Genomelab gexp start kit protocol

Primers for the gene expression study were designed using the Rattus norvegicus gene sequences from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/nucleotide/) and tagged with an 18-nucleotide universal forward and 19-nucleotide universal reverse sequence, respectively. Primers (Table 2) were supplied by Integrated DNA Technologies (Singapore) and reconstituted in RNAse free water. Extracted RNA (20 ng) from liver and adipose tissues was used for reverse transcription and PCR according to the GenomeLab GeXP Start Kit protocol (Beckman Coulter, USA) using the conditions shown in Table 2. PCR products (1 μL) were analyzed on a GeXP genomelab genetic analysis system (Beckman Coulter, Inc., Miami, FL, USA) after mixing with sample loading solution and DNA size standard 400 as recommended by the manufacturer. Results were analyzed with the Fragment Analysis module of the GeXP system software and normalized on the eXpress Profiler software. Fold changes were calculated by dividing the expression value for the different treatment groups by the expression value for the normal group.

RNA was extracted from liver and adipose tissues using the total RNA isolation kit (RBC Biotech Corp., Taipei, Taiwan) according to the manufacturer's instructions. Reverse transcription (20 ng) and PCR were done according to the GenomeLab GeXP Start Kit protocol (Beckman Coulter, USA), using the conditions shown in Table 3.

RNA was extracted using an RNA isolation kit (RBC Biotech Corp., Taipei, Taiwan) and diluted to 20 ng/mL. Reverse transcription and PCR programs were performed according to the GenomeLab™ GeXP Start Kit protocol (Beckman Coulter, USA), as shown in Table 1.