Structured Review

Qiagen library preparation rna
Comparison between RT-qPCR and <t>RNA-seq</t> . Six genes were selected for validation experiments using a subset of animals. Each panel provides the <t>mPFC</t> expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p
Library Preparation Rna, supplied by Qiagen, used in various techniques. Bioz Stars score: 90/100, based on 6941 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/library preparation rna/product/Qiagen
Average 90 stars, based on 6941 article reviews
Price from $9.99 to $1999.99
library preparation rna - by Bioz Stars, 2020-05
90/100 stars

Images

1) Product Images from "Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex"

Article Title: Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2016.00113

Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p
Figure Legend Snippet: Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p

Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing, Two Tailed Test

Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.
Figure Legend Snippet: Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.

Techniques Used: RNA Sequencing Assay

2) Product Images from "Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke"

Article Title: Wnt canonical pathway activator TWS119 drives microglial anti-inflammatory activation and facilitates neurological recovery following experimental stroke

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-019-1660-8

Experimental outline and schematic diagram of brain section. a Experimental outline: TWS119 or BrdU were administrated intraperitoneally once daily from day 1 to 14 or from day 7 to 14 after stroke. Neurobehavioral tests were performed at days 1, 7, 14, and 21 after stroke. Angiogenesis, neural plasticity, microglial polarization and inflammatory cytokines were detected at the indicated time point. Histological assessment was assessed at day 21. The number of mice in each group for each test were shown in parentheses. b Schematic diagram of brain section. Green squares indicated the region of interest in the ipsilateral peri-infarct cortex, in which immunofluorescence images were collected. Yellow strip (0.5 mm wide) indicated peri-infarct region, in which brain samples for qRT-PCR and ELISA were harvested. stroke, focal cerebral ischemia; IF, immunofluorescence; qRT-PCR, quantitative real time polymerase chain reaction; ELISA, enzyme linked immunosorbent assay
Figure Legend Snippet: Experimental outline and schematic diagram of brain section. a Experimental outline: TWS119 or BrdU were administrated intraperitoneally once daily from day 1 to 14 or from day 7 to 14 after stroke. Neurobehavioral tests were performed at days 1, 7, 14, and 21 after stroke. Angiogenesis, neural plasticity, microglial polarization and inflammatory cytokines were detected at the indicated time point. Histological assessment was assessed at day 21. The number of mice in each group for each test were shown in parentheses. b Schematic diagram of brain section. Green squares indicated the region of interest in the ipsilateral peri-infarct cortex, in which immunofluorescence images were collected. Yellow strip (0.5 mm wide) indicated peri-infarct region, in which brain samples for qRT-PCR and ELISA were harvested. stroke, focal cerebral ischemia; IF, immunofluorescence; qRT-PCR, quantitative real time polymerase chain reaction; ELISA, enzyme linked immunosorbent assay

Techniques Used: Mouse Assay, Immunofluorescence, Stripping Membranes, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

3) Product Images from "Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury"

Article Title: Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065034

Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool. A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells ( A ) or Schwann cells and dorsal root ganglion cells ( B ). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.
Figure Legend Snippet: Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool. A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells ( A ) or Schwann cells and dorsal root ganglion cells ( B ). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.

Techniques Used: Expressing, Over Expression

Main metabolic pathways associated to in vitro calcitriol supplementation. A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes ( Prx, Tspan2, IgF1, Spp1 ) involved in axogenesis and myelination.
Figure Legend Snippet: Main metabolic pathways associated to in vitro calcitriol supplementation. A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes ( Prx, Tspan2, IgF1, Spp1 ) involved in axogenesis and myelination.

Techniques Used: In Vitro, Functional Assay, Real-time Polymerase Chain Reaction

4) Product Images from "Riluzole reduces amyloid beta pathology, improves memory, and restores gene expression changes in a transgenic mouse model of early-onset Alzheimer’s disease"

Article Title: Riluzole reduces amyloid beta pathology, improves memory, and restores gene expression changes in a transgenic mouse model of early-onset Alzheimer’s disease

