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Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human hepatoma Hep3B cells following irradiation to investigate the effects of DNA damage

ABSTRACT: To monitor the changes in transcription and alternative splicing upon UV irradiation, human hepatoma Hep3B cells were Hep3B cells were washed two times with PBS and then irradiated with 40J/m2 UV light; fresh medium was immediately added after the irradiation and the cells were incubated at 37 C and collected 6 hours after the treatment for the RNA preparation. RNAs from three biological replicates of control or irradiated cells were purified, reverse transcribed into cDNA, labelled with Cy5 or Cy3 fluorochromes, and hybridized to a custom splicing-sensitive microarray
cDNA and Cy5-Cy3 labelled cRNA were generated from the total RNA using the Agilent Low RNA Input Fluorescent Linear Amplification kit; the cRNA was purified with the RNeasy Mini kit (Qiagen). 8?g of each cRNA were used for the hybridization with the arrays (Agilent In situ hybridization kit plus). After hybridization, arrays were washed, and scanned images analyzed. Three biological replicates were hybridized, with both direct and dye-reversal hybridizations. General gene expression values represent the average of log2 ratios for all the probes in constitutive exones of a locus. Statystical analyses were carried out with Linear Models for Microarray Data (Limma; Bioconductor Project; Dudoit, et al., 2003). The background correction method used in the analysis was Normexp (Ritchie, et al., 2007). Locally weighted linear regression (LOWESS) analysis was used as a normalization method (Yang, et al., 2002).

ORGANISM(S): Homo sapiens

SUBMITTER: Claudia Ben-Dov

PROVIDER: E-MEXP-2353 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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