Competitive reverse transcription/polymerase chain reaction for the quantification of p53 and mdm2 mRNA expression.
Wild-type p53 (wtp53) is a tumour suppressor gene involved in cell cycle regulation. The mdm2 protein can complex with the p53 protein and influence its function as a regulator of cell growth. To detect and quantify wtp53 and mdm2 mRNA expression, we established the competitive reverse transcription/polymerase chain reaction for these genes and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The target RNA differed from the competitor cRNA by having 183 bp, 205 bp and 173 bp deletions for p53, mdm2 and GAPDH, respectively. Target RNA and known concentrations of competitor cRNA were co-reverse transcribed and co-amplified with the same primers. Target cDNA and the corresponding competitor cDNA were amplified at the same efficiency.
Totzke
G
G
Medizinische Universitäts-Poliklinik, Bonn, Germany.
Sachinidis
A
A
Vetter
H
H
Ko
Y
Y
D004730:Endothelium, Vascular / Q000378:metabolism; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2; D018075:RNA, Complementary / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012015:Reference Standards; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000235:genetics
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D018075:RNA, Complementary; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2