Genegenius automatic gel imaging and analysis system

Total ribonucleic acid (RNA) was extracted from intra‐abdominal adipose tissue or liver tissue using RNAiso Plus Isolation Reagent (TAKARA, Otsu, Shiga, Japan) according to the manufacturer's instructions. Total RNA was reserve transcribed and amplified in a GeneAmp PCR system (Eppendorf, Hamburg, Germany). Primers used in the reverse transcription polymerase chain reaction were of: adipose triglyceride lipase (ATGL; forward TTC AAG TTT CCT TGC AGA GT; reverse CTC CCA AAC TGA CCC TTA AA) in visceral adipose tissue, acetyl‐CoA carboxylase (ACCase; forward GCC AGC AGA ATT TGT TAC TC; reverse AGA CGA TGC AAT CTT ATC CC) in liver tissue, and glyceraldehyde 3‐phosphate dehydrogenase (forward TAT CGG ACG CCT GGT TAC; reverse TGC TGA CAA TCT TGA GGG A). Data analysis was carried out using a GeneGenius automatic gel imaging and analysis system (Syngene, Cambridge, UK), and scanned by densitometry for quantitation. To exclude variations as a result of RNA quantity and quality, the data for genes were adjusted to glyceraldehyde 3‐phosphate dehydrogenase, and the relative expression levels of ATGL and ACCase were calculated as: (relative gray value of the gene / mean of relative gray value in the control) × 100%.

Total ribonucleic acid (RNA) was extracted from BRL‐3A cells, liver tissue, intra‐abdominal adipose tissue or thigh muscle tissue using RNAiso Plus Isolation Reagent (TAKARA, Otsu, Shiga, Japan) following the manufacturer's instructions. Total RNA was reserve transcribed and amplified in a GeneAmp polymerase chain reaction (PCR) system (Eppendorf, Hamburg, Germany); reverse transcription PCR for Tph1, AADC, glycerin‐3‐phosphate acyltransferase 1 (GPAT1), phosphoenolpyruvate carboxykinase‐1 (PEPCK‐1), and glucose transporter 2 (GLUT2) in the liver and BRL‐3A cells, 5‐HT 2A and 2B receptor (5‐HT_2AR and 5‐HT_2BR) in the BRL‐3A cells, Tph1, AADC and adipose triglyceride lipase in the intra‐abdominal adipose, and Tph1 and AADC in the thigh muscle were carried out in the same system. Data analysis was carried out using a GeneGenius automatic gel imaging and analysis system (Syngene, Cambridge, UK), and scanned by densitometry for quantitation. To exclude variations as a result of RNA quantity and quality, the data for all genes were adjusted to glyceraldehyde 3‐phosphate dehydrogenase. The relative expression levels of each gene were calculated as: (relative gray value of each gene / mean of relative gray value in the control) × 100%. PCR primer sequences are listed in Table1.