Ultra-fast simultaneous detection of obesity-related coenzymes in mice using microchip electrophoresis with a LIF detector

Abstract

Hepatic acyl-coenzyme A synthetase (ACS), carnitine palmitoyltransferase-I (CPT-I) and acetyl coenzyme A carboxylase (ACC) are coenzymes associated with the genetic type of obesity in animal models. This paper reports the use of microchip electrophoresis (ME) with a laser-induced fluorescence (LIF) detector based on a reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the amplified DNA fragments of these coenzymes (ACS, CPT-I and ACC) in the mRNA extracted from mice. DNA fragments ranging from 50 to 2652 bp were well resolved using this procedure with a running buffer (1× TBE), 0.5% polyvinylpyrrolidone (Mr 1,000,000) as the coating gel and 0.7% polyethyleneoxide (Mr 8,000,000) as the sieving gel at pH 8.30. The separation of the three RT-PCR products was achieved by ME in a single-run within 17 s using programmed field strength gradients (PFSG) (470 V cm-1 for 9 s, 205.8 V cm-1 for 2 s, 411.6 V cm-1 for 4 s, 117.6 V cm-1 for 2 s and 470.4 V cm-1 for 8 s). The ME-PFSG method was found to be 4 times faster than the method using a constant field and 138 times faster than slab gel electrophoresis. Moreover, the amplified RT-PCR products of the obesity-related coenzymes in C57BL/6J mice were analyzed using only sub-micro liter samples.