Methods for measuring type I collagen synthesis in vitro.
The excess accumulation of type I collagen within tissues leads to organ dysfunction and occurs as a result of an imbalance between synthesis and degradation. This chapter outlines several methods to assess the in vitro production of type I collagen that are employed in our laboratory. We describe Western immunoblotting of intact alpha1(I) collagen using antibodies directed to alpha1(I) collagen amino and carboxyl propeptides. The measurement of alpha1(I) collagen mRNA levels using real-time polymerase chain reaction is then outlined. Finally, methods to determine the transcriptional regulation of alpha1(I) collagen using a nuclear run-on assay are described.
Rishikof
David C
DC
The Pulmonary Center, Boston University Schol of Mediicne, The Boston VA Medical Center, Boston, MA, USA.
Kuang
Ping-Ping
PP
Subramanian
Mangalalaxmy
M
Goldstein
Ronald H
RH
D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D024042:Collagen Type I / Q000032:analysis* / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008168:Lung / Q000196:embryology; D008967:Molecular Biology / Q000379:methods*; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D024042:Collagen Type I; D018076:DNA, Complementary; D012333:RNA, Messenger; D003609:Dactinomycin; D012313:RNA