Journal: Translational Psychiatry

doi: 10.1038/s41398-018-0201-z

Treatment of 5XFAD mice with riluzole rescues gene expression changes in the hippocampus. a Venn diagram illustrating the overlap of 247 genes that were changed in 5XFAD compared to wild type and in 5XFAD mice treated with riluzole compare to untreated mice. b Scatter plot illustrating the 247 overlapping genes showing fold change by 5XFAD ( x -axis) against fold change with riluzole treatment on 5XFAD mice ( y -axis). 86% of overlapping 5XFAD genes is reversed by riluzole treatment. The upper left quadrant represents 26 genes that had decreased expression with 5XFAD and increased expression after riluzole treatment. Conversely, the lower-right quadrant illustrates 189 genes that were increased with 5XFAD and decreased by riluzole. c The mRNA expression of Mouse APP from RNA-Seq data revealed no significant difference among WT, 5XFAD, and 5XFAD-Riluzole groups. d Histograms illustrating significantly enriched pathways based on genes differentially expressed by overlapping gene. (enrichment score > 1.3). e , f Histograms illustrating significantly enriched pathways with the highest sum enrichment scores across opposite conditions (Increased in 5XFAD and Decreased with riluzole treatment or vice-versa) based on differentially expressed genes (enrichment score > 1.3). Similar pathways and enrichment scores were observed when comparing genes decreased by 5XFAD and increased by riluzole, as well as for genes increased with age and decreased by riluzole. The RNA-Seq data is based on hippocampal tissue pooled into three replicate sequencing libraries/group from WT, n = 10 (pooled 3, 3, 4 mice); 5XFAD, n = 7 (pooled 2, 2, 3 mice); and 5XFAD-Riluzole, n = 8 (pooled 2, 3, 3 mice)
Figure Legend Snippet: Treatment of 5XFAD mice with riluzole rescues gene expression changes in the hippocampus. a Venn diagram illustrating the overlap of 247 genes that were changed in 5XFAD compared to wild type and in 5XFAD mice treated with riluzole compare to untreated mice. b Scatter plot illustrating the 247 overlapping genes showing fold change by 5XFAD ( x -axis) against fold change with riluzole treatment on 5XFAD mice ( y -axis). 86% of overlapping 5XFAD genes is reversed by riluzole treatment. The upper left quadrant represents 26 genes that had decreased expression with 5XFAD and increased expression after riluzole treatment. Conversely, the lower-right quadrant illustrates 189 genes that were increased with 5XFAD and decreased by riluzole. c The mRNA expression of Mouse APP from RNA-Seq data revealed no significant difference among WT, 5XFAD, and 5XFAD-Riluzole groups. d Histograms illustrating significantly enriched pathways based on genes differentially expressed by overlapping gene. (enrichment score > 1.3). e , f Histograms illustrating significantly enriched pathways with the highest sum enrichment scores across opposite conditions (Increased in 5XFAD and Decreased with riluzole treatment or vice-versa) based on differentially expressed genes (enrichment score > 1.3). Similar pathways and enrichment scores were observed when comparing genes decreased by 5XFAD and increased by riluzole, as well as for genes increased with age and decreased by riluzole. The RNA-Seq data is based on hippocampal tissue pooled into three replicate sequencing libraries/group from WT, n = 10 (pooled 3, 3, 4 mice); 5XFAD, n = 7 (pooled 2, 2, 3 mice); and 5XFAD-Riluzole, n = 8 (pooled 2, 3, 3 mice)

Techniques Used: Mouse Assay, Expressing, RNA Sequencing Assay, Sequencing

5) Product Images from "ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity, molecularly defining a human dys-metabolic obese phenotype"

Article Title: ASK1 (MAP3K5) is transcriptionally upregulated by E2F1 in adipose tissue in obesity, molecularly defining a human dys-metabolic obese phenotype

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2017.05.003

Effect of BMI on E2F1 binding to the ASK1 promoter and associations between Om- ASK1 expression levels and different clinical characteristics. (A) Formaldehyde cross-linked chromatin from 16 paired human (Om and Sc) ATs was subjected to ChIP experiments. Immunoprecipitation of E2F1 containing complexes was performed using anti-E2F1 antibody. Anti-POLR2 (polymerase RNA II), was used as positive control and anti-ICAM1 (intercellular adhesion molecule 1) was used as negative control. After isolation of bound DNA, end-point PCR and quantitative real time PCR were performed for a 300 bp region of the endogenous human ASK1 promoter. Quantitative real time PCR results were analyzed using linear regression. Inputs indicate PCR performed on DNA (diluted 1:300) without any immunoprecipitation. (B) Multi variate models to assess associations between Om- ASK1 mRNA levels and parameters shown in Figure 1 as continuous variables. Values are the β coefficient of association, with model 1 adjusted for age and sex, model 2 for age, sex, and BMI, and model 3 adjusted for age, sex, BMI, and Om- E2F1 mRNA expression. * Associations with p values
Figure Legend Snippet: Effect of BMI on E2F1 binding to the ASK1 promoter and associations between Om- ASK1 expression levels and different clinical characteristics. (A) Formaldehyde cross-linked chromatin from 16 paired human (Om and Sc) ATs was subjected to ChIP experiments. Immunoprecipitation of E2F1 containing complexes was performed using anti-E2F1 antibody. Anti-POLR2 (polymerase RNA II), was used as positive control and anti-ICAM1 (intercellular adhesion molecule 1) was used as negative control. After isolation of bound DNA, end-point PCR and quantitative real time PCR were performed for a 300 bp region of the endogenous human ASK1 promoter. Quantitative real time PCR results were analyzed using linear regression. Inputs indicate PCR performed on DNA (diluted 1:300) without any immunoprecipitation. (B) Multi variate models to assess associations between Om- ASK1 mRNA levels and parameters shown in Figure 1 as continuous variables. Values are the β coefficient of association, with model 1 adjusted for age and sex, model 2 for age, sex, and BMI, and model 3 adjusted for age, sex, BMI, and Om- E2F1 mRNA expression. * Associations with p values

Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, Immunoprecipitation, Positive Control, Negative Control, Isolation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

6) Product Images from "Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish"

