PMC:3475485 / 7445-9568 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3475485","sourcedb":"PMC","sourceid":"3475485","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3475485","text":"Semi-quantitative and real-time RT-PCR\nTotal RNA was prepared using a commercial kit (Qiagen) according to the manufacturer's instructions. Single-strand cDNA was synthesized by incubating 5 µg total RNA with 200 units AMV, 100 nM oligo(dT)12-18, 1 mM dNTP mixture, and 40 units RNase inhibitor at 42℃ for 1 h in a final volume of 25 µL. The reaction was terminated by incubation at 70℃ for 15 min. The initial amount of mRNA and reaction conditions were optimized to obtain linearity for mouse 18s rRNA. For semi-quantitative reverse transcription (RT)-PCR, the used primers were: human PKD2, forward 5'-CGTGCCCCAGCCCAGTC-3' and reverse 5'-TTCCAGTACAGCCCATCCAATAAG-3'; mouse Pkd2, forward 5'-TGCGAGGGCTGCGAGGTC-3' and reverse 5'-TGTCAGCTTGCGTGTGGTTGC-3'; mouse 18s rRNA, forward 5'-GTAACCCGTTGCACCCCATT-3' and reverse 5'-CCATCCAATCGGTAGTAGCG-3'. RT-PCR cycling conditions were as follows: 10 min at 95℃, 25 cycles of 50 s at 94℃, 50 s at 57℃, 50 s at 72℃, and 5 min at 72℃. The amplified products were separated on a 1% agarose gel. For real-time PCR, the used primers were: mouse β-actin, (forward 5'-GACGATGCTCCCCGGGCTGTATTC-3' and reverse 5'-TCTCTTGCTCTGGGCCTCGTCACC-3') used as a positive control; mouse peroxisome proliferator-activated receptor γ (PPAR-γ), forward 5'-TCTTAACTGCCGGATCCACAAAAA-3' and reverse 5'-ATCTCCGCCAACAGCTTCTCCTTC-3'; mouse protein kinase α (PKCα), forward 5'-GGGCAGCCTCCGTTTGATGGT-3' and reverse 5'-CGCTTGGCAGGGTGTTTGGTC-3'; mouse Wnt4, forward 5'-GCCATCGAGGAGTGCCAATACC-3' and reverse 5'-GGCCACACCTGCTGAAGAGATG-3'; mouse integrin α4 (ITGα4), forward 5'-GTAGCCCCAGTGGAGAGCCTTGTG-3' and reverse 5'-ATGCCAGTGGGGAGTTTGTTATCG-3'; mouse IL6ST, forward 5'-TGAATCGGACCCACTTGAGAGG-3' and reverse 5'-CAGGAGCGGCTTGTTTGAGGTA-3'; mouse aquaporin 1 (AQP1), forward 5'-GGAGGCGCCGAGACTTAGGT-3' and reverse 5'-GCGGGTGAGCACAGCAGAGC-3'; mouse transforming growth factor-β2 (TGF-β2), forward 5'-TCATCCCGAATAAAAGCGAAGAGC-3' and reverse 5'-AGGGCAACAACATTAGCAGGAGAT-3'. Real-time RT-PCR was performed using the real-time SensiMixPlus SYBR kit as described by the manufacturer's instructions (Quantance, London, UK).","divisions":[{"label":"Title","span":{"begin":0,"end":38}}],"tracks":[]}