TY - JOUR
T1 - Separation and quantitation of reverse transcriptase polymerase chain reaction fragments of basic fibroblast growth factor by capillary electrophoresis in polymer networks
AU - Gelfi, C.
AU - Leoncini, F.
AU - Righetti, P. G.
AU - Cremonesi, L.
AU - Di Blasio, A. M.
AU - Carniti, C.
AU - Vignali, M.
PY - 1995
Y1 - 1995
N2 - In human ovarian carcinomas (epithelial and endometrial tumors) the presence of mRNA coding for the basic fibroblast growth factor (bFGF) was previously demonstrated by reverse transcription-polymerase chain reaction (PCR). For quantitation purposes, competitive PCR is adopted, using as a competing fragment a sequence of a highly homologous bovine bFGF. However, separation and detection of the PCR products by stab gel electrophoresis and ethidium bromide staining gives poor quantitative data due to the nonstoichiometric binding of the dye. Thus, the only possible quantitation that can be obtained is via autoradiography with 32P-labeled primers. We report here a capillary electrophoresis protocol, in 6% linear, liquid polyacrylamide as a sieving system, able to fully resolve and quantify the undigested (354 bp) bovine and the digested (295 bp and 59 bp) human fragments, with peak ratios in good agreement with the autoradiographic data.
AB - In human ovarian carcinomas (epithelial and endometrial tumors) the presence of mRNA coding for the basic fibroblast growth factor (bFGF) was previously demonstrated by reverse transcription-polymerase chain reaction (PCR). For quantitation purposes, competitive PCR is adopted, using as a competing fragment a sequence of a highly homologous bovine bFGF. However, separation and detection of the PCR products by stab gel electrophoresis and ethidium bromide staining gives poor quantitative data due to the nonstoichiometric binding of the dye. Thus, the only possible quantitation that can be obtained is via autoradiography with 32P-labeled primers. We report here a capillary electrophoresis protocol, in 6% linear, liquid polyacrylamide as a sieving system, able to fully resolve and quantify the undigested (354 bp) bovine and the digested (295 bp and 59 bp) human fragments, with peak ratios in good agreement with the autoradiographic data.
KW - Capillary electrophoresis
KW - DNA
KW - Fibroblast growth factor
KW - Liquid polyacrylamide
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M3 - Article
C2 - 7588561
AN - SCOPUS:0029069702
VL - 16
SP - 780
EP - 783
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 5
ER -