Project description:We did microarray to compare the gene expression profile of peripheral blood mononuclear cells (PBMC from normal volunteer) and two leukemic cell lines that is K562 (Chronic myelogenous leukemia cell line), HL60 (Promyelocytic leukemia cell line) in order to find differentially expressed genes in these samples.
Project description:MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post-transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack ability to identify novel miRNAs and accurately determine expression at a range of concentration. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts. The results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 (chronic myelogenous leukemia) and HL60 (acute promyelogenous leukemia) are presented. Custom computation pipelines were used to generate expression profiles of known and for identification of novel miRNAs. Some of the highly expressed miRNAs in the leukocytes include several members of the let 7 family, mir-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 cells or HL60 revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by realtime RT-PCR and or RNAase protection assay. The small RNA population from four samples - Two Normal Peripheral blood mononuclear cells (PBMC) samples, K562 cell line (This cell line is used as a model to study Chronic Myelogenous Leukemia), HL60 (This cell line is used to study acute promyelogenous leukemia) was sequenced using Solexa technology.
Project description:As part of our study in understanding the role of SP140 in inflammatory pathways in macrophages, we inhibited SP140 mRNA using siRNA. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors (from Sanquin Institute Amsterdam or from GSK Stevenage Blood Donation Unit) by Ficoll density gradient (Invitrogen). CD14+ monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec). CD14+ cells were differentiated with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) (R&D systems) for 3 days followed by 3 days of polarization into classically activated (inflammatory) M1 macrophages (100 ng/mL IFN-γ; R&D systems). M1 macrophages were transfected with siGENOME human smartpool SP140 siRNA or non-targeting scrambled siRNA for 48h with DharmaFECT™ transfection reagents according to manufacturer’s protocol (Dharmacon). The cells were left unstimulated or stimulated with 100 ng/mL LPS (E. coli 0111:B4; Sigma) for 4h (for qPCR) or 24h (for Elisa). The cells were lysed (ISOLATE II RNA Lysis Buffer RLY-Bioline) for RNA extraction.150 ng total RNA was labelled using the cRNA labelling kit for Illumina BeadArrays (Ambion) and hybridized with Ref8v3 BeadArrays (Illumina). Arrays were scanned on a BeadArray 500GX scanner and data were normalized using quantile normalization with background subtraction (GenomeStudio software; Illumina). This submission only contains processed data