Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells.
Amplification of the mRNA polymerase chain reaction is a very sensitive technique to detect low-abundance transcripts. We describe in this paper conditions necessary to make this technique quantitative. Quantification is performed in the exponential phase of the amplification process and the results are standardized with respect to those obtained with an exogenous mRNA which is co-reverse-transcribed and co-amplified in the same reaction as the analyzed transcripts. The primers are chosen in different exons to distinguish the amplification of mRNA fragments from the amplification of contaminating DNA. Analysis of the kinetics of amplification and parameters influencing this kinetics shows that: (a) in the exponential phase of amplification, the amount of amplified fragments is proportional to the initial amount of transcripts; (b) in a certain range of length fragment, the yield of amplification is inversely proportional to the length of the amplified fragments. Using this method we have demonstrated that the dystrophin gene is already activated at the myoblastic stage. A quantitative estimation of the transcript showed that the expression of this gene increases strongly in the course of in vitro myogenesis. In primary culture of mouse brain cells, the dystrophin gene was found to be more expressed in neuronal than in glial cells.
Chelly
J
J
Unité de Recherches en Génétique et Pathologie Moléculaires, Unité 129 INSERM, CHU Cochin, Paris, France.
Montarras
D
D
Pinset
C
C
Berwald-Netter
Y
Y
Kaplan
J C
JC
Kahn
A
A
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003720:Densitometry; D016189:Dystrophin; D005784:Gene Amplification; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics*; D009474:Neurons / Q000032:analysis; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*
D016189:Dystrophin; D005904:Glial Fibrillary Acidic Protein; D009124:Muscle Proteins; D012333:RNA, Messenger