Semiquantitative reverse transcription-polymerase chain reaction to evaluate the expression patterns of genes involved in the oestrogen pathway.
The increasing number of factors to be taken into account in the oestrogen transcriptional process has created a need to develop a rapid screening method to evaluate their role in physiology and pathology. Molecular biology techniques enable gene expression studies at the mRNA level with small amounts of tissues. We therefore developed a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique using fluorescent oligonucleotides to analyse simultaneously a large panel of interrelated genes involved in the oestrogen transcriptional pathway using a moderately expressed housekeeping gene, the hypoxanthine phosphoribosyltransferase gene (HPRT), as the reference gene. Expression levels of oestrogen receptors (ERalpha, ERbeta), cofactors AIB1, RIP140, SMRT and the Fas-associated protein-tyrosine phophatase-1 (FAP-1) genes were evaluated in breast, endometrial and ovarian cancer cell lines and in three ERalpha-positive and three ERalpha-negative breast cancer tumours. This technique provides a rapid and reliable way to quantitate simultaneously numerous mRNAs of genes involved in the oestrogen pathway from small amounts of tissues.
Rey
J M
JM
Laboratoire de Biologie Cellulaire et Hormonale, CHU Arnaud de Villeneuve, 371 av. du Doyen G Giraud, 34295 Montpellier Cedex 5, France.
Pujol
P
P
Callier
P
P
Cavailles
V
V
Freiss
G
G
Maudelonde
T
T
Brouillet
J P
JP
D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D004967:Estrogens / Q000378:metabolism*; D015870:Gene Expression; D051548:Histone Acetyltransferases; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D056985:Nuclear Receptor Co-Repressor 2; D056920:Nuclear Receptor Coactivator 1; D000078723:Nuclear Receptor Interacting Protein 1; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004967:Estrogens; C539130:NCOR2 protein, human; C000627127:NRIP1 protein, human; D009687:Nuclear Proteins; D056985:Nuclear Receptor Co-Repressor 2; D000078723:Nuclear Receptor Interacting Protein 1; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D012097:Repressor Proteins; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; D007041:Hypoxanthine Phosphoribosyltransferase; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases