Journal: Epigenetics & Chromatin

Article Title: The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner

doi: 10.1186/1756-8935-5-8

Figure Lengend Snippet: Regulation of testis-specific genes by CTCFL. A Heatmap representation of microarray data. We compared five samples from heterozygous and three samples from homozygous Ctcfl knockout mice. Depicted are the top 27 deregulated genes, where the log2-transformed fold change compared to the average expression in heterozygous testis is shown. B Expression analyses in Ctcfl mutant testes. Real-time RTPCR expression analyses on testis RNA from Ctcfl mutant mice relative to wild type using Ccna1 as reference. Genes were examined in 90-day-old testes. C Reduced PRSS50 expression in Ctcfl mutant testis. Immunofluorescence analysis of testis sections from heterozygous (del/+) and homozygous (del/del) Ctcfl knockout mice, using antibodies against PRSS50.

Article Snippet: Labeled cRNA was hybridized to the GeneChip Mouse Genome 430 2.0 array oligonucleotide microarray (Affymetrix) according to manufacturer’s recommendations; 300 ng ES cell RNA was used for production of end-labeled biotinylated ssDNA.

Techniques: Microarray, Knock-Out, Transformation Assay, Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence

Journal: Epigenetics & Chromatin

Article Title: The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner

doi: 10.1186/1756-8935-5-8

Figure Lengend Snippet: Genome-wide analysis of CTCFL expression in ES cells. A Inducible expression of CTCFL-V5-GFP in ES cells. Notice the nuclear localization of CTCFL-V5-GFP in cells expressing the protein. B Flow chart of experiments. ES cells with a Tet-on inducible expression of a CTCFLV5-GFP transgene were sorted for GFP and used for microarray and ChIP-Seq analyses. C CTCFL expression and DNA binding are associated with elevated gene expression levels. We plotted gene expression levels, as determined by microarray analysis of induced ( ind ) or non-induced ES cells, for all genes ( all ), or those bound by CTCF, or CTCFL, to the respective promoter region (−2 k to +1 kb around TSS). Differences are highly significant ( p -value CTCF-ind versus CTCFL-ind: 5.1 × e -14 ; p -value CTCF versus CTCFL: 5.9 × e -13 ). D Transcript analyses in ES cells expressing CTCFL-V5-GFP. Real-time RT-PCR expression analyses of CTCFL-V5-GFP-induced and GFP-sorted ES cells, relative to non-induced ES cells, for the indicated genes, referenced to Cdk2 expression. E Venn diagram of DNA-binding sites for CTCFL and CTCF. F Clustered heatmap representation of three classes of CTCF/CTCFL-binding sites. Shown are the binding profiles of CTCFL and CTCF ( 1 : our own data; 2 : ) across all CTCF/CTCFL-binding sites identified in mES cells. Sites are grouped into CTCFL-only, CTCF-only, and combined CTCFL and CTCF sites. Within the three classes, data sets were sorted decreasingly from top to bottom for average binding across the interval from 2 kb to +2 kb around the identified binding peak center positions. Additionally the occurrences of predicted CTCFL motifs within these intervals are plotted. G Motif comparison of CTCF and CTCFL. DNA-binding motif for CTCFL-only ( top panel ), CTCF + CTCFL ( middle panel ) and CTCF-only binding sites ( bottom panel ).

Article Snippet: Labeled cRNA was hybridized to the GeneChip Mouse Genome 430 2.0 array oligonucleotide microarray (Affymetrix) according to manufacturer’s recommendations; 300 ng ES cell RNA was used for production of end-labeled biotinylated ssDNA.

Techniques: Genome Wide, Expressing, Microarray, ChIP-sequencing, Binding Assay, Quantitative RT-PCR

Journal: Genome Biology

Article Title: Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

doi: 10.1186/gb-2005-6-7-r61

Figure Lengend Snippet: Relating yeast spike-in RNA control copy number to qPCR measurements and microarray signal intensity. (a) To verify abundances of yeast sequence RNA transcripts in a control mixture, cDNA was transcribed from the control mixture alone (open boxes), as well as E12.5 whole-mouse embryo total RNA (open diamonds) and Universal Mouse RNA (filled triangles) with added spike-in control mixture. The cDNA was used as template for real-time PCR quantitation of each yeast sequence RNA, using a separately prepared standard of cDNA transcribed from the yeast sequences. Expected and measured copy numbers are closely matched ( r 2 ≥ 0.99), with maximum measured/observed ratios of 1.5, 1.5, and 2.6, respectively. (b) Expression profiles were generated for triplicate total RNA samples from E12.5 embryo (filled circles), E12.5 placenta (open circles), ES cells (filled boxes), and TS cells (open boxes) with yeast sequence control transcripts spiked-in prior to target labeling. For the seven control transcripts, mean log 10 [intensity] is shown for each tissue type, as well as the mean across all samples (filled triangles), and these data were used to perform linear regression analysis and relate signal intensity to transcript copy number, allowing abundance estimation for endogenous transcripts. The regression line for the average of all tissues (dashed line) and its equation is shown. Intensity-copy number correlations for individual tissues were very strong, with r 2 values of 0.98 - 0.99.

Article Snippet: In designing a mouse whole-genome microarray, we began by examining existing designs - the NIA Mouse 22K Microarray v1.1 (Development 60-mer Oligo) [ ], which became commercially available from Agilent as the Agilent Mouse (Development) Oligonucleotide Microarray (see Additional data files 1 and 2), and the National Institute of Environmental Health Sciences (NIEHS) Toxicogenomics Consortium mouse array (Agilent Mouse Microarray).

Techniques: Microarray, Sequencing, Real-time Polymerase Chain Reaction, Quantitation Assay, Expressing, Generated, Labeling

Journal: BMC Genomics

Article Title: Altered expression patterns of lipid metabolism genes in an animal model of HCV core-related, nonobese, modest hepatic steatosis

doi: 10.1186/1471-2164-9-109

Figure Lengend Snippet: Gene functional analysis for selected genes from microarray analyses

Article Snippet: Correspondingly, fragmented labeled cRNA was then pooled and hybridized to a mouse oligonucleotide microarray containing 20,871 unique mouse genes (Agilent Technologies) at 60°C for 17 h. After washing and drying by nitrogen gun blowing, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3 and 625 nm for Cy5.

Techniques: Functional Assay, Microarray, Activity Assay