Journal: BMC Genomics

Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

doi: 10.1186/1471-2164-12-326

Figure Lengend Snippet: Microarray interplatform analysis . (A) Overlap of unique and named genes shared among the 3 microarray platforms used in this study. The pool of 17070 shared genes was used for further cross-platform analysis. The total numbers of genes for each platform and for all platforms combined are indicated. (B) Overlap of significantly regulated genes at 6 h after EGF treatment considering each of the 3 microarray platforms independently.

Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

Techniques: Microarray

Journal: BMC Genomics

Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

doi: 10.1186/1471-2164-12-326

Figure Lengend Snippet: GSEA analysis on significantly regulated gene sets across microarray platforms . Profile of the Running ES Score & Positions of Gene Set Members on the Rank Ordered List using 6 h EGF treatment data according to each of the three microarray platforms. In each panel, the vertical black lines indicate the position of each of the genes of the tested gene set in the reference data set (ranked by average of the three respective EGF versus control log2ratios of replicate experiments). The green curve plots the ES (enrichment score), which is the running sum of the weighted enrichment score obtained from GSEA software. Within each queried gene set, the farther the position of a gene to the left (red) implies a higher correlation with EGF up-regulated genes in the reference platform, and the farther to the right (blue) implies a higher correlation with genes down-regulated upon EGF treatment in the reference platform. Studied gene sets correspond to lists of up- or down-regulated genes in each platform at 6 h of EGF treatment. Significantly enriched data sets are defined according to GSEA default settings (p < 0.001 and a false discovery rate (FDR) < 0.25). R.L.M = ranked list metric.

Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

Techniques: Microarray, Software

Journal: BMC Genomics

Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

doi: 10.1186/1471-2164-12-326

Figure Lengend Snippet: Microarray versus DGE analysis . (A) Overlap of unique and named genes shared among the 3 microarray platforms and genes detected by DGE. The pool of 14645 shared genes was used for further cross-platform analysis. The total numbers of genes for each platform and for all platforms combined are indicated. (B) Overlap of significantly regulated genes considering the 3 microarray platforms at 6 h after EGF treatment and the genes found regulated after assessing significance by grouping microarray and DGE data in a RankProd analysis. Left panels show up-regulated genes and right panels show down-regulated genes.

Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

Techniques: Microarray

Journal: BMC Genomics

Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

doi: 10.1186/1471-2164-12-326

Figure Lengend Snippet: Correlation between microarrays and Illumina GA-I sequencing . (A) Comparison of estimated log2ratios from DGE ( Y -axis) and the mean of all microarray platforms ( X -axis). We consider only genes that were interrogated using all platforms and genes with a mean number of counts across lanes greater than 0. Genes with counts greater than 32 reads (colored red or green) or less than (black) 32 reads in at least one sample are shown. (Red dots) Genes called differentially expressed based on DGE data at an 10% FDR by RankProd. (Green dots) Genes not called as differentially expressed but above 32 counts. (Inset box) Correlation between technologies is higher when considering genes above the 32 count detection level (0.57) than when all genes are included (0.49). (B-C) Concordance at the top (CAT) plots of the different platforms with the 500 top genes from a reference platform, shown for Agilent in (B) and DGE in (C). See inset box for color codes identifying each platforms compared to the remaining platform used as reference. (D) Correlation plots with regression lines between log2ratios of the five high content platforms measurements (Y-axis) and quantitative real time PCR results using SYBR green assays (X-axis), based on measurements for 21 genes at the 6 h time point (see Additional file , Table S1).

Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

Techniques: Sequencing, Microarray, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: BMC Genomics

Article Title: Multiple platform assessment of the EGF dependent transcriptome by microarray and deep tag sequencing analysis

doi: 10.1186/1471-2164-12-326

Figure Lengend Snippet: Top regulated genes derived from meta-analysis . RankProd analysis of the combination of microarray and Illumina GA-I ultrasequencing data sets. Heatmap of the top 50 up and down-regulated genes detected in all four platforms ordered by Median Fold Change (all have RankProd adjusted p-values < 0.0001). IL11, IL8, PLAUR, ANXA10 and FOS were validated by RT-qPCR showing concordant results (See Additional file , Table S1). The full RankProd matrix from these experiments is accessible in Additional file , Table S5. The list of all 1164 significantly regulated genes (median |FC| > 1.2 and RankProd q-value < 0.05) is given in Additional file , Table S6.

Article Snippet: Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer.

