| 18539180 |
Lee HG, Kumar KS, Soh JR, Cha YS, Kang SH: Ultra-fast simultaneous detection of obesity-related coenzymes in mice using microchip electrophoresis with a LIF detector. Anal Chim Acta. 2008 Jun 30;619(1):94-100. Epub 2008 Jan 15.
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 (1x TBE), 0.5% polyvinylpyrrolidone (M (r) 1,000,000) as the coating gel and 0.7% polyethyleneoxide (M (r) 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 470.4V 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. |
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