Ultra-fast simultaneous detection of obesity-related coenzymes in mice using microchip electrophoresis with a LIF detector.
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.
Lee
Hee Gu
HG
Cellomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea.
Kumar
K S
KS
Soh
Ju-Ryoun
JR
Cha
Youn-Soo
YS
Kang
Seong Ho
SH
D000818:Animals; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D046209:Electrophoresis, Microchip / Q000379:methods*; D005453:Fluorescence; D007834:Lasers; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009765:Obesity / Q000201:enzymology / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D003067:Coenzymes; D012333:RNA, Messenger; D011092:Polyethylene Glycols; D004247:DNA