Quantitation of RT-PCR products of bFGF-mRNA by capillary electrophoresis and laser-induced fluorescence.
The numerous challenges of modern biology and medical science require the development of analytical methodologies of extreme sensitivity and resolving power. Because it deals with the extraordinary complexity of biological mixtures and minute samples, capillary electrophoresis combined with laser-induced fluorescence (LIF) is now one of the most popular analytical biotechniques. Using polymer matrices and very high voltages, it allows the separation of very low quantities of microsamples and the selective detection of fluorescent species at a level of a few thousand molecules. This method of analysis has been used to quantitate reverse transcription-polymerase chain reaction (RT-PCR) products. In the authors' laboratory, they were looking for a means of analyzing mRNAs of cytokines. In the first attempt, they chose to quantitate RT-PCR products of basic fibroblast growth factor (bFGF)-mRNA, which could be involved in atherosclerosis. The results on two patients confirm the lower quantity of bFGF expressed in internal mammary athery (IMA) biopsies compared to human aortic cells. This study shows the usefulness of CE-LIF to identify and quantitate RT-PCR products and its ability to be used in clinical studies, to find very low levels of bFGF expression in atheroma biopsies.
Caussé
E
E
Laboratoire de Biochimie, Centre Louis Bugnard, CHU Rangueil, Toulouse, France.
Siméon
N
N
Nertz
M
M
Salvayre
R
R
Bayard
F
F
Valdiguié
P
P
Couderc
F
F
D001158:Arteries / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D016222:Fibroblast Growth Factor 2 / Q000032:analysis*; D005453:Fluorescence; D006801:Humans; D007834:Lasers; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity
D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2