Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA . ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Reverse Transcription, Purification, Amplification, In Vitro

Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Experimental procedure utilized for the analysis of 10-year-old matched frozen and formalin-fixed paraffin embedded breast cancer samples. ( a ) Five micrograms of RNA extracted from the 10-year-old frozen portion of the sample, is reverse-transcribed and the cDNA is double stranded (dsDNA), in four individual reactions. The dsDNA of three reactions undergoes IVT-amplification (MessageAmpII, Ambion), which gives rise to complementary RNA (cRNA) for cDNA microarray analyses. The dsDNA of one reaction is used for PCR experiments. ( b ) Five micrograms of RNA extracted from the 10-year-old FFPE portion of the sample underwent the exact same process. ( c ) Single-stranded DNA (ssDNA) obtained by RT of 5 μg of FFPE-RNA is purified and hybridized to the sense-RNA template library. The restored ssDNA is double stranded and purified. Three of the CT-RT reactions undergo IVT-amplification, while the dsDNA of one reaction is used for PCR experiments.

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Formalin-fixed Paraffin-Embedded, Reverse Transcription, Amplification, Microarray, Purification

Journal: Nucleic Acids Research

Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

doi: 10.1093/nar/gkm510

Figure Lengend Snippet: Signal intensity and heat-map analysis of the correlation between the log 2 ratios measured by cDNA microarrays. ( a ) Signal intensity of one sample grid in the red channel (Cy5) across all microarrays. Top three panels display the grids obtained from three repeats using cRNA from 10-year-old frozen RNA (Frozen-Amp 1–3). Three mid-panels show the signal of three repeats using cRNA obtained by restoration and IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Restored 1–3). Three bottom panels display the signal of three repeats using cRNA obtained by direct IVT-amplification of RNA from 10-year-old FFPE tissue. ( b ) Heat map displaying the log 2 of expression ratios ranging between 0.5 and 2 for 1044 genes detected in frozen tissue on a 28 032 features cDNA microarray and represented in the UHR library. From left to right are displayed the ratios obtained by IVT-amplification of RNA from 10-year-old frozen tissue (Frozen-Amp 1–3), restoration and IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Restored 1–3) and direct IVT-amplification of RNA from 10-year-old FFPE tissue (FFPE-Amp 1–3). Each column represents an individual hybridization and each line a different feature. Red and blue represent up-regulated and down-regulated genes, respectively.

Article Snippet: Arrays used for the studies were designed and printed at the cDNA Microarray Facility, Albert Einstein College of Medicine (AECOM), Bronx, NY.

Techniques: Amplification, Expressing, Microarray, Hybridization

Journal: Gene Expression

Article Title: Global Gene Expression Profiling of Dimethylnitrosamine-Induced Liver Fibrosis: From Pathological and Biochemical Data to Microarray Analysis

doi:

Figure Lengend Snippet: A schematic illustration of DMN-induced fibrosis in rats. (A) Each rat was either injected with DMN three times per week for 3 consecutive weeks (triangle) or injected with normal saline as a control under the same regime. Rats were weighed and sacrificed each week (starting on day 11, which are referred to as first week to sixth week). Blood samples were collected for biochemical assay (summary in Table 1) and livers were excised and weighed, followed by either fixing in formaldehyde for histopathology or isolation of RNA for microarray analysis. (B) The quantitative real-time PCR result for Tgfbl. The TaqMan® assays were conducted in triplicate for each sample, and a mean value was used for calculation of expression levels. To standardize the quantification of the target genes, 18S rRNA from each sample was quantified at the same time as the target genes.

Article Snippet: Secondly, Kim and his colleagues identified 556 chronic liver disease (CLD)-related genes, which included 273 HCC-associated gene signatures and 283 etiology-associated signatures; this involved a comparison of low-risk and high-risk CLD groups using an Incyte human cDNA microarray ( 26 ).

