**2.3.3 Polymerase chain reaction for PCR**

The cDNA from 2.5 µL of RT product were amplified by PCR in 47.5 µL of PCR mixture containing 5 µL of 10X Taq PCR buffer, 100 nmoles MgCl2, 15 nmoles of deoxynucleoside triphosphate (Promega), 2.5 U Taq DNA polymerase, 15 pmoles corresponding to forward and reverse primers. Gene-specific oligonucleotide primers (20-22 nucleotides) were selected from the cDNA sequences of mouse OAT and rat cyclophilin A. Forward and reverse primers were designed using Primer3 software (v. 0.4.0) (http://frodo.wi.mit.edu/primer3/) and are shown in Table 1. Sequence identity between rat and mouse cyclophilin A was 91% for forward primer and 95% for reverse primer. The PCR program consisted of a denaturation step (2 min at 94°C), X cycles (see Table 1) and a final elongation step (10 min at 72°C). Each cycle included denaturation (1 min at 94°C), primer annealing (1 min at 60°C), and elongation (1 min at 72°C). Preliminary experiments were performed to determine the linear range of PCR amplication for each target mRNA. RT and PCR reactions were performed using a thermocycler (Mastercycler® Personal, Eppendorf, Le Pecq, France).


Abbreviation: ornithine aminotransferase (OAT) and eukaryotic initiation factor eIF4-E (eIF4-E).

Table 1. Gene-specific sequences of PCR primers, predicted PCR products, and parameters for PCR analyses

## **2.3.4 Quantification of RT-PCR products**

Fourteen µL of each PCR products containing 14% of a gel loading buffer (Blue juice 10X) were separated by electrophoresis on a 4% (w/v) agarose (Eurobio) gel prestained with 0.01% ethidium bromide. For quantitation of band intensities, pictures were taken with a Camera DC120 (Kodak) and intensities of the bands were determined with Kodak Digital Science 1D 2.0 (Kodak Scientific Imaging System). The housekeeping rat cyclophilin A gene was used as an internal control to normalize the target gene expression as previously reported (Bitoun et al., 2001).