Techniques: Derivative Assay, Microarray, Quantitative RT-PCR

Journal:

Article Title: Genomic Analysis of Anti-Hepatitis B Virus (HBV) Activity by Small Interfering RNA and Lamivudine in Stable HBV-Producing Cells

doi: 10.1128/JVI.79.22.14392-14403.2005

Figure Lengend Snippet: The fidelity and reproducibility of microarray profiling. (A) The CapitalBio's long oligonucleotide (Operon 70-mer) microarray and Affymetrix's short oligonucleotide (25-mer) HG U133A 2.0 microarray platform were compared, and the correlation coefficient (R value) between the two platforms was 0.787 when the total of 6754 common genes were detected using the two platforms, employing the same batch of RNA extracted from HeLa and HEK293 cell lines. (B) Self-to-self comparison of the gene expression. For each test and control sample, two hybridizations were performed by using a reversal fluorescence strategy. The change of magnitude of self-to-self expression profiling was within twofold, which indicated that a change less than twofold could be considered to represent the noise level of the microarray experiment. The two lines parallel in the graph represent 2- and 0.5-fold changes in expression. (C) The determination of global gene expression of HepG2.2.15 versus HepG2 cells was repeated three times independently. For each test and control sample in one independent experiment, two hybridizations were performed by using a reverse fluorescence strategy. Two sets of the expression ratios were chosen to determine the reproducibility of microarray results. The Pearson linear correlation coefficient value (R) was 0.956. (D) Comparison of expression measurement by quantitative RT-PCR and microarray. The expression changes in the selected 17 genes showed good agreement between the two methods.

Article Snippet: The reason for this is that the fidelity of our microarray is twofold; therefore, genes with a change of less than twofold fell into the “gray zone” of microarray analysis, which means that the calculated up- or down-regulation change of these genes may not be precise, and hence it is reasonable to conclude that parts of these five genes may have some disparities in terms of their regulation changes when the two methodologies were applied. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 7. caption a7 The fidelity and reproducibility of microarray profiling. (A) The CapitalBio's long oligonucleotide (Operon 70-mer) microarray and Affymetrix's short oligonucleotide (25-mer) HG U133A 2.0 microarray platform were compared, and the correlation coefficient (R value) between the two platforms was 0.787 when the total of 6754 common genes were detected using the two platforms, employing the same batch of RNA extracted from HeLa and HEK293 cell lines. (B) Self-to-self comparison of the gene expression.

Techniques: Microarray, Expressing, Fluorescence, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

doi: 10.1093/nar/gkq163

Figure Lengend Snippet: Hybridization based depurination assay. A hybridization based depurination assay was used to measure the extent of the depurination side reaction during synthesis of microarrays using the legacy process. The depurination probes are composed of a depurination sensitive stilt of variable length (A n , n = 10–35) and a common 25mer reporter sequence. The length of each depurination probe is n + 25 and their synthesis was staggered during the 60-nt synthesis cycles used to synthesize the 60mer microarray. Early depurination probes ( A ) are such that their synthesis is initiated as early as possible, i.e. m blank synthesis cycles are performed after the probe synthesis so that m + n + 25 = 60. Late depurination probes ( B ) are such that their synthesis is initiated as late as possible, i.e. m blank synthesis cycles are performed prior to the probe synthesis so that m + n + 25 = 60. All probes are synthesized simultaneously and hybridized to a labeled oligonucleotide complementary to the reported probe. The early depurination probe hybridization signals show a decrease in signal intensity with increasing number of n due to a loss of reporter probe ( C ). A similar profile is observed for late depurination probes.

Article Snippet: First the synthesis of high-quality long oligonucleotides of length up to 150mer is now possible on the Agilent microarray platform while current methods rarely generate oligomers in excess of 100 nt.

Techniques: Hybridization, Sequencing, Microarray, Synthesized, Labeling

Journal: Nucleic Acids Research

Article Title: Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

doi: 10.1093/nar/gkq163

Figure Lengend Snippet: Synthesis of 125, 135 and 150mer oligonucleotides on microarray surfaces and analysis of full-length products. ( A ) Scheme for the 5′-end labeling of oligonucleotides by ligation. Chip synthesized oligonucleotides were phosphorylated, annealed to gene-specific oligonucleotide, and ligated to the labeling duplex. As a result, only full length oligonucleotides are ligated to Cy-3 labeled oligonucleotides. ( B ) Oligonucleotide design. ( C ) Left panel: denaturing gel analysis of 5′-end labeled 125, 135 and 150mer oligonucleotides. Right panel: the same products after UDG treatment. ( D ) Estimation of yield for full-length product.

Article Snippet: First the synthesis of high-quality long oligonucleotides of length up to 150mer is now possible on the Agilent microarray platform while current methods rarely generate oligomers in excess of 100 nt.

