PMID: 9450638Feb 5, 1998Paper

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

Molecular and Cellular Biochemistry
K W KuoW C Leung

Abstract

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 ...Continue Reading

Citations

Jan 17, 2004·Clinical Chemistry·Anders StåhlbergMikael Kubista
Aug 28, 2004·Clinical Chemistry·Anders StåhlbergMichael Pfaffl
Jan 5, 2014·Nature Protocols·Simone PicelliRickard Sandberg
Aug 11, 2017·European Journal of Clinical Investigation·Stephen Bustin, Tania Nolan
Jan 27, 1999·Biochemical and Biophysical Research Communications·K W KuoW C Leung
Jun 30, 2000·Biochemical and Biophysical Research Communications·L C ChangK W Kuo
Jan 30, 2002·Analytical Biochemistry·Andrej-Nikolai Spiess, Richard Ivell
Jul 5, 2003·Journal of Clinical Microbiology·Manfred WeidmannFrank T Hufert

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