ucRNA profiling in human hepatocytes

Genome-wide expression profiling has been performed in malignant HepG2 cells and compared to the expression in normal human hepatocytes. 56 ucRNAs, representing 11% of all ucRNAs analyzed, were aberrantly and significantly (p<0.05) expressed in malignant HepG2 cells compared to non-malignant human hepatocytes. RNA was extracted from three separate biological samples for each analysis using Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA (5 μg) was reverse transcribed using biotin end-labeled random oligonucleotide primers and cDNA was hybridized to a custom microarray (OSU-CCC 4.0), which includes sense and antisense probes to all 481 human ultraconserved reported by Bejerano et al, each spotted in duplicate. Biotin-containing transcripts were detected using streptavidin–Alexa647 conjugate, scanned and analyzed using an Axon 4000B scanner and the GenePix 6.0 software (Axon Instruments, Downingtown, PA). The mean fluorescence intensity of replicate spots were subtracted from background and normalized using the global median method. We selected ucRNAs measured as present in all the three replicates. Differentially expressed ucRNAs were identified using the Class Comparison Analysis of BRB tools version 3.6.0 (http://linus.nci.nih.gov/BRB-ArrayTools.html). The criterion for inclusion of a gene in the gene list was a p-value < 0.05.