Different real-time PCR systems yield different gene expression values.
Most polymerase chain reaction (PCR) systems employ pre-determined settings and proprietary master mixes that differ from one system to another. It is not known whether these differences may affect gene expression values. We compared two major real-time PCR technologies, from Life Technologies (formerly Applied Biosystems; ABI7500) and Roche Applied Science (LC480), using their default settings, proprietary reagents and other potential variables such as ramp rates and magnesium concentrations. We analyzed four genes (IL-8, COX2, ID-1 and CXCR2) in a human breast cancer cell line and found that two of them, though readily detected by ABI, were not detected using the Roche system. By altering some of the parameters and reagents used in the Roche protocol, we were able to detect expression of these two genes, but the level remained far below that detected by ABI, particularly for ID-1. When we tested three additional ID-1 primer pairs, two of these primer pairs yielded higher expression values in the LC system, yet still significantly lower than the values obtained in ABI. These results suggest critical differences in these two PCR systems, which could result in significant discrepancies in results reported by different laboratories.
Lu
Shan
S
California Pacific Medical Center Research Institute, San Francisco, CA 94107, USA.
Smith
Andrew P
AP
Moore
Dan
D
Lee
Nancy M
NM
D045744:Cell Line, Tumor; D019411:Clinical Laboratory Techniques / Q000295:instrumentation / Q000379:methods*; D051546:Cyclooxygenase 2 / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D023063:Receptors, Interleukin-8B / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; C497453:ID1 protein, human; D051798:Inhibitor of Differentiation Protein 1; D016209:Interleukin-8; D023063:Receptors, Interleukin-8B; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human