Ballard Lab: BABS, UNSW, Sydney, Australia

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia

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We hypothesize that increased expression of electron-transport genes may increase survival, possibly by decreasing ROS production. Alzheimer’s is associated with a decrease in COX activity, and Parkinson’s with decreased activity of NADH: ubiqunione oxidoreductase subunit 4.

We have examined the RNA expression levels for the mitochondrial COIII and cytochrome b genes normalized to the expression level of the nuclear encoded ribosomal protein gene, RP49. RP49, encodes ribosomal protein 49, and has been used previously as a control gene in Drosophila studies of expression and longevity. We employ the Taqman® method to quantify expression.

This work was done in collaboration with Dr. Joe Miller.

Taqman® primer/probe combinations were designed using Assays-by-Design (Applied Biosystems). Total RNA was extracted from a single fly thorax using the Qiagen RNeasy kit and was reverse transcribed to single stranded cDNA with a Superscript III First Strand Synthesis Kit for RT-PCR (Invitrogen). Real-time quantitative Taqman“ PCR analysis, as described above, were performed on an ABI7000 Sequence Detection System. Each cDNA sample was amplified in triplicate for each of the three genes. The COIII and cytochrome b averages of these three cDNA replicates was normalized to the RP49 average and are presented as relative RNA copy number.