Deoxyribonucleoside triphosphate

The polymerase chain reaction amplifications and single-strand conformation polymorphism screening for nucleotide sequence variation described below were undertaken in the Gansu Key Laboratory of Herbivorous Animal Biotechnology, Faculty of Animal Science and Technology, Gansu Agricultural University, People’s Republic of China.
On the basis of the putative ovine keratin-associated protein 2-1 gene sequence identified above, we designed two polymerase chain reaction primers (5′-AACAAGGAATGGCATGAGTC-3′ and 5′-GTTGCTTTATAGGAAAGTGGG-3′) to amplify a 565-bp deoxyribonucleic acid fragment that would include the entire coding region of the putative ovine keratin-associated protein 2-1 gene. These primers were synthesized by the Takara Biotechnology Company Limited (Dalian, China).
Amplification reactions contained the genomic deoxyribonucleic acid on a 1.2 mm punch of TFN paper, 150 µM of each deoxyribonucleoside triphosphate (Takara), 2.5 mM Mg²⁺, 0.5 U of Taq DNA polymerase (Takara), and 0.25 µM of each primer. The thermal profile consisted of an initial denaturation for 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 59 °C, and 30 s at 72 °C, with a final extension of 5 min at 72 °C. Amplifications were performed in a 20 µL reaction in S1000 thermal cyclers (Bio-Rad, Hercules, CA, USA).

The differentiating cells were treated with 10 ng/ml ghrelin and IGF-1 for 8 days. Total RNA was extracted using a Trizol RNA kit (Promega, United States), and 2 μg RNA was reverse transcribed with an RT-PCR system including M-Mul reverse transcriptase (Promega, United States), RNasin (Promega, United States), Oligo (dT) primer (Promega, United States) and deoxyribonucleoside Triphosphate (TaKaRa, Japan). 2 μl RT products (cDNA) were amplified with human LPL, PPARγ2, C/EBPα, β-actin primers and mouse LPL, IGF-1 primers as shown in Table 1. 10 μl RT-PCR products were electrophoresed in 2% agarose gel in Tris-acetate-EDTA buffer. The gel was then stained with ethidium bromide and photographed using an Alphalmager M2200 (AlphaInnotech, United States). Expression changes were determined by the density ratio of the target genes to mGAPDH or β-actin in 3T3-L1 and human primary preadipocytes, respectively. Experiments were replicated three times with triplicate samples in each experiment.