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Comparative Study
. 2008 Jul 11:9:328.
doi: 10.1186/1471-2164-9-328.

Cross-platform comparison of SYBR Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC) study

Emi Arikawa  1 , Yanyang Sun, Jie Wang, Qiong Zhou, Baitang Ning, Stacey L Dial, Lei Guo, Jingping Yang
Affiliations
Comparative Study

Cross-platform comparison of SYBR Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC) study

Emi Arikawa et al. BMC Genomics. .

Abstract

Background: The MicroArray Quality Control (MAQC) project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006). The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan Gene Expression PCR Assay, Standardized (Sta) RT-PCRtrade mark and QuantiGene. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profilertrade mark PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency.

Results: The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes.

Conclusion: These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

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Figures

Figure 1
Figure 1
Assay precision of SYBR Green RT2 Profiler PCR Arrays. The plot shows the coefficient of variation for each average CT value. A total of 184 average CT measurements were obtained from Sample A and Sample B. Each average CT value and its coefficient of variation were calculated from the result of 6 replicate assays.
Figure 2
Figure 2
Inter-site comparison of RT2 Profiler PCR Arrays. Threshold cycle measurements and fold-change results have been obtained using the two MAQC reference RNAs analyzed on the Human Drug Metabolism RT2 Profiler PCR Array (APH-002) using different real-time PCR thermal cyclers at two different sites. Five replicate arrays were run for each sample at each site, and the average CT value for each assay is compared between the two sites in panels A and B. The results show a high correlation between CT values obtained from the two sites. Panel C shows the inter-site comparison of fold-change results obtained from these data, indicating a correlation coefficient of 0.976.
Figure 3
Figure 3
Cross-platform comparison between SYBR Green RT2 Profiler PCR Arrays and the three quantitative platforms. The concordance of fold-changes between the PCR Arrays and the three quantitative platforms was evaluated by regression analysis of fold differences in sample B compared to sample A. Data were normalized against POLR2A for RT2 Profiler PCR Arrays and TaqMan, and against beta-actin for StaRT-PCR. The sample B/sample A (B/A) fold changes (log2) for each gene common between RT2 Profiler PCR Arrays and another platform were subjected to bivariate analysis. The dashed line on each graph represents the ideal slope of 1.0. The solid lines show a linear regression fit.
Figure 4
Figure 4
Cross-platform comparison between SYBR Green RT2 Profiler PCR Arrays and microarray platoforms. The concordance of fold-changes between the PCR Arrays and microarray platforms was evaluated by regression analysis of fold differences in sample B compared to sample A. Data were normalized against POLR2A for RT2 Profiler PCR Arrays. The sample B/sample A (B/A) fold changes (log2) for each gene common between RT2 Profiler PCR Arrays and another platform were subjected to bivariate analysis. The dashed line on the graph represents the ideal slope of 1.0. Each solid line represents the linear regression fit. The numbers in brackets indicate the number of common genes between RT2 Profiler PCR Arrays and each microarray platform. The individual scatter plot for each comparison is provided in Additional file 3.
Figure 5
Figure 5
Comparison of the probing locations for the discordant genes between RT2 Profiler PCR Arrays and other gene expression measurement platforms in the MAQC phase 1 study. The diagram illustrates the most 3' interrogative locations of the assays of different platforms for the eight discordant genes discussed in the text and shown in Table 6. The black lines represent, from left to right, the 5' to 3' position of the genes, each indicated with the length of the transcript. The assay location of each platform is indicated by a letter above the black line (A = RT2 Profiler PCR Array; B = TaqMan; C = StaRT-PCR; D = QuantiGene; E = AFX; F = ABI; G = AG1; H = GEH; and I = ILM). Assays with discordant results compared to those of PCR Arrays are highlighted in grey.
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