Lymphoblastoid cell lines of families with Mitochondrial Myopathy and Sideroblastic Anemia

We analyzed samples from two affected brothers (Affected 3 and 4) and their two affected female cousins (Affected 7 and 9) homozygous for the MLASA ? associated C656T mutation in the PUS1 gene; four parents ? heterozygous carriers of the C656T mutation (Parents 1, 2, 5, 6), and an unaffected female carrying wild-type genotype at the PUS1 gene (Control 8). In addition, two females and one male with normal hearing from Arab-Israeli family with nonsyndromic deafness carrying wild-type PUS1 sequence were used as controls (Controls 10, 11, 12). Keywords: Comparison of genome-wide expression in cell lines of patients and controls Lymphoblastoid cell lines established from peripheral blood lymphocytes of study participants and immortalized with Epstein-Barr virus, were grown in suspension in T flasks in RPMI-1640 medium (Invitrogen, Inc., Carlsbad, CA), containing 2 mmol/L L-glutamine, 100 ug/ml streptomycin, and 10% fetal calf serum. Total RNA was extracted using Trizol reagent (Invitrogen, Inc.) according to the manufacturer?s protocol. cRNA amplification and labeling with biotin were performed using Illlumina® TotalPrep RNA amplification kit, manufactured by Ambion, Inc (Austin, TX) according to the manufacturer?s protocol using 100 ng of total RNA as input material. 1ug of labeled cRNAs were hybridized to the three ?whole genome? Sentrix Human-6 Expression BeadChips (Illumina, San Diego, CA). RNA sample from patient Affected 4 was duplicated on the same chip, RNA samples from Affected 7 and Control 8 were duplicated on different chips. All hybridization and scanning steps were performed according to the manufacturers? instructions using reagents and equipment purchased from Illumina, Inc.