Phenol-Chloroform extraction is efficient for extracting NADH.

Six samples of 15 male D. melanogaster adults were homogenized in 250 ul homogenization buffer. Following a 5 min 16000×g centrifugation, the supernatant was then divided into 3 parts. One part was kept as control. The other two parts were treated with equal volume of chloroform and phenol-chloroform respectively. Three parts were then assayed for NAD+ and NADH in duplicates. The concentration of NAD+ and NADH, with S.E.M, are standardized by the concentration of soluble protein measured from the control group. The difference in NAD+ and NADH concentration is tested using two sample t-test. See supplementary material for the method of testing redox ratio difference between groups.