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Multiplex cDNA quantification method that facilitates the standardization of gene expression data.

Gotoh O, Murakami Y, Suyama A - Nucleic Acids Res. (2011)

Bottom Line: Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations.These requirements impose limitations on the extensive comparison of gene expression data.Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Institute of Physics, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.

ABSTRACT
Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h.

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Comparison between GEP-DEAN and qPCR. The reproducibility of GEP-DEAN (a) and that of qPCR (b) are plotted. The solid lines show the ideal lines at an angle of 45°. The dashed lines are 2-fold or 0.5-fold of the ideal lines. The R2-values on a log–log scale were 1.00 and 0.97, respectively. The accuracies of GEP-DEAN (c) and qPCR (d) are plotted. The horizontal lines show the level of no quantification error. Error bars show the SDs of replicated measurements.
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Figure 3: Comparison between GEP-DEAN and qPCR. The reproducibility of GEP-DEAN (a) and that of qPCR (b) are plotted. The solid lines show the ideal lines at an angle of 45°. The dashed lines are 2-fold or 0.5-fold of the ideal lines. The R2-values on a log–log scale were 1.00 and 0.97, respectively. The accuracies of GEP-DEAN (c) and qPCR (d) are plotted. The horizontal lines show the level of no quantification error. Error bars show the SDs of replicated measurements.

Mentions: First, we compared the reproducibilities of the two methods. Independent duplicate analyses demonstrated that the measurement of the absolute amount of DNA by GEP-DEAN was as highly reproducible as that by qPCR (Figure 3a and b). The correlation plot of the duplicate measurement by GEP-DEAN exhibited a regression line with a slope of one and R2-value of 1.00. For the measurement by qPCR, the correlation plot indicated a regression line with a slope of one and an R2-value of 0.97.Figure 3.


Multiplex cDNA quantification method that facilitates the standardization of gene expression data.

Gotoh O, Murakami Y, Suyama A - Nucleic Acids Res. (2011)

Comparison between GEP-DEAN and qPCR. The reproducibility of GEP-DEAN (a) and that of qPCR (b) are plotted. The solid lines show the ideal lines at an angle of 45°. The dashed lines are 2-fold or 0.5-fold of the ideal lines. The R2-values on a log–log scale were 1.00 and 0.97, respectively. The accuracies of GEP-DEAN (c) and qPCR (d) are plotted. The horizontal lines show the level of no quantification error. Error bars show the SDs of replicated measurements.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105393&req=5

Figure 3: Comparison between GEP-DEAN and qPCR. The reproducibility of GEP-DEAN (a) and that of qPCR (b) are plotted. The solid lines show the ideal lines at an angle of 45°. The dashed lines are 2-fold or 0.5-fold of the ideal lines. The R2-values on a log–log scale were 1.00 and 0.97, respectively. The accuracies of GEP-DEAN (c) and qPCR (d) are plotted. The horizontal lines show the level of no quantification error. Error bars show the SDs of replicated measurements.
Mentions: First, we compared the reproducibilities of the two methods. Independent duplicate analyses demonstrated that the measurement of the absolute amount of DNA by GEP-DEAN was as highly reproducible as that by qPCR (Figure 3a and b). The correlation plot of the duplicate measurement by GEP-DEAN exhibited a regression line with a slope of one and R2-value of 1.00. For the measurement by qPCR, the correlation plot indicated a regression line with a slope of one and an R2-value of 0.97.Figure 3.

Bottom Line: Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations.These requirements impose limitations on the extensive comparison of gene expression data.Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Institute of Physics, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.

ABSTRACT
Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h.

Show MeSH