Article Title: Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish

Journal: Scientific Reports

doi: 10.1038/s41598-019-56466-z

A zebrafish model of Farber disease. ( a ) Schematic of asah1a/b −/− zebrafish generation and propagation. ( b ) DNA fragment analysis demonstrating presence of altered alleles. The 30-bp difference in the asah1b spectra (rather than the noted 20-bp deletion) is due to presence of different polymorphisms across the introns of the WT and KO alleles (Fig. S2b , see Fig. S3 for sequencing of full cDNA). ( c ) asah1a/b −/− (DKO) and asah1b −/− (SKO) zebrafish at 3.5 months. ( d ) Length of DKO and SKO zebrafish at 3.5–4 months. Zebrafish length measurements: Student t-test, SEM, combined data from n = 13 SKO and n = 12 DKO fish, fish within each data set (n = 4–5 SKO, n = 4 DKO) are age-matched. ***p
Figure Legend Snippet: A zebrafish model of Farber disease. ( a ) Schematic of asah1a/b −/− zebrafish generation and propagation. ( b ) DNA fragment analysis demonstrating presence of altered alleles. The 30-bp difference in the asah1b spectra (rather than the noted 20-bp deletion) is due to presence of different polymorphisms across the introns of the WT and KO alleles (Fig. S2b , see Fig. S3 for sequencing of full cDNA). ( c ) asah1a/b −/− (DKO) and asah1b −/− (SKO) zebrafish at 3.5 months. ( d ) Length of DKO and SKO zebrafish at 3.5–4 months. Zebrafish length measurements: Student t-test, SEM, combined data from n = 13 SKO and n = 12 DKO fish, fish within each data set (n = 4–5 SKO, n = 4 DKO) are age-matched. ***p

Techniques Used: Sequencing, Fluorescence In Situ Hybridization

7) Product Images from "The Guinea Pig as a Model for Sporadic Alzheimer's Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes"

Article Title: The Guinea Pig as a Model for Sporadic Alzheimer's Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066235

Increased BACE1 RNA and reduced ADAM10 RNA expression levels in brain tissue from guinea pigs fed a high cholesterol diet. Quantitative PCR analysis analysis for (A) ADAM10 and (B) BACE1 expression on total RNA extracted from the frontal cortex and cerebellum of guinea pigs fed the control or cholesterol diets. Data is represented as relative expression to RPS16. Compared to animals fed the control diet, ADAM10 expression is significantly decreased in the frontal cortex ( p
Figure Legend Snippet: Increased BACE1 RNA and reduced ADAM10 RNA expression levels in brain tissue from guinea pigs fed a high cholesterol diet. Quantitative PCR analysis analysis for (A) ADAM10 and (B) BACE1 expression on total RNA extracted from the frontal cortex and cerebellum of guinea pigs fed the control or cholesterol diets. Data is represented as relative expression to RPS16. Compared to animals fed the control diet, ADAM10 expression is significantly decreased in the frontal cortex ( p

Techniques Used: RNA Expression, Real-time Polymerase Chain Reaction, Expressing

Formation of the PS2V Transcript. A) Presenilin structure in lipid bilayers: Arrowhead indicates boundary between protein sequences derived from exon 4 and 5. Dashed line indicates sequence from exon 5. Arrow indicates endoproteolysis site. Filled circle indicates γ-secretase catalytic site. B) PS2V forms when HMGA1a is expressed and binds to exon 5 (lighter shading) of PSEN2 RNA causing ligation of exon 4 to exon 6 and ORF termination. C) Nucleotide sequence alignment of the 3′ end of exon 5 in human PSEN2 RNA (with corresponding encoded residues) and the cognate exon of other species. Red boxes enclose sequences aligned with the HMGA1a-binding sites in human PSEN2 RNA. D) mRNA from guinea brains exposed to control media or to media containing NaN 3 followed by RT-PCR analysis using primers amplifying cDNA spanning exons 3 to 7 of Psen2 . In untreated samples a prominent ∼420 bp band is observed. In NaN 3 treated samples an additional ∼350 bp band is evident representing the cDNA fragment predicted from exclusion of the exon 5 sequence (PS2V). E) qPCR using a primer spanning the exon 4/6 junction PS2V cDNA showed up-regulation of PS2V mRNA in samples treated with NaN 3 .
Figure Legend Snippet: Formation of the PS2V Transcript. A) Presenilin structure in lipid bilayers: Arrowhead indicates boundary between protein sequences derived from exon 4 and 5. Dashed line indicates sequence from exon 5. Arrow indicates endoproteolysis site. Filled circle indicates γ-secretase catalytic site. B) PS2V forms when HMGA1a is expressed and binds to exon 5 (lighter shading) of PSEN2 RNA causing ligation of exon 4 to exon 6 and ORF termination. C) Nucleotide sequence alignment of the 3′ end of exon 5 in human PSEN2 RNA (with corresponding encoded residues) and the cognate exon of other species. Red boxes enclose sequences aligned with the HMGA1a-binding sites in human PSEN2 RNA. D) mRNA from guinea brains exposed to control media or to media containing NaN 3 followed by RT-PCR analysis using primers amplifying cDNA spanning exons 3 to 7 of Psen2 . In untreated samples a prominent ∼420 bp band is observed. In NaN 3 treated samples an additional ∼350 bp band is evident representing the cDNA fragment predicted from exclusion of the exon 5 sequence (PS2V). E) qPCR using a primer spanning the exon 4/6 junction PS2V cDNA showed up-regulation of PS2V mRNA in samples treated with NaN 3 .

