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.将6份各含15只成年雄性黑腹果蝇（Drosophila melanogaster，缩写为D. melanogaster）的样本置于250微升匀浆缓冲液中进行匀浆处理。经5分钟16000×g离心后，收集上清液并均分为三份。其中一份作为对照组留存；剩余两份分别加入等体积的氯仿与苯酚-氯仿混合液进行处理。随后以双重复形式对三份样本的烟酰胺腺嘌呤二核苷酸（NAD+）与还原型烟酰胺腺嘌呤二核苷酸（NADH）进行含量检测。所有NAD+与NADH的浓度及其标准误（S.E.M.）均以对照组中测得的可溶性蛋白浓度作为参照进行标准化校正。采用两独立样本t检验对NAD+与NADH的浓度差异进行统计学检验。各组间氧化还原比值的组间差异检测方法详见补充材料。