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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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Genes expressed differentially in the APAP-administered mouse liver. The Y-axis represents the ratio of the absolute amounts of cDNA in an APAP-administered sample to those in a no-drug-administered one. The X-axis represents the absolute amounts of cDNA in a no drug-administered sample. The dashed line represents the median ratio value. Error bars on both axes show the SDs. The candidates for significantly changed genes (open circle) were determined by a two-sided Welch's t-test (P < 0.05) using quadruplicate data.
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Figure 5: Genes expressed differentially in the APAP-administered mouse liver. The Y-axis represents the ratio of the absolute amounts of cDNA in an APAP-administered sample to those in a no-drug-administered one. The X-axis represents the absolute amounts of cDNA in a no drug-administered sample. The dashed line represents the median ratio value. Error bars on both axes show the SDs. The candidates for significantly changed genes (open circle) were determined by a two-sided Welch's t-test (P < 0.05) using quadruplicate data.

Mentions: Figure 5 shows a plot of calculated from quadruplicate measurements of the cDNA quantities in the APAP administered (after 6 h) and no drug-administered samples against the mean of the absolute cDNA quantity of the i-th gene in a no drug-administered sample. When the amount of total cDNA is equal in all samples, is expected to be one. However, as shown in Figure 5, many genes whose expression levels were expected to be unchanged had a ratio of around 0.82, because the total amount of cDNA prepared from the same amount of total RNA varied among the samples. In the actual comparison of gene expression profiles, the information about the total amount of cDNA is usually unavailable. The values for and VU were thus determined from genes with an value close to the median ratio. These genes are safely assumed to be candidate genes with unchanged expression levels. Using and VU calculated from the mean of and Vi of the candidate genes with of a median value ±10%, 20 of 223 genes were found to be significantly up- or downregulated (P < 0.05). When the range of unchanged-gene candidates was taken as the median value ±5%, the number of significantly regulated genes was 19 and hardly varied with the range. In this test, Bonferroni multi-test corrections were not applied because a t-test with a corrected P < 0.00022 (0.05/223) is not appropriate for screening of APAP-induced genes.Figure 5.


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

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

Genes expressed differentially in the APAP-administered mouse liver. The Y-axis represents the ratio of the absolute amounts of cDNA in an APAP-administered sample to those in a no-drug-administered one. The X-axis represents the absolute amounts of cDNA in a no drug-administered sample. The dashed line represents the median ratio value. Error bars on both axes show the SDs. The candidates for significantly changed genes (open circle) were determined by a two-sided Welch's t-test (P < 0.05) using quadruplicate data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Genes expressed differentially in the APAP-administered mouse liver. The Y-axis represents the ratio of the absolute amounts of cDNA in an APAP-administered sample to those in a no-drug-administered one. The X-axis represents the absolute amounts of cDNA in a no drug-administered sample. The dashed line represents the median ratio value. Error bars on both axes show the SDs. The candidates for significantly changed genes (open circle) were determined by a two-sided Welch's t-test (P < 0.05) using quadruplicate data.
Mentions: Figure 5 shows a plot of calculated from quadruplicate measurements of the cDNA quantities in the APAP administered (after 6 h) and no drug-administered samples against the mean of the absolute cDNA quantity of the i-th gene in a no drug-administered sample. When the amount of total cDNA is equal in all samples, is expected to be one. However, as shown in Figure 5, many genes whose expression levels were expected to be unchanged had a ratio of around 0.82, because the total amount of cDNA prepared from the same amount of total RNA varied among the samples. In the actual comparison of gene expression profiles, the information about the total amount of cDNA is usually unavailable. The values for and VU were thus determined from genes with an value close to the median ratio. These genes are safely assumed to be candidate genes with unchanged expression levels. Using and VU calculated from the mean of and Vi of the candidate genes with of a median value ±10%, 20 of 223 genes were found to be significantly up- or downregulated (P < 0.05). When the range of unchanged-gene candidates was taken as the median value ±5%, the number of significantly regulated genes was 19 and hardly varied with the range. In this test, Bonferroni multi-test corrections were not applied because a t-test with a corrected P < 0.00022 (0.05/223) is not appropriate for screening of APAP-induced genes.Figure 5.

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