Techniques Used: Derivative Assay, Sequencing, Ligation, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

PS2V is up-regulated under cholesterol-fed conditions. Quantitative PCR analysis shows that, in comparison to animals fed a control diet, guinea pigs fed a cholesterol rich diet showed a significant increase in PSEN2 and PS2V transcripts in ( A ) frontal cortex ( p = 0.004; t = 3.429, d.f. = 14 and p
Figure Legend Snippet: PS2V is up-regulated under cholesterol-fed conditions. Quantitative PCR analysis shows that, in comparison to animals fed a control diet, guinea pigs fed a cholesterol rich diet showed a significant increase in PSEN2 and PS2V transcripts in ( A ) frontal cortex ( p = 0.004; t = 3.429, d.f. = 14 and p

Techniques Used: Real-time Polymerase Chain Reaction

8) Product Images from "The Guinea Pig as a Model for Sporadic Alzheimer's Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes"

Article Title: The Guinea Pig as a Model for Sporadic Alzheimer's Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066235

Formation of the PS2V Transcript. A) Presenilin structure in lipid bilayers: Arrowhead indicates boundary between protein sequences derived from exon 4 and 5. Dashed line indicates sequence from exon 5. Arrow indicates endoproteolysis site. Filled circle indicates γ-secretase catalytic site. B) PS2V forms when HMGA1a is expressed and binds to exon 5 (lighter shading) of PSEN2 RNA causing ligation of exon 4 to exon 6 and ORF termination. C) Nucleotide sequence alignment of the 3′ end of exon 5 in human PSEN2 RNA (with corresponding encoded residues) and the cognate exon of other species. Red boxes enclose sequences aligned with the HMGA1a-binding sites in human PSEN2 RNA. D) mRNA from guinea brains exposed to control media or to media containing NaN 3 followed by RT-PCR analysis using primers amplifying cDNA spanning exons 3 to 7 of Psen2 . In untreated samples a prominent ∼420 bp band is observed. In NaN 3 treated samples an additional ∼350 bp band is evident representing the cDNA fragment predicted from exclusion of the exon 5 sequence (PS2V). E) qPCR using a primer spanning the exon 4/6 junction PS2V cDNA showed up-regulation of PS2V mRNA in samples treated with NaN 3 .
Figure Legend Snippet: Formation of the PS2V Transcript. A) Presenilin structure in lipid bilayers: Arrowhead indicates boundary between protein sequences derived from exon 4 and 5. Dashed line indicates sequence from exon 5. Arrow indicates endoproteolysis site. Filled circle indicates γ-secretase catalytic site. B) PS2V forms when HMGA1a is expressed and binds to exon 5 (lighter shading) of PSEN2 RNA causing ligation of exon 4 to exon 6 and ORF termination. C) Nucleotide sequence alignment of the 3′ end of exon 5 in human PSEN2 RNA (with corresponding encoded residues) and the cognate exon of other species. Red boxes enclose sequences aligned with the HMGA1a-binding sites in human PSEN2 RNA. D) mRNA from guinea brains exposed to control media or to media containing NaN 3 followed by RT-PCR analysis using primers amplifying cDNA spanning exons 3 to 7 of Psen2 . In untreated samples a prominent ∼420 bp band is observed. In NaN 3 treated samples an additional ∼350 bp band is evident representing the cDNA fragment predicted from exclusion of the exon 5 sequence (PS2V). E) qPCR using a primer spanning the exon 4/6 junction PS2V cDNA showed up-regulation of PS2V mRNA in samples treated with NaN 3 .

Techniques Used: Derivative Assay, Sequencing, Ligation, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

9) Product Images from "Pharmacological targeting of the ?-amyloid precursor protein intracellular domain"

Article Title: Pharmacological targeting of the ?-amyloid precursor protein intracellular domain

Journal: Scientific Reports

doi: 10.1038/srep04618

Effect of in vivo CHF5074 treatment on AICD occupancy, histone H3 acetylation and transcriptional activity of the KAI1 gene. (a) Chromatin immunoprecipitation assays performed on brain cortices from Tg2576 mice treated for 4 weeks with standard ( black bars ) or CHF5074-supplemented diet (375 ppm; ≈60 mg/kg/day; white bars ) using anti-APP C-terminus and anti-acetyl H3 as test antibodies and an anti-IgG as a negative control antibody. DNA extracted from immunoprecipitated chromatin was analyzed by qRT-PCR, setting C T values relative to untreated samples to 1.0 (see ‘Methods' for details). (b) KAI1 mRNA levels determined by qRT-PCR analysis of reverse-transcribed total RNA extracted from brain cortices of Tg2576 mice fed on standard ( black bars ) and CHF5074-supplemented ( white bars ) diet as above. Data are presented as relative values determined with the comparative C T method using β-actin as house-keeping gene reference; bars are the mean ± s.e.m. of three independent experiments (**, p
Figure Legend Snippet: Effect of in vivo CHF5074 treatment on AICD occupancy, histone H3 acetylation and transcriptional activity of the KAI1 gene. (a) Chromatin immunoprecipitation assays performed on brain cortices from Tg2576 mice treated for 4 weeks with standard ( black bars ) or CHF5074-supplemented diet (375 ppm; ≈60 mg/kg/day; white bars ) using anti-APP C-terminus and anti-acetyl H3 as test antibodies and an anti-IgG as a negative control antibody. DNA extracted from immunoprecipitated chromatin was analyzed by qRT-PCR, setting C T values relative to untreated samples to 1.0 (see ‘Methods' for details). (b) KAI1 mRNA levels determined by qRT-PCR analysis of reverse-transcribed total RNA extracted from brain cortices of Tg2576 mice fed on standard ( black bars ) and CHF5074-supplemented ( white bars ) diet as above. Data are presented as relative values determined with the comparative C T method using β-actin as house-keeping gene reference; bars are the mean ± s.e.m. of three independent experiments (**, p

