**2.3.5 Polymerase chain reaction for qPCR**

118 Basic and Clinical Endocrinology Up-to-Date

frozen (-80°C). RNA concentration was determined from the absorbance at 260 nm and RNA purity the absorbance ratio at 260 and 280 nm (ratio ≈ 2) using a BioPhotometer (Eppendorf France S.A.R.L., Le Pecq, France). RNA integrity was checked by electrophoresis on 1%

Total RNA (1 µg) was denatured for 5 min at 65°C and mRNAs were reverse-transcripted (60 min at 37°C) in 25 µL final volume containing 5 µL of M-MLV RT 5X buffer, 100 U moloney murine leukemia virus reverse transcriptase (Invitrogen, France), 15 nmoles of deoxynucleotide triphosphate and 1 µg oligo dT (PCR) or 100 ng random primer (qPCR, Invitrogen). After RT, inactivation step was 15 min at 70°C. RT products were frozen

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,

Gene Accession Primer Sequences (5' -> 3') PCR Cycle number product size (pb) number OAT NM\_016978.2 Forward TCCAGGATACCTGACGGGAGTT 330 27

eIF4-E NM\_007917.3 Forward AGCAGAGTGGACTGCACTGA 394 27

Cyclophilin A NM\_017101 Forward GTGGCAAGTCCATCTACGGAG 265 24 Reverse CCACAGTCGGAGATGGTGATC 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

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

Reverse ATCTTGTCTGCGTTTTCAGCAA

Reverse GCAAGGACAATGCTGTGAAA

agarose gel (Eurobio).

(-30°C).

**2.3.2 Reverse transcription** 

Eppendorf, Le Pecq, France).

for PCR analyses

**2.3.4 Quantification of RT-PCR products** 

**2.3.3 Polymerase chain reaction for PCR** 

Real-time PCR was performed in a MyiQ thermal cycler (Bio-Rad, Marnes La Coquette, France) using IQ SYBR Green Supermix (Bio-Rad). Primers specific to the mouse sequence of OAT and HPRT were designed using Primer3 software. The following qPCR conditions were used: 3 min at 95°C, followed by 40 cycles of denaturation for 10 s at 95°C and annealing/extension for 45 s at 60°C, according to the manufacturer's instructions. All samples were run in duplicate along with dilutions of known amounts of target sequence to quantify the initial cDNA copy number (Concentration = EfficiencyΔCt). The results are expressed as the ratio of the target gene over HPRT mRNA concentration which was verified to exhibit non-significant variation between the different groups of cDNAs.


Abbreviation: ornithine aminotransferase (OAT) and hypoxanthine guanine phosphoribosyl transferase (HPRT).

Table 2. Gene-specific sequences of PCR primers and predicted PCR products for qPCR analyses.
