|Cao, Fangping -|
Submitted to: In Vitro Cellular and Developmental Biology - Animals
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 17, 2013
Publication Date: August 16, 2013
Citation: Cao, H., Cao, Fangping, Roussel, A-M., Anderson, R.A. 2013. Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages. In Vitro Cellular and Developmental Biology - Animal. 49:759–770. Interpretive Summary: 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 assays are two frequently used qPCR techniques. TaqMan qPCR uses a fluorogenic single-stranded oligonucleotide probe that binds only the DNA sequence between the two PCR primers. Therefore, only specific PCR product can generate fluorescent signal in TaqMan PCR. SYBR Green qPCR is widely used because of the ease in designing the assays and its relatively low setup and running costs. Unlike TaqMan fluorescent probes, SYBR Green dye intercalates into double-stranded DNA to monitor the amplification of target gene specifically initiated by gene-specific primers. One drawback of the SYBR Green assay is that the dye is non-specific which can generate false positive signals if non-specific products or primer-dimers are present in the assay. The other drawback of the SYBR Green assay is that the length of the amplicon also affects the intensity of the amplification. There is a lack of literature on direct comparison of TaqMan and SYBR Green qPCR assays using the same biological samples and the same PCR primers. We compared both qPCR assays for quantifying 24 mRNAs using cDNAs from mouse cells and rat tissues. Our data showed that both assays were reliable for determining the expression of these genes in animal cells and tissues. The surprising finding was that the TaqMan qPCR generated lower calculated expression levels than the SYBR Green qPCR regardless of the tissues and gene targets tested with a few exceptions. Therefore, interpretation of qPCR results between these two assays requires caution.
Technical Abstract: Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate falsepositive signals and that the length of the amplicon affects the intensity of the amplification. Previous results demonstrate that TaqMan assay is more sensitive but generates lower calculated expression levels than SYBR Green assay in quantifying seven mRNAs in tung tree tissues. The objective of this study is to expand the analysis using animal cells. We compared both qPCR assays for quantifying 24 mRNAs including those coding for glucose transporter (Glut) and mRNA-binding protein tristetraprolin (TTP) in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. The results showed that SYBR Green and TaqMan qPCR were reliable for quantitative gene expression in animal cells. This result was supported by validation analysis of Glut and TTP family gene expression. However, SYBR Green qPCR overestimated the expression levels in most of the genes tested. Finally, both qPCR instruments (Bio-Rad’s CFX96 real-time system and Applied Biosystems’ Prism 7700 real-time PCR instrument) generated similar gene expression profiles in the mouse cells. These results support the conclusion that both qPCR assays (TaqMan and SYBR Green qPCR) and both qPCR instruments (Bio-Rad’s CFX96 real-time system and Applied Biosystems’ Prism 7700 real-time PCR instrument) are reliable for quantitative gene expression analyses in animal cells but SYBR Green qPCR generally overestimates gene expression levels than TaqMan qPCR.