U.S. Department of Agriculture-Agricultural Research Service, Southern Regional Research Center, 1100 Robert E. Lee Blvd, New Orleans, LA, 70124, USA,
In Vitro Cell Dev Biol Anim. 2013 Dec;49(10):759-70. doi: 10.1007/s11626-013-9671-8. Epub 2013 Aug 16.
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 false-positive 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.
实时荧光定量 PCR（qPCR），如 TaqMan 和 SYBR Green qPCR，广泛用于基因表达分析。SYBR Green 检测法的缺点是染料会与任何双链 DNA 结合，从而产生假阳性信号，并且扩增子的长度会影响扩增的强度。先前的结果表明，在定量分析油桐组织中的 7 个 mRNA 时，TaqMan 检测法比 SYBR Green 检测法更灵敏，但生成的计算表达水平更低。本研究的目的是使用动物细胞扩展分析。我们比较了两种 qPCR 检测法，用于定量分析 3T3-L1 脂肪细胞和 RAW264.7 巨噬细胞中的 24 个 mRNA，包括编码葡萄糖转运蛋白（Glut）和 mRNA 结合蛋白 tristetraprolin（TTP）的 mRNA。结果表明，SYBR Green 和 TaqMan qPCR 可用于动物细胞中定量基因表达。这一结果得到了 Glut 和 TTP 家族基因表达验证分析的支持。然而，SYBR Green qPCR 高估了大多数测试基因的表达水平。最后，两种 qPCR 仪器（Bio-Rad 的 CFX96 实时系统和 Applied Biosystems 的 Prism 7700 实时 PCR 仪器）在小鼠细胞中生成了相似的基因表达谱。这些结果支持以下结论：两种 qPCR 检测法（TaqMan 和 SYBR Green qPCR）和两种 qPCR 仪器（Bio-Rad 的 CFX96 实时系统和 Applied Biosystems 的 Prism 7700 实时 PCR 仪器）都可用于动物细胞中的定量基因表达分析，但 SYBR Green qPCR 通常会高估基因表达水平，而 TaqMan qPCR 则不会。