Techniques Used: In Vivo, Activity Assay, Chromatin Immunoprecipitation, Mouse Assay, Negative Control, Immunoprecipitation, Quantitative RT-PCR

10) Product Images from "Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish"

Article Title: Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish

Journal: Scientific Reports

doi: 10.1038/s41598-019-56466-z

A zebrafish model of Farber disease. ( a ) Schematic of asah1a/b −/− zebrafish generation and propagation. ( b ) DNA fragment analysis demonstrating presence of altered alleles. The 30-bp difference in the asah1b spectra (rather than the noted 20-bp deletion) is due to presence of different polymorphisms across the introns of the WT and KO alleles (Fig. S2b , see Fig. S3 for sequencing of full cDNA). ( c ) asah1a/b −/− (DKO) and asah1b −/− (SKO) zebrafish at 3.5 months. ( d ) Length of DKO and SKO zebrafish at 3.5–4 months. Zebrafish length measurements: Student t-test, SEM, combined data from n = 13 SKO and n = 12 DKO fish, fish within each data set (n = 4–5 SKO, n = 4 DKO) are age-matched. ***p
Figure Legend Snippet: A zebrafish model of Farber disease. ( a ) Schematic of asah1a/b −/− zebrafish generation and propagation. ( b ) DNA fragment analysis demonstrating presence of altered alleles. The 30-bp difference in the asah1b spectra (rather than the noted 20-bp deletion) is due to presence of different polymorphisms across the introns of the WT and KO alleles (Fig. S2b , see Fig. S3 for sequencing of full cDNA). ( c ) asah1a/b −/− (DKO) and asah1b −/− (SKO) zebrafish at 3.5 months. ( d ) Length of DKO and SKO zebrafish at 3.5–4 months. Zebrafish length measurements: Student t-test, SEM, combined data from n = 13 SKO and n = 12 DKO fish, fish within each data set (n = 4–5 SKO, n = 4 DKO) are age-matched. ***p

Techniques Used: Sequencing, Fluorescence In Situ Hybridization

11) Product Images from "Bone marrow-derived and peritoneal macrophages have different inflammatory response to oxLDL and M1/M2 marker expression – implications for atherosclerosis research"

Article Title: Bone marrow-derived and peritoneal macrophages have different inflammatory response to oxLDL and M1/M2 marker expression – implications for atherosclerosis research

Journal: Scientific Reports

doi: 10.1038/srep35234

M1/M2 marker expression in BMDMs and PEMs. ( a–f ) Flow cytometry of BMDMs and PEMs after incubation with 0 μg/mL oxLDL (macrophage, Mø) or 25 μg/mL oxLDL (foam cells, Fc) for 24 hours (n = 3 mice for BMDMs and 3 pools of 4–5 mice for PEMs). For both single positive (CD68 − F4/80 + ) and double positive (CD68 + F4/80 + ) cells, the percentage of CD45 + cells ( a,d ), and MFI for CD206 ( b,e ) and CD11c ( c,f ) expression were detected. MFI: Mean Fluorescent Intensity. *p
Figure Legend Snippet: M1/M2 marker expression in BMDMs and PEMs. ( a–f ) Flow cytometry of BMDMs and PEMs after incubation with 0 μg/mL oxLDL (macrophage, Mø) or 25 μg/mL oxLDL (foam cells, Fc) for 24 hours (n = 3 mice for BMDMs and 3 pools of 4–5 mice for PEMs). For both single positive (CD68 − F4/80 + ) and double positive (CD68 + F4/80 + ) cells, the percentage of CD45 + cells ( a,d ), and MFI for CD206 ( b,e ) and CD11c ( c,f ) expression were detected. MFI: Mean Fluorescent Intensity. *p

Techniques Used: Marker, Expressing, Flow Cytometry, Cytometry, Incubation, Mouse Assay

BMDMs and PEMs have a different inflammatory response to oxLDL stimulation. ( a–d ) Cholesterol or MCP-1 content (% of control (0 μg/mL oxLDL)) in BMDMs ( a,c ) and PEMs ( b,d ) after stimulation with 0–50 μg/mL oxLDL for 24 hours (each graph represents 3 separate experiments (n = 3–4 wells per experiment)). *p
Figure Legend Snippet: BMDMs and PEMs have a different inflammatory response to oxLDL stimulation. ( a–d ) Cholesterol or MCP-1 content (% of control (0 μg/mL oxLDL)) in BMDMs ( a,c ) and PEMs ( b,d ) after stimulation with 0–50 μg/mL oxLDL for 24 hours (each graph represents 3 separate experiments (n = 3–4 wells per experiment)). *p

Techniques Used:

