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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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Schematic representation of GEP-DEAN. (a) Outline of the assay procedures. (b) Structure of molecular translation table (MTT) that connects the target cDNA with a target specific sequence TSSi to the corresponding two-digit DNA-coded number DCNj composed of the common start digit (SD), the first digit D1j1, the second digit D2j2 and the common end digit (ED). (c) Encoding step. The encoding reaction for the target cDNA strands with TSSi starts with ligation of 5′-MTTi,j and 3′-MTTi,j strands that hybridize adjacently with TSSi. After ligation of the MTT strands, the reaction mixture is subjected to incubation at high temperature and a subsequent quick cooling to dissociate the target cDNA strands and to make 3′-biotinylated cCS (cCS-b) hybridize. The ligation products with cCS-b bound are captured by streptavidin-coupled magnetic beads (SA-beads) and then excess free MTT strands and the dissociated target cDNA strands are washed out. After elution of the ligation products, a primer pair of 5′-b-CS and cED strands is added to the reaction mixture and the ligation products are amplified by PCR to further remove the free 3′-MTT strands. The amplified ligation products are then captured by SA-beads and converted to single strands by alkali wash. (d) Amplification step. The DCN region of the single-stranded ligation products is amplified by PCR with a primer pair of 5′-biotinylated SD and cED. The amplified products are then captured by SA-beads and converted to single strands by alkali wash. (e) Decoding step. The two-digit DCNs are decoded with respect to the second digit (D2). The solution of the single-stranded amplified products of the sample and that of the reference are divided into ND2 tubes. To the j2-th tube, a fluorescence-labeled (Cy5- and Cy3-labeled for sample and reference, respectively) cD2j2 strand and the mixture of all cD1 strands are added. When a DCNj strand corresponding to the target cDNA with TSSi is present, a cD2j2 and a cD1j1 strand are joined together by DCN-templated ligation to produce a decoding product cD2j2–cD1j1. The decoding products of the sample and reference are thus labeled with different fluorescence colors. (f) Quantification step. The decoding products of the sample and those of the reference in the j2-th tube are competitively hybridized in the j2-th capillary of a DNA capillary array (DCA), which is a kind of DNA microarray composed of an array of capillaries with DNA probes immobilized on their inner surface. All capillaries have a common set of DNA probes D1j1 ( j 1 = 0, 1, 2 , … , ND1 − 1). The absolute amount of the target cDNA with TSSi is obtained from the Cy5/Cy3 fluorescence intensity ratio measured for a spot of D1j1 probe in the j2-th capillary.
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Figure 1: Schematic representation of GEP-DEAN. (a) Outline of the assay procedures. (b) Structure of molecular translation table (MTT) that connects the target cDNA with a target specific sequence TSSi to the corresponding two-digit DNA-coded number DCNj composed of the common start digit (SD), the first digit D1j1, the second digit D2j2 and the common end digit (ED). (c) Encoding step. The encoding reaction for the target cDNA strands with TSSi starts with ligation of 5′-MTTi,j and 3′-MTTi,j strands that hybridize adjacently with TSSi. After ligation of the MTT strands, the reaction mixture is subjected to incubation at high temperature and a subsequent quick cooling to dissociate the target cDNA strands and to make 3′-biotinylated cCS (cCS-b) hybridize. The ligation products with cCS-b bound are captured by streptavidin-coupled magnetic beads (SA-beads) and then excess free MTT strands and the dissociated target cDNA strands are washed out. After elution of the ligation products, a primer pair of 5′-b-CS and cED strands is added to the reaction mixture and the ligation products are amplified by PCR to further remove the free 3′-MTT strands. The amplified ligation products are then captured by SA-beads and converted to single strands by alkali wash. (d) Amplification step. The DCN region of the single-stranded ligation products is amplified by PCR with a primer pair of 5′-biotinylated SD and cED. The amplified products are then captured by SA-beads and converted to single strands by alkali wash. (e) Decoding step. The two-digit DCNs are decoded with respect to the second digit (D2). The solution of the single-stranded amplified products of the sample and that of the reference are divided into ND2 tubes. To the j2-th tube, a fluorescence-labeled (Cy5- and Cy3-labeled for sample and reference, respectively) cD2j2 strand and the mixture of all cD1 strands are added. When a DCNj strand corresponding to the target cDNA with TSSi is present, a cD2j2 and a cD1j1 strand are joined together by DCN-templated ligation to produce a decoding product cD2j2–cD1j1. The decoding products of the sample and reference are thus labeled with different fluorescence colors. (f) Quantification step. The decoding products of the sample and those of the reference in the j2-th tube are competitively hybridized in the j2-th capillary of a DNA capillary array (DCA), which is a kind of DNA microarray composed of an array of capillaries with DNA probes immobilized on their inner surface. All capillaries have a common set of DNA probes D1j1 ( j 1 = 0, 1, 2 , … , ND1 − 1). The absolute amount of the target cDNA with TSSi is obtained from the Cy5/Cy3 fluorescence intensity ratio measured for a spot of D1j1 probe in the j2-th capillary.

Mentions: DCNs are 92-base DNA-tag sequences that are composed of four sections, designated as SD, D1j1 (j1 = 0, 1, 2 , … , ND1 − 1), D2j2 (j2 = 0, 1, 2 , … , ND2 − 1) and ED (Figure 1b). The characteristics of DCNs were described previously (18). Briefly, these sequences are designed to have a uniform length, a uniform melting temperature and have no potential for mishybridizations or stable self-folded structures. The sequences are listed in the Supplementary Table 4.Figure 1.


