Quantitative analysis of alpha 1 (I) procollagen transcripts in vivo by competitive polymerase chain reaction.
Due to the limited amount of RNA obtainable from punch biopsies, few data exist on the human alpha 1 (I) procollagen mRNA steady state level in vivo. Therefore, we established a competitive PCR method to quantitate this mRNA in human biopsies. In our approach, the target template and the standard share the same sequence except for a 69 bp deletion, thus competing for the same primer pair and subsequently amplifying at the same rate. Titration of a competitor DNA dilution series against an unknown cDNA sample then allows quantitation of the separated and nonradioactively detected PCR products after densitometrical analysis. Almost identical results were obtained by quantitations of the same total RNA by competitive PCR as well as Northern blot analysis.
Thur
J
J
Institute for Biochemistry, Medical Faculty, University of Cologne.
Nischt
R
R
Krieg
T
T
Hunzelmann
N
N
D001483:Base Sequence; D001706:Biopsy; D002478:Cells, Cultured; D017931:DNA Primers; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011347:Procollagen / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D012867:Skin / Q000166:cytology / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D011347:Procollagen; D012333:RNA, Messenger