No clear M1/M2 gene expression profile in BMDMs and PEMs. ( a–c ) mRNA levels (2 −ΔCT ) of M2 markers ( a ), M1 markers ( b ), or genes related to lipid metabolism ( c ) in PEMs depicted as a function of mRNA levels (2 −ΔCT ) in BMDMs (n = 3 mice for BMDMs and 3 pools of 4–5 mice for PEMs). Each grey circle represents expression of a given gene in macrophages (Mø, 0 μg/mL oxLDL 24 h), whereas each square represents expression of a given gene in foam cells (Fc, 25 μg/mL oxLDL 24 h) as measured by PCR array analyses. The full line indicate genes, with similar expression levels in both cell types, while data points placed above or below the dotted lines are more than 2 fold up- or down-regulated, respectively, in PEMs as compared to BMDMS. The specific genes of interest, their fold change, and statistics can be found in Table 1 .
Figure Legend Snippet: No clear M1/M2 gene expression profile in BMDMs and PEMs. ( a–c ) mRNA levels (2 −ΔCT ) of M2 markers ( a ), M1 markers ( b ), or genes related to lipid metabolism ( c ) in PEMs depicted as a function of mRNA levels (2 −ΔCT ) in BMDMs (n = 3 mice for BMDMs and 3 pools of 4–5 mice for PEMs). Each grey circle represents expression of a given gene in macrophages (Mø, 0 μg/mL oxLDL 24 h), whereas each square represents expression of a given gene in foam cells (Fc, 25 μg/mL oxLDL 24 h) as measured by PCR array analyses. The full line indicate genes, with similar expression levels in both cell types, while data points placed above or below the dotted lines are more than 2 fold up- or down-regulated, respectively, in PEMs as compared to BMDMS. The specific genes of interest, their fold change, and statistics can be found in Table 1 .

Techniques Used: Expressing, Mouse Assay, Polymerase Chain Reaction

12) Product Images from "Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines"

Article Title: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

Journal: BMC Research Notes

doi: 10.1186/s13104-017-2959-0

Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p
Figure Legend Snippet: Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Synthesized, Expressing, Construct, Activated Clotting Time Assay

Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method
Figure Legend Snippet: Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method

Techniques Used: Isolation, Purification

13) Product Images from "Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions"

Article Title: Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky929

The presence of pathological prefibrillar oligomerized forms of Tau protein repressed the expression of Tau-interacting genes. Relative gene expression was analyzed by RT-qPCR with respect to three reference genes ( Ppib, Rplp0, Hmbs ). Total RNA was collected from the CA1 region of 6 month old mice either wild-type (WT) or THY-Tau22 expressing Tau oligomers, not submitted (CTRL) or after being submitted to HS (HS). ( A ) Representative images of sagital CA1 sections from 6-month-old WT and THY-Tau22 mice under CTRL and HS conditions labeled with the TOC1 antibody directed against prefibrillar Tau oligomers ( 32 , 33 ) and analyzed by laser scanning confocal microscopy. The nuclei were identified using DAPI staining. The scale bars represent 20 μm. ( B ) The labeling intensity of Tau-oligomers was quantified within the nuclei (based on DAPI detection) of CA1 cells. The data shown are the means ± s.d of 60 to 160 nuclei. *** P -values
Figure Legend Snippet: The presence of pathological prefibrillar oligomerized forms of Tau protein repressed the expression of Tau-interacting genes. Relative gene expression was analyzed by RT-qPCR with respect to three reference genes ( Ppib, Rplp0, Hmbs ). Total RNA was collected from the CA1 region of 6 month old mice either wild-type (WT) or THY-Tau22 expressing Tau oligomers, not submitted (CTRL) or after being submitted to HS (HS). ( A ) Representative images of sagital CA1 sections from 6-month-old WT and THY-Tau22 mice under CTRL and HS conditions labeled with the TOC1 antibody directed against prefibrillar Tau oligomers ( 32 , 33 ) and analyzed by laser scanning confocal microscopy. The nuclei were identified using DAPI staining. The scale bars represent 20 μm. ( B ) The labeling intensity of Tau-oligomers was quantified within the nuclei (based on DAPI detection) of CA1 cells. The data shown are the means ± s.d of 60 to 160 nuclei. *** P -values

Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Labeling, Confocal Microscopy, Staining

14) Product Images from "GDNF is not required for catecholaminergic neuron survival in vivo"

Article Title: GDNF is not required for catecholaminergic neuron survival in vivo

Journal: Nature neuroscience

doi: 10.1038/nn.3941

Dopamine system analysis after striatal Gdnf reduction by AAV5- Cre injection in adult mice. ( a ) Experimental timeline for analysis, time points for AAV5 encoding for synapsin promoter driven Cre injection (AAV5-Cre), RNA (R); protein (P); histology (H); striatal dopamine level measurements (DA) and behavior by corridor test (C) and after amphetamine (Amphet) or apomorphine (Apo) injections are indicated. ( b–c ) Relative levels of Gdnf mRNA determined by qPCR in the intact and AAV5 -Cre injected striata. ( d–f ) Measurements of rotations (Rot) after amphetamine (Amphet) and apomorphine (Apo) injection at the indicated time-points after AAV5 -Cre injection. ( g ) Corridor test at the indicated timepoints after AAV5 -Cre injection. ( h ) Unbiased stereological cell counts of substanti nigra tyrosine hydroxylase (TH) immunoreactive neurons 7 months (210 days) after Gdnf reduction. ( i ) Striatal tissue dopamine (DA) levels 7 months after Gdnf reduction. §-experiments were independently performed in Lund University, Sweden and University of Helsinki, Finland; § H -experiment performed in Helsinki; § L- experiment performed in Lund. F-floxed ; C-control containing only wt alleles; n = number of animals analyzed in each experiment; n.a. = not applicable. Error bars are mean ± s.e.m.
Figure Legend Snippet: Dopamine system analysis after striatal Gdnf reduction by AAV5- Cre injection in adult mice. ( a ) Experimental timeline for analysis, time points for AAV5 encoding for synapsin promoter driven Cre injection (AAV5-Cre), RNA (R); protein (P); histology (H); striatal dopamine level measurements (DA) and behavior by corridor test (C) and after amphetamine (Amphet) or apomorphine (Apo) injections are indicated. ( b–c ) Relative levels of Gdnf mRNA determined by qPCR in the intact and AAV5 -Cre injected striata. ( d–f ) Measurements of rotations (Rot) after amphetamine (Amphet) and apomorphine (Apo) injection at the indicated time-points after AAV5 -Cre injection. ( g ) Corridor test at the indicated timepoints after AAV5 -Cre injection. ( h ) Unbiased stereological cell counts of substanti nigra tyrosine hydroxylase (TH) immunoreactive neurons 7 months (210 days) after Gdnf reduction. ( i ) Striatal tissue dopamine (DA) levels 7 months after Gdnf reduction. §-experiments were independently performed in Lund University, Sweden and University of Helsinki, Finland; § H -experiment performed in Helsinki; § L- experiment performed in Lund. F-floxed ; C-control containing only wt alleles; n = number of animals analyzed in each experiment; n.a. = not applicable. Error bars are mean ± s.e.m.