Techniques: Injection, Histopathology, Isolation, Microarray, Real-time Polymerase Chain Reaction, Expressing

Journal: Gene Expression

Article Title: Global Gene Expression Profiling of Dimethylnitrosamine-Induced Liver Fibrosis: From Pathological and Biochemical Data to Microarray Analysis

doi:

Figure Lengend Snippet: The 256 gene expression patterns of experimental samples. (A) Hierarchical clustering results of these gene expression patterns. The results are shown in a diagram format, in which rows represent individual transcripts and columns represent time course sample. The color in each cell reflected the expression level of the corresponding sample, relative to its mean expression level. The scale extends from fluorescence ratios of 0.25 to 4 relative to the mean level for all samples. (B) Gene ontology results of 256 genes. The plots of different category of these genes by gene ontology database (http://fatigo.bioinfo.cnif.es/). (C) The hierarchical clustering results of the three biology processes are: metabolism, cell growth and/or maintenance and response stimulus. These diagrams are formatted as rows representing individual transcripts and columns representing time course sample. The color in each cell reflects the expression level of the corresponding sample relative to its mean expression level and the scale extends from fluorescence ratios of 0.25 to 4 relative to the mean level for all samples. (D) The comparison of Timpl expression between the Q-RT-PCR results and microarray data. The TaqMan® assays were conducted in triplicate for each sample, and a mean value was used for calculation of expression levels (marked by the square). To standardize the quantification of the Timpl, 18S rRNA from each sample was quantified at the same time as the target gene and a log scale was used as indicated on the right side of plot. For the two Timpl transcripts, rc_AI169327_at and rc_AI169327_g_at (marked by circle and triangle), the expression levels of the microarray data were relative to the mean of all gene expression levels and the scale is indicated on the left side of plot. The Pearson correlation coefficients (r), which compared the Q-RT-PCR result and the microarray data of two Timpl transcripts (rc_AI169327_at and rc_AI169327_g_at), were 0.79 and 0.92, respectively. (E) Endogenous Spp1 protein expression pattern in DMN-induced rat liver samples. Rat liver samples were lysed and 50 itg protein lysates were subjected to immunoblot analysis with antibody against Spp1 and Actb. Spp1 was significantly overexpressed at the protein level after the fourth week of DMN treatment.

Article Snippet: Secondly, Kim and his colleagues identified 556 chronic liver disease (CLD)-related genes, which included 273 HCC-associated gene signatures and 283 etiology-associated signatures; this involved a comparison of low-risk and high-risk CLD groups using an Incyte human cDNA microarray ( 26 ).

Techniques: Expressing, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Microarray, Western Blot

Journal: Gene Expression

Article Title: Global Gene Expression Profiling of Dimethylnitrosamine-Induced Liver Fibrosis: From Pathological and Biochemical Data to Microarray Analysis

doi:

Figure Lengend Snippet: SUMMARY OF MICROARRAY DATASETS COMPARISON

Article Snippet: Secondly, Kim and his colleagues identified 556 chronic liver disease (CLD)-related genes, which included 273 HCC-associated gene signatures and 283 etiology-associated signatures; this involved a comparison of low-risk and high-risk CLD groups using an Incyte human cDNA microarray ( 26 ).

Techniques: Microarray, Mouse Assay, Marker, Binding Assay, Translocation Assay

Journal: Gene Expression

Article Title: Global Gene Expression Profiling of Dimethylnitrosamine-Induced Liver Fibrosis: From Pathological and Biochemical Data to Microarray Analysis

doi:

Figure Lengend Snippet: THE GENE EXPRESSION PATTERNS OF FIBROSIS MARKERS BY QUANTITATIVE REAL-TIME PCR AND MICROARRAY DATA

Article Snippet: Secondly, Kim and his colleagues identified 556 chronic liver disease (CLD)-related genes, which included 273 HCC-associated gene signatures and 283 etiology-associated signatures; this involved a comparison of low-risk and high-risk CLD groups using an Incyte human cDNA microarray ( 26 ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Microarray, Reverse Transcription Polymerase Chain Reaction