PANC-1 Comparison of Real-time PCR fluorescent and non-fluorescent quenchers in standard amplification plots of delta-6 desaturase gene in PANC-1 cell line culture

authors:

avatar M Mehdi , avatar M Darabi , avatar SH Biagowi , avatar R Najafipour , avatar T NaserpoorFarivar , avatar M Darabi Amin , *

Journal of Inflammatory Diseases: Vol.17, issue 5; 17-24
article type: issue_documents_importer

how to cite: Mehdi M, Darabi M, Biagowi S, Najafipour R, NaserpoorFarivar T, et al. PANC-1 Comparison of Real-time PCR fluorescent and non-fluorescent quenchers in standard amplification plots of delta-6 desaturase gene in PANC-1 cell line culture. J Inflamm Dis. 2014;17(5):e155778. 

Abstract

Abstract AIM: The present study was undertaken to determine the difference between florescence and non-florescence quenchers and their efficiency in Real-time PCR. MATERIALS AND METHODS: PANC-1 cells were cultured in 75cm flasks, 3x106 cells were seeded in 6-well plates and treated with specific signaling drugs, their effect on the expression of delta6 desaturase gene were examined with Real-time PCR in equal conditions, once with florescence and another time with non-florescence quencher. RESULTS: different quenchers absorbances of the fluorescence emission from the reporter, cause different results in Real-time PCR. The amplification plot is more precisely when using non-florescence quencher and its base line is lower. Therefore the signal to noise ratio(S/N) decrease. Also non-florescence quencher with raising the melting temperature cause to decrease in non-specific matching and this led to lower CT ratio. CONCLUSION: Data showing increased performance of using non-florescence quencher compared to florescence quencher and it can be a better replace for common quencher specially in Allelic discrimination and SNP(single nucleotide polymorphism) studies.