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

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

Schematic representation of GEP-DEAN. (a) Outline of the assay procedures. (b) Structure of molecular translation table (MTT) that connects the target cDNA with a target specific sequence TSSi to the corresponding two-digit DNA-coded number DCNj composed of the common start digit (SD), the first digit D1j1, the second digit D2j2 and the common end digit (ED). (c) Encoding step. The encoding reaction for the target cDNA strands with TSSi starts with ligation of 5′-MTTi,j and 3′-MTTi,j strands that hybridize adjacently with TSSi. After ligation of the MTT strands, the reaction mixture is subjected to incubation at high temperature and a subsequent quick cooling to dissociate the target cDNA strands and to make 3′-biotinylated cCS (cCS-b) hybridize. The ligation products with cCS-b bound are captured by streptavidin-coupled magnetic beads (SA-beads) and then excess free MTT strands and the dissociated target cDNA strands are washed out. After elution of the ligation products, a primer pair of 5′-b-CS and cED strands is added to the reaction mixture and the ligation products are amplified by PCR to further remove the free 3′-MTT strands. The amplified ligation products are then captured by SA-beads and converted to single strands by alkali wash. (d) Amplification step. The DCN region of the single-stranded ligation products is amplified by PCR with a primer pair of 5′-biotinylated SD and cED. The amplified products are then captured by SA-beads and converted to single strands by alkali wash. (e) Decoding step. The two-digit DCNs are decoded with respect to the second digit (D2). The solution of the single-stranded amplified products of the sample and that of the reference are divided into ND2 tubes. To the j2-th tube, a fluorescence-labeled (Cy5- and Cy3-labeled for sample and reference, respectively) cD2j2 strand and the mixture of all cD1 strands are added. When a DCNj strand corresponding to the target cDNA with TSSi is present, a cD2j2 and a cD1j1 strand are joined together by DCN-templated ligation to produce a decoding product cD2j2–cD1j1. The decoding products of the sample and reference are thus labeled with different fluorescence colors. (f) Quantification step. The decoding products of the sample and those of the reference in the j2-th tube are competitively hybridized in the j2-th capillary of a DNA capillary array (DCA), which is a kind of DNA microarray composed of an array of capillaries with DNA probes immobilized on their inner surface. All capillaries have a common set of DNA probes D1j1 ( j 1 = 0, 1, 2 , … , ND1 − 1). The absolute amount of the target cDNA with TSSi is obtained from the Cy5/Cy3 fluorescence intensity ratio measured for a spot of D1j1 probe in the j2-th capillary.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Schematic representation of GEP-DEAN. (a) Outline of the assay procedures. (b) Structure of molecular translation table (MTT) that connects the target cDNA with a target specific sequence TSSi to the corresponding two-digit DNA-coded number DCNj composed of the common start digit (SD), the first digit D1j1, the second digit D2j2 and the common end digit (ED). (c) Encoding step. The encoding reaction for the target cDNA strands with TSSi starts with ligation of 5′-MTTi,j and 3′-MTTi,j strands that hybridize adjacently with TSSi. After ligation of the MTT strands, the reaction mixture is subjected to incubation at high temperature and a subsequent quick cooling to dissociate the target cDNA strands and to make 3′-biotinylated cCS (cCS-b) hybridize. The ligation products with cCS-b bound are captured by streptavidin-coupled magnetic beads (SA-beads) and then excess free MTT strands and the dissociated target cDNA strands are washed out. After elution of the ligation products, a primer pair of 5′-b-CS and cED strands is added to the reaction mixture and the ligation products are amplified by PCR to further remove the free 3′-MTT strands. The amplified ligation products are then captured by SA-beads and converted to single strands by alkali wash. (d) Amplification step. The DCN region of the single-stranded ligation products is amplified by PCR with a primer pair of 5′-biotinylated SD and cED. The amplified products are then captured by SA-beads and converted to single strands by alkali wash. (e) Decoding step. The two-digit DCNs are decoded with respect to the second digit (D2). The solution of the single-stranded amplified products of the sample and that of the reference are divided into ND2 tubes. To the j2-th tube, a fluorescence-labeled (Cy5- and Cy3-labeled for sample and reference, respectively) cD2j2 strand and the mixture of all cD1 strands are added. When a DCNj strand corresponding to the target cDNA with TSSi is present, a cD2j2 and a cD1j1 strand are joined together by DCN-templated ligation to produce a decoding product cD2j2–cD1j1. The decoding products of the sample and reference are thus labeled with different fluorescence colors. (f) Quantification step. The decoding products of the sample and those of the reference in the j2-th tube are competitively hybridized in the j2-th capillary of a DNA capillary array (DCA), which is a kind of DNA microarray composed of an array of capillaries with DNA probes immobilized on their inner surface. All capillaries have a common set of DNA probes D1j1 ( j 1 = 0, 1, 2 , … , ND1 − 1). The absolute amount of the target cDNA with TSSi is obtained from the Cy5/Cy3 fluorescence intensity ratio measured for a spot of D1j1 probe in the j2-th capillary.
Mentions: DCNs are 92-base DNA-tag sequences that are composed of four sections, designated as SD, D1j1 (j1 = 0, 1, 2 , … , ND1 − 1), D2j2 (j2 = 0, 1, 2 , … , ND2 − 1) and ED (Figure 1b). The characteristics of DCNs were described previously (18). Briefly, these sequences are designed to have a uniform length, a uniform melting temperature and have no potential for mishybridizations or stable self-folded structures. The sequences are listed in the Supplementary Table 4.Figure 1.

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