Techniques Used: Injection, Mouse Assay, Real-time Polymerase Chain Reaction

15) Product Images from "Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain"

Article Title: Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain

Journal: American Journal of Human Genetics

doi: 10.1016/j.ajhg.2010.03.019

Real-Time qPCR Analysis of Tuba8 Expression Levels in Mouse and Human Brain (A) Relative expression levels of Tuba1a , Tubb2b , Tubb3 , and Tuba8 in the developing mouse brain (E10.5 [head], E12.5, E14.5, E16.5, E18.5, P0, and P6). Tuba8 is expressed at very low levels at all developmental stages. (B) In the adult mouse brain, there are regional expression differences when comparing Tuba1a , Tubb2b , Tubb3 , and Tuba8 . The expression of Tuba8 is again low, with the exception of the olfactory bulbs and cerebellum, where it is expressed at modest levels. (C) Expression of TUBA1A , TUBB2B , TUBB3 , and TUBA8 in the developing human brain at GW13 (frontal lobe) and GW22 (total fetal brain). Again, TUBA8 is expressed at low levels. Error bars indicate the SEM.
Figure Legend Snippet: Real-Time qPCR Analysis of Tuba8 Expression Levels in Mouse and Human Brain (A) Relative expression levels of Tuba1a , Tubb2b , Tubb3 , and Tuba8 in the developing mouse brain (E10.5 [head], E12.5, E14.5, E16.5, E18.5, P0, and P6). Tuba8 is expressed at very low levels at all developmental stages. (B) In the adult mouse brain, there are regional expression differences when comparing Tuba1a , Tubb2b , Tubb3 , and Tuba8 . The expression of Tuba8 is again low, with the exception of the olfactory bulbs and cerebellum, where it is expressed at modest levels. (C) Expression of TUBA1A , TUBB2B , TUBB3 , and TUBA8 in the developing human brain at GW13 (frontal lobe) and GW22 (total fetal brain). Again, TUBA8 is expressed at low levels. Error bars indicate the SEM.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

16) Product Images from "Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury"

Article Title: Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065034

Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool. A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells ( A ) or Schwann cells and dorsal root ganglion cells ( B ). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.
Figure Legend Snippet: Analysis of the main functions altered by vitamin D supplementation using the Ingenuity Pathway Analysis Tool. A. List of functions for the genes involved in “nervous system development and function” whose expression was altered after addition of calcitriol to Schwann cells ( A ) or Schwann cells and dorsal root ganglion cells ( B ). Red arrows indicate an over-expression; green arrows, an under-expression. C. Twenty-five nervous system-related genes were used to generate a network representation. The genes shaded red are upregulated and those that are green are downregulated. The intensity of the shading shows to what degree each gene was up- or downregulated. The genes in white colour were not significantly changed in the analysis and can be considered as “missing links”. Orange solid lines represent a known direct interaction between calcitriol and the genes present in the network.

Techniques Used: Expressing, Over Expression

Main metabolic pathways associated to in vitro calcitriol supplementation. A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes ( Prx, Tspan2, IgF1, Spp1 ) involved in axogenesis and myelination.
Figure Legend Snippet: Main metabolic pathways associated to in vitro calcitriol supplementation. A. Venn diagram showing the functional pathways affected by the addition of calcitriol in cultures of Schwann cells or in co-cultures of DRG/Schwann cells. Five of the fifteen metabolic calcitriol-regulated pathways are affected in both cell types. B. Validation by qPCR of four selected up-regulated genes ( Prx, Tspan2, IgF1, Spp1 ) involved in axogenesis and myelination.

Techniques Used: In Vitro, Functional Assay, Real-time Polymerase Chain Reaction

17) Product Images from "Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex"

Article Title: Transcription Profile of Aging and Cognition-Related Genes in the Medial Prefrontal Cortex

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2016.00113

Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p
Figure Legend Snippet: Comparison between RT-qPCR and RNA-seq . Six genes were selected for validation experiments using a subset of animals. Each panel provides the mPFC expression determined by RT-qPCR (left, ΔΔCT values) and RNA-seq (right, counts). Two-tailed t -tests confirmed increased expression of Arc, Fos, Egr1, Egr2, and Egr4 in AI, relative to AU rats. Gene expression for young animals is provided for comparison to aged animals. For two genes, Lin7b and Egr4 , age differences were confirmed ( *** p

Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay, Expressing, Two Tailed Test

Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.
Figure Legend Snippet: Region of the mPFC and white matter (WM) collected for RNA-seq . The right panel provides a schematic of a coronal slice +2.7 anterior to bregma diagram as adapted from Paxinos and Watson ( 1986 ) and illustrates the region of the mPFC and white matter collected for RNA-seq. The left panel shows a coronal slice from this same region.

