAPC). We subjected these cells to differentiation towards smooth muscle cell as previously reported using TGF-ß1. The differentiation process reuires 6 days with media change every 2 days followed by RNA harvest. RNA was isolated using commercially available kits (Qiagen RNA easy micro kit). RNA integrity and quality was assessed prior to labeling and hybridization. As a control RNA from rat aortic smooth muscle cells was commercially obtained. Two biological replicate clones of rMAPC cells were used for the differentiation to smooth muscle like cells. The RNA was harvested at days 0, 2, 4 and 6 in triplicates. The RNA from primary smooth muscle cells was commercially obtained and was used in duplicates as control.