Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes

Authors

1 Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Research Center; Physiology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

3 Physiology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: Real-time polymerase chain reaction (PCR) is based on the revolutionary method of PCR. This technique is the result of PCR enormous sensitivity and real-time monitoring combination. In quantitative gene expression analysis, two methods have more popularity, SYBR Green and TaqMan, SYBR Green is relatively cost benefit and easy to use and technically based on binding the fluorescent dye to double-stranded deoxyribonucleic acid (dsDNA) where TaqMan method has more expensive and based on dual labeled oligonucleotide and exonuclease activity of Taq polymerase enzyme. Specificity is the most important concern with the usage of any non-specific dsDNA-binding Dyes such as SYBR Green whiles more specificity showed by labeled oligonucleotide method such as TaqMan. In this study, we compared two common RT PCR methods, TaqMan and SYBR Green in measurement gene expression profile of adenosine receptors.
Materials and Methods: Gene expression profiles of A1, A2A, A2B and A3 Adenosine receptors were analyzed by optimized TaqMan and SYBR Green quantitative RT PCR in breast cancer tissues. Primary expression data was normalizing by B. actin reference gene.
Results: Efficiencies were calculated more than 95% for TaqMan and SYBR Green methods in all genes. The correlations between means of normalized data of each gene in two methods were positive and significant (P < 0.05).
Conclusion: Data analysis showed that with the use of high performance primer and by use proper protocols and material we can make precise data by SYBR Green as TaqMan method. In other word by optimization of SYBR Green method, its performance and quality could be comparable to TaqMan method.

Keywords

1. Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am 1990;262:56-61, 64.  Back to cited text no. 1
    
2. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 1987;155:335-50.  Back to cited text no. 2
    
3. Tan X, Sun X, Gonzalez-Crussi FX, Gonzalez-Crussi F, Hsueh W. PAF and TNF increase the precursor of NF-kappa B p50 mRNA in mouse intestine: Quantitative analysis by competitive PCR. Biochim Biophys Acta 1994;1215:157-62.  Back to cited text no. 3
    
4. Huang SK, Xiao HQ, Kleine-Tebbe J, Paciotti G, Marsh DG, Lichtenstein LM, et al. IL-13 expression at the sites of allergen challenge in patients with asthma. J Immunol 1995;155:2688-94.  Back to cited text no. 4
    
5. Huang SK, Yi M, Palmer E, Marsh DG. A dominant T cell receptor beta-chainin response to a short ragweed allergen, Amb a 5. J Immunol 1995;154:6157-62.  Back to cited text no. 5
    
6. Manzin A, Solforosi L, Bianchi D, Gabrielli A, Giostra F, Bruno S, et al. Viral load in samples from hepatitis C virus (HCV)-infected patients with various clinical conditions. Res Virol 1995;146:279-84.  Back to cited text no. 6
    
7. Connor RI, Mohri H, Cao Y, Ho DD. Increased viral burden and cytopathicity correlate temporally with CD4+T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals. J Virol 1993;67:1772-7.  Back to cited text no. 7
    
8. Piatak M Jr, Saag MS, Yang LC, Clark SJ, Kappes JC, Luk KC, et al. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science 1993;259:1749-54.  Back to cited text no. 8
    
9. Furtado MR, Kingsley LA, Wolinsky SM. Changes in the viral mRNA expression pattern correlate with a rapid rate of CD4+T-cell number decline in human immunodeficiency virus type 1-infected individuals. J Virol 1995;69:2092-100.  Back to cited text no. 9
    
10. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986-94.  Back to cited text no. 10
    
11. Rossi JJ. Capture, mutate, quantitate: Variations of a central PCR scheme. Genet Anal Tech Appl 1992;9:101-2.  Back to cited text no. 11
    
12. Alyapkina YS, Romanova YM, Alekseeva NV, Kovalev YN, Gaintseva AV, Gintsburg AL. Development of a quantitative PCR technique and its application to the evaluation of gene expression. Russ J Genet 2000;36:821-5.  Back to cited text no. 12
    
13. Raeymaekers L. A commentary on the practical applications of competitive PCR. Genome Res 1995;5:91-4.  Back to cited text no. 13
    
14. Ferre F. Quantitative or semi-quantitative PCR: Reality versus myth. PCR Methods Appl 1992;2:1-9.  Back to cited text no. 14
    
15. Fasco MJ, Treanor CP, Spivack S, Figge HL, Kaminsky LS. Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence. Anal Biochem 1995;224:140-7.  Back to cited text no. 15
    
16. Lazar JG. Advanced methods in PCR product detection. PCR Methods Appl 1994;4:S1-14.  Back to cited text no. 16
    
17. Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ 2005;29:151-9.  Back to cited text no. 17
    
18. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230:1350-4.  Back to cited text no. 18
    
19. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 1993;11:1026-30.  Back to cited text no. 19
    
20. Klein D. Quantification using real-time PCR technology: Applications and limitations. Trends Mol Med 2002;8:257-60.  Back to cited text no. 20
    
21. Orlando C, Pinzani P, Pazzagli M. Developments in quantitative PCR. Clin Chem Lab Med 1998;36:255-69.  Back to cited text no. 21
    
22. Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques 2005;39:75-8.  Back to cited text no. 22
    
23. Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 1997;22:130-1, 134.  Back to cited text no. 23
    
24. Mackay IM. Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 2004;10:190-212.  Back to cited text no. 24
    
25. Wilhelm J, Pingoud A, Hahn M. Real-time PCR-based method for the estimation of genome sizes. Nucleic Acids Res 2003;31:e56.  Back to cited text no. 25
    
26. Apte A, Daniel S. PCR primer design. Cold Spring Harb Protoc 2009;2009:pdb.ip65.  Back to cited text no. 26