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Comparison of TaqMan and SYBR Green qPCR Methods for Quantitative Gene Expression in Animals
Author(s) -
Cao Heping,
Roussel AnneMarie,
Anderson Richard A.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.574.5
Subject(s) - taqman , amplicon , sybr green i , real time polymerase chain reaction , microbiology and biotechnology , biology , gene expression , primer (cosmetics) , complementary dna , gene , primer dimer , rna , polymerase chain reaction , chemistry , genetics , multiplex polymerase chain reaction , organic chemistry
Quantitative real‐time‐PCR (qPCR) such as TaqMan and SYBR Green qPCR assays are widely used for gene expression analysis. The drawbacks of SYBR Green assays include that the dye is nonspecific which can generate false positive signals if non‐specific products or primer‐dimers are present in the assay and that the length of the amplicon affects the intensity of the amplification. However, little was reported in the literature on direct comparison of both methods using the same pairs of primers and biological samples. We therefore compared both assays using 24 RNAs from mouse 3T3‐L1 adipocytes, RAW264.7 macrophages, and rat adipocytes, brain, kidney, liver, and muscle tissues. High quality RNA with high rRNA ratio and RNA integrity number was isolated from these animal cells and tissues and used for cDNA synthesis and qPCR amplification. The surprising finding was that SYBR Green qPCR over‐estimated the expression levels by several folds in most of the genes and tissues tested. This was unlikely due to non‐specific PCR products or the length of PCR products. The results demonstrate that both assays are reliable for determining gene expression levels in cells and tissues, but that TaqMan assay generally generates lower calculated expression levels than SYBR Green assay. This study suggests that any discussion of gene expression levels requires specification of qPCR methods used in the analysis.