Intrinsic secondary structure of human TNFR-I mRNA influences the determination of gene expression by RT-PCR.

The secondary structure of human tumor necrosis factor receptor I (TNFR-I) mRNA based on its lowest folding energy was predicted. Three combinations of primers selected from open-regions and four combinations of primers from closed-regions of TNFR-I mRNA structure were employed for single-tube reverse transcription-polymerase chain reaction (RT-PCR) for the determination of TNFR-I gene expression in U937 cell. All the primers were designed with the same criteria. However, the different primers generated distinct quantities of RT-PCR products from the same concentration of TNFR-I mRNA, implying that the determination of gene expression by RT-PCR was affected by the mRNA secondary structure. In addition, the sensitivity of the open-region RT-PCR was approximately one hundred-fold higher than that in the closed-regions of TNFR-I mRNA. 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 suggest that consideration of the influence of intrinsic mRNA structure of a gene is essential prior to the determination of gene expression by quantitative RT-PCR, and this open-region strategy of primer design may yield an efficient primer for in vitro amplification of cDNA by RT-PCR.