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Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 11/25/2012 Publication Date: 11/26/2012 Citation: Cao, H., Shockey, J.M. 2012. Comparison of TaqMan and SYBR Green qPCR methods for quantitative gene expression in tung tree tissues. Journal of Agricultural and Food Chemistry. 60(50):12296-12303. Interpretive Summary: Tung tree (Vernicia fordii) is a tropical plant with a very limited growing area in the southeastern United States. Tung tree produces large seeds containing about 50-60% oil (dry weight basis) with approximately 80 mole % a-eleostearic acid (9cis, 11trans, 13trans octadecatrienoic acid). Tung oil is readily oxidized due to the three unique conjugated double bonds in eleostearic acid. Dried tung oil is impervious to heat, moisture, dust, and many chemical challenges, and unlike other drying oils, does not darken with age. These properties underlie the value of tung oil as a drying ingredient in paints, varnishes, and other coatings and finishes. Most of the tung orchards were destroyed in the southern United States by hurricanes, including Hurricanes Betsy in 1965, Camille in 1969, and Katrina and Rita in 2005. These losses spurred interest in trying to preserve a reliable domestic source of tung oil by transferring the genetic components of the tung oil biosynthetic pathway into traditional, temperate oilseeds. Our project focuses on engineering oilseed crops to produce tung oil, which are rich in novel fatty acids that impart valuable properties to their respective oils. A number of genes have been identified for tung oil biosynthesis. However, less is known about the expression patterns of many other genes in tung seeds. As a first step to profile gene expression in tung oil biosynthesis, we validated 25 TaqMan and SYBR Green qPCR assays, optimized these assays, and compared both assays using cDNAs from tung seeds, leaves, and flowers. The assay development will help to identify key factors for tung oil biosynthesis and provide potential targets for genetic engineering value-added oil crops. Technical 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 post-amplification 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 was isolated from tung tissues, as indicated by high rRNA ratio and RNA integrity number. qPCR primers and TaqMan probes were optimized. Under optimized conditions, both qPCR gave high correlation co-efficiency and similar amplification efficiency, but TaqMan qPCR 11 generated higher Y-intercepts than SYBR Green qPCR, which over-estimated 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 TaqMan assay is more sensitive but generates lower calculated expression levels than 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. |
