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

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SAHMANI M.; DARABI M.; BYAGOWI SH.; NAJAFIPOUR R.; NASERPOUR FARIVAR T.; DARABI AMIN M.

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Journal: Journal of Inflammatory Diseases Year:2014 | Volume:17 | Issue:5 (70) Page(s): 17-24

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Title

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

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Keywords

REAL-TIME PCRQ1

DELTA-6 DESATURASEQ2

CELL LINEQ2

Abstract

Background: Employing non-fluorescent quenchers in REAL-TIME PCR is appropriate for gene expression examination.Objective: The objective of this study was to compare REAL-TIME PCR fluorescent and nonfluorescent quenchers in standard amplification plots of DELTA-6 DESATURASE gene in PANC-1 CELL LINE culture.Methods: This analytical study was conducted in the Reference Laboratory affiliated to Qazvin University of Medical Sciences in 2012. Human pancreatic cancer cells (PANC-1) were cultured in 75 cm2 flasks, 3x106 cells were seeded in 6-well plates and were treated with specific intracellular signaling drugs. Changes in expression of DELTA-6 DESATURASE gene were examined with fluorescent and non-fluorescent quenchers using REAL-TIME PCR in equal conditions, separately. Data were analyzed using student T-test and gene expression results were analyzed using ΔΔCT method with the assumption of 100% efficiency.Findings: Employed quenchers showed different absorption of the fluorescence emitted by the reporter and caused different results in REAL-TIME PCR. Using non-fluorescent quencher, the amplification plot was more precise and its baseline was lower. Therefore the signal to noise ratio (S/N) was decreased. Also, the Threshold cycle (Ct) value was lower because of increased Tm (melting temperature) and decreased non-specific bindings.Conclusion: With regards to the results, non-fluorescent quencher is more appropriate compared to fluorescent quencher and can be a better alternative for current quenchers especially in allelic discrimination and SNP (single nucleotide polymorphism) studies.

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APA: Copy

SAHMANI, M., DARABI, M., BYAGOWI, SH., NAJAFIPOUR, R., NASERPOUR FARIVAR, T., & DARABI AMIN, M.. (2014). 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. JOURNAL OF INFLAMMATORY DISEASES (THE JOURNAL OF QAZVIN UNIVERSITY OF MEDICAL SCIENCES), 17(5 (70)), 17-24. SID. https://sid.ir/paper/49297/en

Vancouver: Copy

SAHMANI M., DARABI M., BYAGOWI SH., NAJAFIPOUR R., NASERPOUR FARIVAR T., DARABI AMIN M.. 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. JOURNAL OF INFLAMMATORY DISEASES (THE JOURNAL OF QAZVIN UNIVERSITY OF MEDICAL SCIENCES)[Internet]. 2014;17(5 (70)):17-24. Available from: https://sid.ir/paper/49297/en

IEEE: Copy

M. SAHMANI, M. DARABI, SH. BYAGOWI, R. NAJAFIPOUR, T. NASERPOUR FARIVAR, and M. DARABI AMIN, “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,” JOURNAL OF INFLAMMATORY DISEASES (THE JOURNAL OF QAZVIN UNIVERSITY OF MEDICAL SCIENCES), vol. 17, no. 5 (70), pp. 17–24, 2014, [Online]. Available: https://sid.ir/paper/49297/en

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