The mRNA expression of ANP and its receptors (NPR-A and NPR-C) wa

The mRNA expression of ANP and its receptors (NPR-A and NPR-C) was determined by real time-PCR. The gene expression of ANP was evaluated in the RA and LA, and the NPR-A and NPR-C expression was determined in the right kidney. The cDNA was synthesized by the reverse transcription of mRNA. For this process, 1 μl of mRNA from each sample was mixed in plastic tubes with HKI 272 a solution containing the following compounds: diethyl pyrocarbonate water (DEPC), the reverse primer of the target gene (ANP, NPR-A, or NPR-C) or the reverse primer of the normalizing gene or housekeeping gene (ribosomal

subunit s26), oligo dT, triphosphate deoxyribonucleotide (dNTP), dithiothreitol (DTT), specific buffer (10×) and a solution containing the Moloney murine leukemia virus (MMLV) reverse transcriptase enzyme, according to the manufacturer’s guidelines (Invitrogen, CA, USA). After this

process, the plastic tubes were heated at a temperature of 40 °C for 60 min. After reaching room temperature, the tubes were stored at −20 °C. For the atria, prior to reverse transcription, the samples were subjected to DNAse treatment. The treatment was performed by mixing 0.5 μg of total mRNA from TSA HDAC ic50 the atria with 4 μl of water and 1 μl of mix buffer containing DNAse (1:1). This mixture was incubated for 15 min at room temperature; after this period, EDTA was added, which stopped the reaction. The samples were then heated to FER 65 °C for 10 min. After

building the cDNA, a PCR was performed to amplify the cDNA for ANP, NPR-A and NPR-C, using specific primers (ANP: GGA TTT CAA GAA CCT GCT AGA CTT and CAT CGG TCT GCT CGC TCA, NPR-A: ATC ACA GTG AAT CAC CAG CAG TTC AGA and AGA TGT TAA CTC TGC TTC CCT G, NPR-C: CCT ACA TTA TCG ACG AGA CCA AA and ACT CGC TCA TGG ATG CTG CCC TA). For this procedure, 2 μl of cDNA was added into wells of specific plates for real-time PCR, followed by 1.5 μl of sense primer (1 pmol/μl), 1.5 μl of anti-sense primer (1 pmol/μl), 10 μl of Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington WA, UK) and 5 μl of DEPC water. Afterward, the plates were sealed and taken to the apparatus for the real-time measurement of gene expression in the thermocycler (ABI Prism 7000 SDS; Applied Biosystems, Warrington WA, UK) using the following thermal cycles: [stage 1], a cycle of 52 °C/2 min; [stage 2], a cycle of 95 °C/10 min; [stage 3], 40 cycles of 95 °C/0.15 min and 50 °C/1 min. The ANP receptor autoradiography has been described in detail [2] and [6]. Briefly, the rats were killed by decapitation, and the mesenteric adipose tissue was rapidly isolated, snap-frozen in isopentane at −18 °C, mounted on cryostat chucks and cut into 15-μm-thick sections at −30 °C. The sections were thaw mounted on pre-cleaned gelatin-coated slides and then stored at −80 °C until they were used.

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