Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome of murine DC-like XS52 cells stably overexpressing miRNA 223 compared to control XS52 cells

ABSTRACT: Cells of the DC-like cell line XS52 (Xu et al., 1995, J Invest Dermatol. 1995 Dec;105(6):831-6) were lentivirally transduced with a copGFP reporter (pGreenFire) or the mmu-miR-223 gene locus (both from SBI Biosciences, Palo Alto, CA) and cultured in the presence of puromycin to select for transductants as described (Bollmann et al., 2014, Nucleic Acids Res. 42, 12555-12569). Each 20 µg of total RNA derived from the two transduced XS52 sublines was reverse-transcribed using the SuperScript Plus Direct cDNA Labeling System (Invitrogen, Carlbad, CA), with Alexa Fluor 555-aha-dUTP or Alexa Fluor 647-aha-dUTP to differentially label cDNA of either group. Derived fluorescence-labeled cDNA was purified and analysed for quality and yield (Nanodrop ND-100; Peqlab, Erlangen, Germany). Prehybridized (5X SSPE, 0.1% SDS, and 1% BSA) microarrays (Mouse Whole Genome OneArray® Microarray v2; Phalanx Biotech, San Diego) were hybridized with each 20 pmol of either fluorescence-labeled cDNA probe using OneArray Hybridization Buffer with 18% formamide for 16 h (Lucidea SlidePro Hybridizer; GE Healthcare, Munich, Germany) as recommended by the manufacturer. Washed microarrays were analysed using a ScanArray 4000 system equipped with ScanArray 4.0 software (both Perkin-Elmer, Rodgau-Jügesheim, Germany) at 10 pixel size. Laser intensity (90-100%) and photomultiplier tube sensitivity (60-70%) were varied to optimize signal to background ratios. Derived images were analyzed using the ScanArray 4.0 easyquant software module. Intensity data were normalized (loess normalization; MIDAS-2.19) and subjected to significance analysis (MEV 4.3) using appropriate modules of the TM4 microarray software suite (Saeed et al., 2006, Methods Enzymol. 411, 134-193). Changes in gene expression with p-values < 5% and ratios (mmu-miR-223/control) >2 or <0.5 were used for subsequent analyses.

ORGANISM(S): Mus musculus

SUBMITTER: Hartmut Kleinert

PROVIDER: E-MTAB-6369 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcriptome of murine DC-like XS52 cells stably overexpressing miRNA 223 compared to control XS52 cells

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