Role of the exon 6 of human PDGF A-chain mRNA in the determination of gene expression by reverse transcription-polymerase chain reaction.

Alternative-spliced PDGF A mRNA is constituted with exon 6-excluded (PDGFA [-6]) and exon 6-included (PDGFA[+6]) transcripts. To define the role of exon 6 in the gene expression of alternative-spliced PDGF A in U937 cell, the exon-junction primers were employed for RT-PCR. The PDGFA[-6] mRNA could be amplified with specific primers at annealing temperatures of 60 degrees C, 65 degrees C, 70 degrees C and 75 degrees C, whereas PDGFA[+6] mRNA could be detected only at 70 degrees C, suggesting that the RNA secondary structure of PDGFA[+6] might affect the RT-PCR reactions. In addition, PDGFA[+6] mRNA was incapable of competing with PDGFA[-6] mRNA for the common primers. A highly complementary and double-stranded RNA structure was formed for PDGF A when the exon 6 was included in the sequence. Since accessibility of a RNA template for in vitro amplification is related to RNA secondary structure, the results derived from RT-PCR and mRNA folding of PDGFA[+6] are essentially consistent. Thus, these results suggest that the exon 6 may affect the determination of gene expression by constricting the RNA secondary structure of PDGF A. The requirement of imperatively high stringency in the hybridization conditions for PDGFA[+6] mRNA may account for the low detection of the transcript in cells by conventional methods.