Third-Generation Human Mitochondria-Focused cDNA Microarray and Its Bioinformatic Tools for Analysis of Gene Expression

Abstract

To facilitate profiling mitochondrial transcriptomes, we developed a third-generation human mitochondria-focused cDNA microarray (hMitChip3) and its bioinformatic tools. hMitChip3 consists of the 37 mitochondrial DNA-encoded genes, 1098 nuclear DNA-encoded and mitochondria-related genes, and 225 controls, each in triplicate. The bioinformatic tools included data analysis procedures and customized database for interpretation of results. The database associated 645 molecular functions with 946 hMitChip3 genes, 612 biological processes with 930 genes, 172 cellular components with 869 genes, 107 biological chemistry pathways with 476 genes, 23 reactome events with 227 genes, 320 genetic disorders with 237 genes, and 87 drugs targets with 55 genes. To test these tools, hMitChip3 was used to compare expression profiles between human melanoma cell lines UACC903 (rapidly dividing) and UACC903(+6) (slowly dividing). Our results demonstrated internal gene-set consistency (correlation R > or = 0.980 +/- 0.005) and interexperimental reproducibility (R > or = 0.931 +/- 0.013). Expression patterns of 16 genes, involved in DNA, RNA, or protein biosyntheses in mitochondrial and other organelles, were consistent with the proliferation rates of both cell lines, and the patterns of 6 tested genes were verified by quantitative reverse transcription PCR (RT-PCR). Thus, hMitChip3 and its bioinformatics software provide an integrated tool for profiling mitochondria-focused gene expression.

Mitochondrial Gene Expression Profiles and Metabolic Pathways in the Amygdala Associated with Exaggerated Fear in an Animal Model of PTSD

“…The 1,500 test genes (including 80 “housekeeping” genes as positive controls) and 36 negative controls (non-rat DNA) were printed, each in triplicates, onto the N -hydroxysuccinimide ester reactive group-coated glass slides (CodeLink Activated Slide, SurModics, Eden Prairie, MI, USA). DNA probes in the print buffer (50 mM sodium phosphate), at a final concentration of 20 μM of 5′-amino-C6 modified 50-mers, were printed in the Class 100 super-clean environment as described previously (7, 16, 17), using 100 μM pins and the GeneMachine OmniGrid 100 Microarrayer (Genomic Solutions, Ann Arbor, MI, USA).…”

“…Database construction, data filtering, selection, exclusion and inclusion procedures, and criteria were performed as described previously (7, 8). The quantile normalization method (18) in software R version 2.15.1 (The R Foundation for Statistical Computing) was used to normalize background-subtracted mean intensities across all intra- and inter-slides of informative microarray data.…”

“…Amygdala tissues were collected from rats 14 days after stress, a time point at which exaggerated fear manifests. We then screened for 1500 mitochondrial-function-associated genes, including 37 mitochondrial DNA (mtDNA)-encoded genes, 1,098 nuclear DNA (nDNA)-encoded and mitochondria-focused genes, and 365 neuron-related genes (7, 8). …”

The oncogene c-Myc coordinates regulation of metabolic networks to enable rapid cell cycle entry

“…30 We reasoned that the key processes required for Myc-dependent mitochondrial biogenesis would include the following: transcription and translation from mitochondrial DNA, transcription, translation and import of nuclear encoded proteins into the mitochondria, assembly of electron transport chain protein complexes, and production of mitochondrial membranes. In addition, we included metabolic pathways requiring the contribution of mitochondria, including purine and pyrimidine biosynthesis, lipid, glutamate and folate/C1 metabolism, and the tricarboxylic acid cycle.…”

Pathways related to mitochondrial dysfunction in cartilage of endemic osteoarthritis patients in China

“…The 705 mitochondria-related genes were identified as mitochondria-related genes according to hMitChip 3.0 [12]. The expression level of these genes, which is from the whole human genome transcripts, was assessed in this study.…”