A key enzyme in mitochondrial β¬oxidation, carnitine palmitoyltransferase (CPT) I, is transcriptionally regulated in mammals, but this enzyme also experiences allosteric modulations (Harano et al., 1985; Murthy and Pande, 1987; Bezaire et al., 2004). CPT I is located on the inner side of the outer mitochondrial membrane and catalyses the conversion of acyl-CoA to fatty acylcarnitine (Kerner and Hoppel, 2000; Price et al., 2000). Quantitative RT-PCR is a reliable technique for measuring transcripts in small amounts of tissue (Spriewald et al., 2000). With this technique, multiple mRNAs can be assayed simultaneously in a relatively short period of time. Here we describe the standard procedure, optimized in our laboratory, to assess CPT I levels with β¬actin as an internal control in rainbow trout, and all the necessary controls to ensure a quantitative analysis. RNA Extraction and Reverse Transcription Total cellular RNA was isolated from liver of rainbow trout using RNX reagent (Cinnagen-Iran). To obtain cDNA, 1 µg of
total RNA was subjected to reverse transcription polymerase chain reaction (RT-PCR) with MuLV reverse transcriptase using the RevertAidTM M¬MuLV Reverse Transcriptase Kit (Fermentase Life Science, Germany) and random hexamer primer. Reaction conditions in the reverse transcription step are mostly dependent on the enzyme and the primers of choice. Whereas other protocols to require the use of specific primers, we prefer to reverse transcribe the total RNA population with random hexamers so that different PCR analyses could be performed on the same cDNA sample
