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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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Parallel quantifications of cDNA prepared from mouse liver cells. (a) Reproducibility of two independent quantifications of cDNA equivalent to 2 μg of total RNA. The slope and R2-value of the regression line were 1.00 and 0.98 on a log–log scale. (b) Standard curves used in a. (c–f) Quantification of cDNA equivalent to 2 μg of total RNA was compared to that of serially dilution samples containing a quantity of cDNA equivalent to 1 (c), 0.5 (d), 0.25 (e) and 0.125 μg (f) of total RNA. The solid lines at an angle of 45° are the expected lines calculated from the quantifications of cDNA equivalent to 2 μg of total RNA and the dilution factors. The dashed lines are 2- or 0.5-fold of the solid lines. The R2-values of the regression lines with a slope of one were 0.98, 0.95, 0.89 and 0.77, respectively. Error bars show the SDs of triplicate measurements.
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Figure 4: Parallel quantifications of cDNA prepared from mouse liver cells. (a) Reproducibility of two independent quantifications of cDNA equivalent to 2 μg of total RNA. The slope and R2-value of the regression line were 1.00 and 0.98 on a log–log scale. (b) Standard curves used in a. (c–f) Quantification of cDNA equivalent to 2 μg of total RNA was compared to that of serially dilution samples containing a quantity of cDNA equivalent to 1 (c), 0.5 (d), 0.25 (e) and 0.125 μg (f) of total RNA. The solid lines at an angle of 45° are the expected lines calculated from the quantifications of cDNA equivalent to 2 μg of total RNA and the dilution factors. The dashed lines are 2- or 0.5-fold of the solid lines. The R2-values of the regression lines with a slope of one were 0.98, 0.95, 0.89 and 0.77, respectively. Error bars show the SDs of triplicate measurements.

Mentions: Parallel quantifications of cDNA prepared from mouse liver cells were performed to examine the reproducibility, accuracy and sensitivity of gene expression profiling by GEP-DEAN. cDNA strands for 273 mouse genes (Supplementary Table) with various expression levels were quantified in parallel by using Equation (3), in which was replaced with . The values of α, δ and were determined using the standard curves constructed with synthetic 30-base DNA strands of known quantities added to the samples. The standard curves were constructed for each D2 value because the value of α varied with the tube in which the decoding step was performed (Figure 4b).Figure 4.


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

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

Parallel quantifications of cDNA prepared from mouse liver cells. (a) Reproducibility of two independent quantifications of cDNA equivalent to 2 μg of total RNA. The slope and R2-value of the regression line were 1.00 and 0.98 on a log–log scale. (b) Standard curves used in a. (c–f) Quantification of cDNA equivalent to 2 μg of total RNA was compared to that of serially dilution samples containing a quantity of cDNA equivalent to 1 (c), 0.5 (d), 0.25 (e) and 0.125 μg (f) of total RNA. The solid lines at an angle of 45° are the expected lines calculated from the quantifications of cDNA equivalent to 2 μg of total RNA and the dilution factors. The dashed lines are 2- or 0.5-fold of the solid lines. The R2-values of the regression lines with a slope of one were 0.98, 0.95, 0.89 and 0.77, respectively. Error bars show the SDs of triplicate measurements.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105393&req=5

Figure 4: Parallel quantifications of cDNA prepared from mouse liver cells. (a) Reproducibility of two independent quantifications of cDNA equivalent to 2 μg of total RNA. The slope and R2-value of the regression line were 1.00 and 0.98 on a log–log scale. (b) Standard curves used in a. (c–f) Quantification of cDNA equivalent to 2 μg of total RNA was compared to that of serially dilution samples containing a quantity of cDNA equivalent to 1 (c), 0.5 (d), 0.25 (e) and 0.125 μg (f) of total RNA. The solid lines at an angle of 45° are the expected lines calculated from the quantifications of cDNA equivalent to 2 μg of total RNA and the dilution factors. The dashed lines are 2- or 0.5-fold of the solid lines. The R2-values of the regression lines with a slope of one were 0.98, 0.95, 0.89 and 0.77, respectively. Error bars show the SDs of triplicate measurements.
Mentions: Parallel quantifications of cDNA prepared from mouse liver cells were performed to examine the reproducibility, accuracy and sensitivity of gene expression profiling by GEP-DEAN. cDNA strands for 273 mouse genes (Supplementary Table) with various expression levels were quantified in parallel by using Equation (3), in which was replaced with . The values of α, δ and were determined using the standard curves constructed with synthetic 30-base DNA strands of known quantities added to the samples. The standard curves were constructed for each D2 value because the value of α varied with the tube in which the decoding step was performed (Figure 4b).Figure 4.

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
Related in: MedlinePlus