Determination of amplification level of the c-erbB-2 proto-oncogene in human breast carcinomas: a comparative study between non-radioactive and radioactive labelling.
A quantitative method of polymerase chain reaction (PCR) using both digoxigenin and radioactive labelled probes has been used for the detection of the c-erbB-2 proto-oncogene amplification in breast carcinomas with formalin-fixed paraffin-embedded tissue sections. c-erbB-2 proto-oncogene amplification has been demonstrated in 14 infiltrating ductal carcinomas. The technique consisted of the co-amplification of c-erbB-2 and IFN-gamma (interferon-gamma) genes. The latter was considered as a single copy gene per genome-equivalent. The aim of this study was to compare two quantitative PCR techniques based on the incorporation of either digoxigenin-11-dUTP or 32P-dCTP, during amplification. For the colorigenic method, using the Dig system, after electrophoresis and transfer, the specific bands were revealed with a chromogenic substrate of phosphatase. Their intensity estimated by scanning photometry following blot transparisation. After electrophoresis, the radioactive gel was submitted to radioautography and the band intensities evaluated by scanning spectrophotometry. For the 14 samples, a good agreement between both methods was noted. The colorigenic method is a valuable alternative to radiolabelling due to: i) time saving, ii) reagent conservation, iii) safe manipulation and iv) sensitivity of the same order for both methods.
Edorh
A
A
Laboratoire de Recherche, Centre Régional de Lutte contre le Cancer (Centre Alexis Vautrin), Vandoeuvre-lés-Nancy, France.
Parache
R M
RM
N'Sossani
B
B
Bottin
M C
MC
Merlin
J L
JL
Colosetti
P
P
Guillemin
F
F
Le Faou
A
A
Rihn
B
B
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D018270:Carcinoma, Ductal, Breast / Q000235:genetics; D003124:Colorimetry; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D004076:Digoxigenin; D005069:Evaluation Studies as Topic; D005260:Female; D005784:Gene Amplification; D018734:Genes, erbB-2; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods*; D000090063:Proto-Oncogene Mas
D017931:DNA Primers; D004273:DNA, Neoplasm; C000715470:MAS1 protein, human; D015335:Molecular Probes; D010761:Phosphorus Radioisotopes; D000090063:Proto-Oncogene Mas; D007371:Interferon-gamma; D004076:Digoxigenin