Transforming growth factor-beta1 is a well-known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor-beta1 gene expression was evaluated in small skin samples. Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription-polymerase chain reaction technique was used. Quantitation of gene expression by reverse transcription-polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards. We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor-beta1 starting with a gene specific polymerase chain reaction product. After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen alpha1(I) cDNA with compatible ends was inserted into the polymerase chain reaction-DNA fragment. The recombinant cDNA was re-amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters. Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription-polymerase chain reaction together with the tissue or cellular RNA. After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide. The bands for internal standard and transforming growth factor-beta1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription-polymerase chain reaction. The number of mRNA molecules/cell was calculated. We examined the transforming growth factor-beta1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five-fold more transforming growth factor-beta1 mRNA than normal skin per unit of wet weight. We used this procedure to quantitate transforming growth factor-beta1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin. The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor-beta1 than normal cells (116 +/- 6 vs. 97 +/- 7, p = 0.017, n = 5). These results were supported by Northern analysis for transforming growth factor-beta1 mRNA in the cells and enzyme-linked immunosorbent assay for TGF-beta1 protein in fibroblast-conditioned medium. In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor-beta1, which may be important in hypertrophic scar formation. The construction of the gene specific internal standard for reverse transcription-polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.
