**2.2 Sampling of kidneys and blood**

The renal pedicle of each kidney was clamped and the kidney was rapidly removed, decapsulated; the blood contained in each kidney was removed with blotting paper (freeblood). The kidney was placed in a sterilized Eppendorf tube, frozen, and conserved at -80°C.

Blood was collected in the vena cava of all mice with a 25-gauge needle (Neolus, VWR, Limonest, France) mounted on a 1-mL syringe (Terumo, VWR) prealably heparinized (Heparin, Roche, Meylan, France). Blood was immediately transferred in a cold BD Vacutainer tube, centrifuged at 4,000 x *g* for 20 min at 4°C. Plasma was frozen and stored in liquid nitrogen until testosterone and corticosterone measurement.

## **2.3 RNA extraction and semiquantitative RT-PCR**

The steady state levels of OAT and cyclophilin A transcripts were estimated by semiquantitative polymerase chain reaction (PCR) and quantitative PCR (qPCR) as described further in the text.

## **2.3.1 RNA extraction**

Total RNA was extracted from whole kidney by using Trizol® according to the supplier's procedure. Briefly, kidneys were mixed in the proportion of 150 mg tissue per 1 mL Trizol® at 4°C with a Ultra-Turrax T10 (VWR, Fontenay-sous-Bois, France). RNAs were extracted with chloroform, purified by isopropanol precipitation, and washed with 70% ethanol. RNA pellets were resuspended in sterilized water (Eurobio, Courtaboeuf, France) and stored

Orchidectomy Upregulates While Testosterone Treatment Downregulates

reported (Bitoun et al., 2001).

(HPRT).

analyses.

**2.3.5 Polymerase chain reaction for qPCR** 

**2.4 Protein extraction and western blot** 

(Levillain et al., 2004, 2005).

the Expression of Ornithine Aminotransferase Gene in the Mouse Kidney 119

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

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.

Gene Accession Primer Sequences (5' -> 3') qPCR

OAT NM\_016978.2 Forward GGGCTCTTGTGAAACTCTGC 195

HPRT NM\_013556 Forward GTAATGATCAGTCAACGGGGGAC 177 Reverse CCAGCAAGCTTGCAACCTTAACCA

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

Frozen kidneys were mixed at 4°C with a Turrax homogenizer at 100 mg frozen tissue per 2 mL of lysing buffer containing protease inhibitor (Laemmli, 1970). After centrifugation, the concentration of soluble proteins was determined using the Bradford protein assay (Bradford, 1976). Fifty or 100 µg samples of soluble proteins were subjected to 10% PAGE containing 0.1% SDS using 6 Watts per gel. Protein transfer and equal loading of proteins were visualized on the membranes with Ponceau S solution. Blocked immunoblots were incubated with primary polyclonal rabbit antibodies raised against rat/mouse-OAT (dilution 1:1,000), rat-aldose reductase (AR; EC 1.1.1.21, dilution 1:3,000) (Lambert-Langlais et al, 2009), and monoclonal mouse β-actin (dilution 1:2,000) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; EC 1.2.1.12, dilution 1:170) antibodies, and then with a peroxidase-conjugated anti-rabbit or antimouse IgG secondary antibodies (dilution 1:10,000) as previously described (Levillain et al., 2004, 2005). Antibody binding was revealed using chemiluminescence (ECL) Western Blotting Kit. Details for ECL detection and quantitation of the bands have been mentioned earlier

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

Reverse AGATGGGTCCGTTTCTCCTT

number product size (pb)

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% agarose gel (Eurobio).
