Relative quantification of collagen mrna in fibroblasts by a radioactive polymerase chain reaction technique

✍ Scribed by Edward J. Goetzl; Christoph W. Turck; Jeffrey A. Golden; Alexandra A. J. Migchielsen; Yong Li Yu

• Publisher: John Wiley and Sons
• Year: 1991
• Tongue: English
• Weight: 535 KB
• Volume: 5
• Category: Article
• ISSN: 0887-8013
• DOI: 10.1002/jcla.1860050407

✦ Synopsis

A radioactive polymerase chain reaction (PCR) method has been developed for the relative quantification of the human a-2 chain of type I collagen [hu a-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 a-2(1) cDNA primed by two sequence-specific synthetic oligonucleotides. The distinctive 390 bp hu a-2(1) cDNA and two Aval fragments of 220 and 170 bp are identified by agarose gel electrophoresis. CX-~*P-~CTP 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 a-2(1) mRNA. The amount of hu a-2(1) mRNA expressed in as few as 111 fibroblasts was determined reliably. In contrast, thehua-2(1)mRNAfromatleast5 x 105fibroblasts was required for detection by Northern blot analysis developed with the same cDNA probe radiolabelled with u -~~P -~C T P by random priming. Human bronchoalveolar lavage (BAL) fluids of six patients with fibrosing lung diseases stimulated the level of expression of hu a-2(1) mRNA in cultured human fibroblasts as determined by this technique. The radioactive PCR method thus quantifies hu a-2(1) mRNA in fibroblasts with sufficient sensitivity to study fibroblast activation in vitro and detect fibroblast stimuli in human clinical samples.