Techniques: Microarray, End Labeling, Ligation, Synthesized, Labeling

Journal: Nucleic Acids Research

Article Title: Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

doi: 10.1093/nar/gkq163

Figure Lengend Snippet: Oligonucleotides synthesized on microarray surfaces are uniformly represented. ( A ) Scheme of the labeling PCR product by ligation. Approximately 100 mol of individually CPG-synthesized oligonucleotides, normalized pool of the CPG-synthesized oligonucleotides, or 1 µl of 1000-fold diluted Chip oligo pool were amplified by 35 cycles of PCR and digested with BstXI restriction endonuclease. Aliquots of PCR amplified DNA and digestion reaction fragments were analyzed by agarose gel electrophoresis ( B ). Digested PCR products were labeled by ligation and analyzed by denaturating gel electrophoresis ( C ). Fragment profiles were generated for each multiplex PCR analysis ( D ).

Article Snippet: First the synthesis of high-quality long oligonucleotides of length up to 150mer is now possible on the Agilent microarray platform while current methods rarely generate oligomers in excess of 100 nt.

Techniques: Synthesized, Microarray, Labeling, Ligation, Amplification, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Generated, Multiplex Assay

Journal: Nucleic Acids Research

Article Title: Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

doi: 10.1093/nar/gkq163

Figure Lengend Snippet: Identification of synthesis integrity using the legacy process. Oligonucleotides of length 25, 45 and 60mer were obtained from synthesis on CPG or from synthesis on microarray surfaces using the legacy process. The oligonucleotides were labeled with 32 P at their 5′-end and analyzed using denaturing gel electrophoresis. Profiles from the oligonucleotides synthesized on microarray surfaces show the presence of full length product (full arrow), n − 1 product 32 in the 25 and 45mer samples (lanes 4 and 5), and discrete bands (indicated by dotted arrows). The sizes of the discrete bands match the A nucleobase positions indicated in bold in the sequences and the pattern is characteristic of depurination side reactions during synthesis. The offset between the actual migration length on the gel (indicated by numbers below the sequences) and the position of the A nucleobases relative to the 5′ end of the sequence (−2 bases) is attributed to the chemical functionality of the 3′-end of the chain cleaved by depurination.

Article Snippet: First the synthesis of high-quality long oligonucleotides of length up to 150mer is now possible on the Agilent microarray platform while current methods rarely generate oligomers in excess of 100 nt.

Techniques: Microarray, Labeling, Nucleic Acid Electrophoresis, Synthesized, Migration, Sequencing

Journal: Nucleic Acids Research

Article Title: Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

doi: 10.1093/nar/gkq163

Figure Lengend Snippet: Visualization of the flow of detritylation solution on the microarray surface. Visualization of the flow of the detritylation solution film on the microarray surface following draining of the bulk detritylation solution from the flowcell. Frames A , B and C were taken at 300 ms intervals and show that detritylation solution is trapped on the feature after the receding front has passed. This phenomenon is due to the difference between the hydrophilic nature of the features (growing oligonucleotide chain) and the hydrophobic nature of the substrate surface. The formation of droplets on every feature is followed by rapid evaporation of the toluene solvent carrier leading to a high DCA concentration and significant depurination side reactions.

Article Snippet: First the synthesis of high-quality long oligonucleotides of length up to 150mer is now possible on the Agilent microarray platform while current methods rarely generate oligomers in excess of 100 nt.

Techniques: Microarray, Evaporation, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

doi: 10.1093/nar/gkq163

Figure Lengend Snippet: PCR analysis of oligonucleotides synthesized on microarray surfaces. Six hundred and twenty-six oligonucleotides were released in 100 µl of TE buffer and diluted 1/1000. An aliquot (1 µl) of the diluted pool was used for PCR with a specific set of PCR primers. PCR products with correct lengths were produced after amplification with primers corresponding to A3 oligonucleotide of 78 nt (lane 1) and B3 oligonucleotide of 104 nt (lane 2). Synthesis of a dT 6 linker at either the 5′ (lane 3) or 3′-end (lane 4) of oligonucleotides 114 nucleobase in length had no effect on amplification. PCR products were also detected after amplification with corresponding primers which were designed to simultaneously amplify six (lanes 5 and 6), or 18 (lane 7) sequences. The lengths of oligonucleotides vary from 80 to 99 nt; 104 to 120 nt; and 106 to 120 nt, respectively. M-Low Molecular Weight DNA Ladder DNA (New England Biolabs Inc.) was used as size marker.

Article Snippet: First the synthesis of high-quality long oligonucleotides of length up to 150mer is now possible on the Agilent microarray platform while current methods rarely generate oligomers in excess of 100 nt.

Techniques: Synthesized, Microarray, Produced, Amplification, Molecular Weight, Marker