Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and mac
- PDF / 769,286 Bytes
- 12 Pages / 595.276 x 790.866 pts Page_size
- 28 Downloads / 176 Views
antitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages Heping Cao & Fangping Cao & Anne-Marie Roussel & Richard A. Anderson
Received: 23 April 2013 / Accepted: 17 July 2013 / Published online: 16 August 2013 / Editor: T. Okamoto # The Society for In Vitro Biology 2013
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
H. Cao (*) U.S. Department of Agriculture-Agricultural Research Service, Southern Regional Research Center, 1100 Robert E. Lee Blvd, New Orleans, LA 70124, USA e-mail: [email protected] F. Cao Beijing Forestry University, 35 Qinghua Donglu, Haidian District, Beijing 100083, People’s Republic of China A.Zfp36l1>Zfp36>Zfp36l3 (Table 3). Identical order of Zfp36 family mRNA levels in the cultured adipocytes was obtained by SYBR Green qPCR (Table 3). Similar patterns of Zfp36 family genes were obtained by both qPCR assays in mouse RAW264.7 macrophages (Table 3). Comparison of Bio-Rad’s CFX96 real-time system and Applied Biosystems’ ABI Prism 7700 real time PCR instrument. The reliability of qPCR data generated by TaqMan methods in this study using Bio-Rad’s CFX96 real-time system is validated by results from previous studies using Applied Biosystems’ ABI Prism 7700 real time PCR instrument (Table 4). TaqMan qPCR assay using both instruments showed that Glut1 and Glut4 genes were almost equally expressed in mouse 3T3-L1 adipocytes (Cao et al. 2010) and Glut1 mRNA being the dominant Glut form in RAW264.7 macrophages (Cao et al. 2008a), whereas Glut3 mRNA levels Table 3. Comparison of TaqMan and SYBR qPCR on relative expression of Zfp36 family genes in mouse cells qPCR assays and mRNAs TaqMan Zfp36 (TTP) Zfp36l1 (Tis11b) Zfp36l2 (Tis11d) Zfp36l3 SYBR Green Zfp36 (TTP) Zfp36l1 (Tis11b) Zfp36l2 (Tis11d) Zfp36l3
3T3-L1 adipocytes
RAW264.7 macrophages
1.00
1.00
3.35±0.53 7.35±3.26 0.003±0.001
0.25±0.02 3.61±0.03 0.01±0.004
1.00 1.48±
Data Loading...
