Relative quantification of collagen mRNA in fibroblasts by a radioactive polymerase chain reaction technique.
A radioactive polymerase chain reaction (PCR) method has been developed for the relative quantification of the human alpha-2 chain of type I collagen [hu alpha-2(I)] in cells. cDNAs generated by reverse transcription from the total pool of cytoplasmic RNA serve as a template for polymerase chain reaction amplification of a hu alpha-2(I) cDNA primed by two sequence-specific synthetic oligonucleotides. The distinctive 390 bp hu alpha-2(I) cDNA and two Aval fragments of 220 and 170 bp are identified by agarose gel electrophoresis. alpha-32P-dCTP of defined specific activity is included in the PCR reaction and the 390 bp cDNA is excised from the electrophoresis gel to permit direct radioactive quantification of hu alpha-2(I) mRNA. The amount of hu alpha-2(I) mRNA expressed in as few as 111 fibroblasts was determined reliably. In contrast, the hu alpha-2(I) mRNA from at least 5 x 10(5) fibroblasts was required for detection by Northern blot analysis developed with the same cDNA probe radiolabelled with alpha-32P-dCTP by random priming. Human bronchoalveolar lavage (BAL) fluids of six patients with fibrosing lung diseases stimulated the level of expression of hu alpha-2(I) mRNA in cultured human fibroblasts as determined by this technique. The radioactive PCR method thus quantifies hu alpha-2(I) mRNA in fibroblasts with sufficient sensitivity to study fibroblast activation in vitro and detect fibroblast stimuli in human clinical samples.
Yu
Y L
YL
Howard Hughes Medical Institute, Department of Medicine, Medical Center, University of California, San Francisco.
Golden
J A
JA
Migchielsen
A A
AA
Goetzl
E J
EJ
Turck
C W
CW
D001345:Autoradiography; D015152:Blotting, Northern; D001992:Bronchoalveolar Lavage Fluid / Q000235:genetics; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics*; D005347:Fibroblasts; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D003094:Collagen