Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages.
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.
U.S. Department of Agriculture-Agricultural Research Service, Southern Regional Research Center, 1100 Robert E. Lee Blvd, New Orleans, LA, 70124, USA, Heping.Cao@ars.usda.gov.
Heping
H
Cao
Fangping
F
Cao
Anne-Marie
AM
Roussel
Richard A
RA
Anderson
D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D052160:Benzothiazoles; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D020869:Gene Expression Profiling / Q000379:methods*; D051246:Glucose Transport Proteins, Facilitative / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D051816:Tristetraprolin / Q000378:metabolism*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D051246:Glucose Transport Proteins, Facilitative; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; D051816:Tristetraprolin; C098022:SYBR Green I; D019914:Taq Polymerase