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Comparison of TaqMan and SYBR Green qPCR Methods for Quantitative Gene Expression in Tung Tree Tissues

Author:
Cao Heping, Shockey Jay M.
Source:
Journal of agricultural and food chemistry 2012 v.60 no.50 pp. 12296-12303
ISSN:
1520-5118
Subject:
DNA, DNA primers, Vernicia fordii, dyes, fluorescence, gene amplification, gene expression, hybridization, nucleotide sequences, oligonucleotide probes, plant tissues, polymerase chain reaction, quantitative analysis, trees
Abstract:
Quantitative real-time-PCR (qPCR) is widely used for gene expression analysis due to its large dynamic range, tremendous sensitivity, high sequence specificity, little to no postamplification processing, and sample throughput. TaqMan and SYBR Green qPCR are two frequently used methods. However, direct comparison of both methods using the same primers and biological samples is still limited. We compared both assays using seven RNAs from the seeds, leaves, and flowers of tung tree (Vernicia fordii), which produces high-value industrial oil. High-quality RNA were isolated from tung tissues, as indicated by a high rRNA ratio and RNA integrity number. qPCR primers and TaqMan probes were optimized. Under optimized conditions, both qPCR gave high correlation coefficiency and similar amplification efficiency, but TaqMan qPCR generated higher y-intercepts than SYBR Green qPCR, which overestimated the expression levels regardless of the genes and tissues tested. This is validated using well-known Dgat2 and Fadx gene expression in tung tissues. The results demonstrate that both assays are reliable for determining gene expression in tung tissues and that the TaqMan assay is more sensitive but generates lower calculated expression levels than the SYBR Green assay. This study suggests that any discussion of gene expression levels needs to be linked to which qPCR method is used in the analysis.
Agid:
57437
Handle:
10113/57437