biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsStephen Hendersontranscription profiling by arrayHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-16356Kaposi sarcoma is the most common cancer in AIDS patients and is typified by red skin lesions. The disease is caused by the KSHV virus (HHV8) and is recognisable by its distinctive red skin lesions. The lesions are KSHV-infected spindle cells, most commonly the lymphatic endothelial and blood vessel endothelial cells (LEC and BEC), plus surrounding stroma. The KSHV virus expresses multiple MAF-downregulating microRNA. Here we test the effects of MAF silencing by siRNA in LEC cells using Affymetrix hgu133plus2 chips. Experiment Overall Design: There are n=3 of 1. LEC control cells transfected with a non-targeting siRNA, 2. LEC transfected with a MAF-targeting siRNAbiostudies-arrayexpressNucleic Acid Extraction - TRIzol solution (Invitrogen) extraction followed by RNeasy Mini kit (Qiagen) purification. Assessed RNA integrity and quantity using RNA 6000 Nano chips (Agilent).Labeling - 1 ug of total RNA was used to generate cDNA using T7-linked oligo(dT)primer and the custom SuperScript dscDNA synthesis kit (invitrogen). After second-strand syntheis, in vitro transcription was carried with biotinylated UTP and CTP using GeneChip® IVT Labeling Kit (http://www.affymetrix.com/support/technical/technotes/ivt_technote.pdf)Growth Protocol - Human lymphatic endothelial cells (LECs) were separated from MVEC cells using a polyclonal antibody of podoplanin, a LEC-specific marker, and were cultured in Endothelial Cell Growth Medium MV (C-22020, PromoCell, Heidelberg, Germany) plus 10 ng/ml of VEGF-C, in fibronectin-coated plates. Transfected LEC were grown under the same conditions. All cells were mycoplasma free.Sample Processing - Lymphatic endothelial cells (LECs) were plated in six well plates (5x10^4cells/well) overnight. The cells were transfected with siRNA against MAF or non-targeting control siRNA (Dharmacon). Two days after transfection, cultures were used for RNA extraction. Typically six wells of a 6-well plate were used per Affymetrix chip.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsStephen HendersonAssay Data Transformation - Data was preprocessed and normalised using the Bioconductor 'affy' package, specifically the robust multiarray algorithm ('RMA') .falseRNAi knock down of MAFin human lymphatic endothelial cellsKaposi sarcoma is the most common cancer in AIDS patients and is typified by red skin lesions. The disease is caused by the KSHV virus (HHV8) and is recognisable by its distinctive red skin lesions. The lesions are KSHV-infected spindle cells, most commonly the lymphatic endothelial and blood vessel endothelial cells (LEC and BEC), plus surrounding stroma. The KSHV virus expresses multiple MAF-downregulating microRNA. Here we test the effects of MAF silencing by siRNA in LEC cells using Affymetrix hgu133plus2 chips. Experiment Overall Design: There are n=3 of 1. LEC control cells transfected with a non-targeting siRNA, 2. LEC transfected with a MAF-targeting siRNA2009-10-10T00:00:00Z2023-08-14T16:16:37.717Z2022-03-03T15:56:45.044ZE-GEOD-16356GSE16356EFO_0002768