Semi-quantitative RT-PCR for comparison of mRNAs in cells with different amounts of housekeeping gene transcripts.
A simple method is reported here for the semi-quantitative assay of mRNAs in the presence of an exogenous mRNA (pBR322 transcript) as a standard. This method uses the co-reverse transcription and co-amplification (co RT-PCR) of the target and standard mRNAs. This procedure enables transcripts to be compared when the differentiation process affects the transcription pattern of the beta-actin housekeeping gene, a commonly used internal standard. This method is sensitive and avoids constructing internal competitive RNA standards. As an example, it is shown that beta-actin transcription decreases and type V adenylyl cyclase transcription increases in adipocytes, when compared to preadipocytes.
Serazin-Leroy
V
V
Laboratoire de Biochimie de la Faculté de Médecine Paris-Ouest, INSERM CJF 94-02, Université René, Descartes Paris V, France.
Denis-Henriot
D
D
Morot
M
M
de Mazancourt
P
P
Giudicelli
Y
Y
D000199:Actins / Q000235:genetics*; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary; D004822:Epididymis; D008297:Male; D010957:Plasmids; D011786:Quality Control; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D014158:Transcription, Genetic
D000199:Actins; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger