INTRINSIC SECONDARY STRUCTURE OF HUMAN TNFR-I MESSENGER-RNA INFLUENCES THE DETERMINATION OF GENE-EXPRESSION BY RT-PCR

The secondary structure of human tumor necrosis factor receptor I (TNF R-I) mRNA based on its lowest folding energy was predicted. Three comb inations of primers selected from open-regions and four combinations o f primers from closed-regions of TNFR-I mRNA structure were employed f or single-tube reverse transcription-polymerase chain reaction (RT-PCR ) for the determination of TNFR-I gene expression in U937 cell. All th e primers were designed with the same criteria. However, the different primers generated distinct quantities of RT-PCR products from the sam e concentration of TNFR-I mRNA, implying that the determination of gen e expression by RT-PCR was affected by the mRNA secondary structure. I n addition, the sensitivity of the open-region RT-PCR was approximatel y one hundred-fold higher than that in the closed-regions of TNFR-I mR NA. The low efficiency of the closed-region RT-PCR was not correlated with the G/C content of the TNFR-I mRNA structure. These results sugge st that consideration of the influence of intrinsic mRNA structure of a gene is essential prior to the determination of gene expression by q uantitative RT-PCR, and this open-region strategy of primer design may yield an efficient primer for in vitro amplification of cDNA by RT-PC R.