biostudies-arrayexpressUnknownTranscriptomicsEirini Gkogkoutranscription profiling by arrayHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17124Stress Induced Premature Senescence in HDFs after altering proteasome activity (acronyms) GA = HDFs growth arrested at G0 PROT = senescent HDFs after proteasome Inhibition PbJ = HDFs transient transfection with the vector X = transient transfection with the B5 proteasome subunitbiostudies-arrayexpressNucleic Acid Extraction - Total RNA was extracted by using the RNeasyﾮ Mini kit (Qiagen Inc.), DNase digested, and the RNA quality evaluated using the Agilent Bioanalyzer 2100 (Agilent Technologies Inc., Palo Alto, CA); 2-5 μg of total RNA were linearly amplified using the AminoAllyl Message Amp aRNA kit (Ambion #1750, #1752). 5 μg aaRNA were incubated with random primer (Invitrogen, Karlsruhe, Germany) for cDNA synthesis. (Parameters: Extracted product = total_RNA, Amplification = RNA polymerases)Hybridization - For chip hybridization, slides were immersed at 42oC in 6 X standard saline citrate (SSC)/0.5% sodium dodecyl sulfate (SDS)/1% bovine serum albumin (BSA) for 45 minutes and carefully washed in ddH2O at room temperature. The attached PCR products were then denatured at 95οC in ddH2O for 2 minutes and then air dried. Prior to hybridization, the purified Cy3/Cy5-labeled cDNA probes were mixed together. Then, 20 μg poly-d-A and 20 μg human Cot1DNA were added and the mixture evaporated in the Vacuum Concentrator 5301 at 60οC to complete dryness. The pellet was dissolved in 50 μl hybridization buffer (50% formamide/6 X SSC/0.5% SDS/5 X Denhardtﾒs) and denatured by incubating at 95oC for 2 minutes. The microarray consists of two slides, which are hybridized on top of each other with the labelled probe in between. The probe was hybridized with the microarray in a humid chamber in a water bath at 42oC for 20 hours. Hybridized slides were washed at 42oC for 5 min in 2 X SSC and 0.1% SDS and for 10 min in 0.1 X SSC and 0.1% SDS, rinsed by dipping twice in 0.1 X SSC and 0.1% SDS twice, dipped in 100% isopropanol, and dried by a brief centrifugation at 715 X g in a microtiter plate centrifuge (Z320; Hermle, Wehingen, Germany). (Parameters: Chamber type = OTHER: Humid chamber, Quantity of label target used = 5, Mass unit = Micro gram, time = 20, Tiny time unit = hours, Volume = 50, Volume unit = Micro litre, temperature = 42)Growth Protocol - Human diploid fibroblast cell line IMR-90 cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% Foetal Bovine Serum (FBS) and 1% Non Essential Amino Acids (NEAA), cultured in incubators at 37oC, 5% CO2, 95% humidity.Labeling - 5 μg aaRNA were incubated with random primer (Invitrogen, Karlsruhe, Germany) for cDNA synthesis which was then labeled by amino-allyl-coupling using the Atlas Glass Fluorescent Labeling Kit (Clontech, Palo Alto, CA, #K1037-1) and Cy3/Cy5-monofunctional reactive dye (Amersham Biosciences, Little Chalfont, United Kingdom, Cy3 #PA23001, Cy5 # PA25001). (Parameters: Amount of nucleic acid labeled = 5, Amplification = none, Mass unit = Micro gram)MIAME ScoreOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsEirini GkogkouData Transformation - Dried slides were scanned immediately at a 10-μm resolution using a GenePix 4000B microarray scanner with GenePix software (Axon Instruments–Molecular Devices, Union City, CA). Individual laser power and photomultiplier settings were used, allowing all signals to remain within the linear range of the scanner. Separate images for Cy3 and Cy5 were produced and analyzed using ChipSkipper microarray data evaluation software (http://angiogenesis.dkfz.de/software). Integration grids were either automatically or manually centered to the images. The generated images then were analyzed in ChipSkipper and quantified using histogram segmentation.For each spot total intensity and local background were calculated. Raw ratios were derived from background reduced signals. Flagged spots and controls were removed, and quantified spots were normalized using a framed median ratio centering (frame = 500 genes). The resulting data from the two slides of the microarray were merged, and were filtered by using a custom developed database, Hybmart.falseTranscriptional Rewiring of IMR90 lung fibroblasts under Opposite Proteasome StatesStress Induced Premature Senescence in HDFs after altering proteasome activity (acronyms) GA = HDFs growth arrested at G0 PROT = senescent HDFs after proteasome Inhibition PbJ = HDFs transient transfection with the vector X = transient transfection with the B5 proteasome subunit2025-12-31T00:00:00Z2026-06-05T17:08:03.388Z2026-06-05T17:07:47.784ZE-MTAB-17124EFO_0002944EFO_0003813EFO_0003789EFO_0003816EFO_0003815