Techniques Used: RNA Sequencing Assay

18) Product Images from "Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines"

Article Title: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

Journal: BMC Research Notes

doi: 10.1186/s13104-017-2959-0

Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p
Figure Legend Snippet: Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Synthesized, Expressing, Construct, Activated Clotting Time Assay

Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method
Figure Legend Snippet: Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method

Techniques Used: Isolation, Purification

19) Product Images from "KCNH2-3.1 expression impairs cognition and alters neuronal function in a model of molecular pathology associated with schizophrenia"

Article Title: KCNH2-3.1 expression impairs cognition and alters neuronal function in a model of molecular pathology associated with schizophrenia

Journal: Molecular psychiatry

doi: 10.1038/mp.2015.219

KCNH2-3.1 transgene expression (a) A schematic of the KCNH2 -3.1 transgene construct. The tetracycline transactivator (tTA) is under the control of the neuron-specific enolase (NSE) promoter. The KCNH2 -3.1 gene is under the control of the tetracycline-responsive promoter, TetOp. Administration of a tetracycline antibiotic turns off expression of the KCNH2 -3.1 gene. (b) qRT-PCR data for KCNH2 -3.1 mRNA taken from the prefrontal cortex. The four lanes on the left (control) are samples from mice that did not have both transgenes and thus were not expected to express KCNH2 -3.1 mRNA. These lanes confirm that there is no endogenous expression of KCNH2 -3.1 mRNA in mice and no “leaky” expression of KCNH2 -3.1 mRNA without the NSE-tTA promoter. The four lanes on the right ( KCNH2 -3.1) are samples from mice that had both transgenes and would be expected to express KCNH2 -3.1 mRNA. The first and third lanes show the expected bands corresponding to KCNH2 -3.1 mRNA. The second and fourth lanes in this group do not have this band because of the absence of reverse transcriptase (RT−) meaning there was no genomic DNA contamination in the RNA samples. (c) Relative expression of KCNH2 -3.1 mRNA in specific regions of the brain of KCNH2 -3.1 transgenic mice measured by qRT-PCR. N = 5. Values are normalized to expression levels in the cerebellum. (d) KCNH2 -3.1 expression in the PFC in mice treated with doxycycline. Doxycycline treatment significantly reduced the expression of KCNH2 -3.1 in KCNH2 -3.1 transgenic mice [ t (10) = 1.88, p = 0.04]. n = 5/non-treated control; n = 5/non-treated KCNH2 -3.1 transgenic; n = 3/treated control; n = 7/treated KCNH2 -3.1 transgenic. * p
Figure Legend Snippet: KCNH2-3.1 transgene expression (a) A schematic of the KCNH2 -3.1 transgene construct. The tetracycline transactivator (tTA) is under the control of the neuron-specific enolase (NSE) promoter. The KCNH2 -3.1 gene is under the control of the tetracycline-responsive promoter, TetOp. Administration of a tetracycline antibiotic turns off expression of the KCNH2 -3.1 gene. (b) qRT-PCR data for KCNH2 -3.1 mRNA taken from the prefrontal cortex. The four lanes on the left (control) are samples from mice that did not have both transgenes and thus were not expected to express KCNH2 -3.1 mRNA. These lanes confirm that there is no endogenous expression of KCNH2 -3.1 mRNA in mice and no “leaky” expression of KCNH2 -3.1 mRNA without the NSE-tTA promoter. The four lanes on the right ( KCNH2 -3.1) are samples from mice that had both transgenes and would be expected to express KCNH2 -3.1 mRNA. The first and third lanes show the expected bands corresponding to KCNH2 -3.1 mRNA. The second and fourth lanes in this group do not have this band because of the absence of reverse transcriptase (RT−) meaning there was no genomic DNA contamination in the RNA samples. (c) Relative expression of KCNH2 -3.1 mRNA in specific regions of the brain of KCNH2 -3.1 transgenic mice measured by qRT-PCR. N = 5. Values are normalized to expression levels in the cerebellum. (d) KCNH2 -3.1 expression in the PFC in mice treated with doxycycline. Doxycycline treatment significantly reduced the expression of KCNH2 -3.1 in KCNH2 -3.1 transgenic mice [ t (10) = 1.88, p = 0.04]. n = 5/non-treated control; n = 5/non-treated KCNH2 -3.1 transgenic; n = 3/treated control; n = 7/treated KCNH2 -3.1 transgenic. * p

Techniques Used: Expressing, Construct, Quantitative RT-PCR, Mouse Assay, Transgenic Assay

20) Product Images from "Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain"

Article Title: Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain

Journal: American Journal of Human Genetics

doi: 10.1016/j.ajhg.2010.03.019

Real-Time qPCR Analysis of Tuba8 Expression Levels in Mouse and Human Brain (A) Relative expression levels of Tuba1a , Tubb2b , Tubb3 , and Tuba8 in the developing mouse brain (E10.5 [head], E12.5, E14.5, E16.5, E18.5, P0, and P6). Tuba8 is expressed at very low levels at all developmental stages. (B) In the adult mouse brain, there are regional expression differences when comparing Tuba1a , Tubb2b , Tubb3 , and Tuba8 . The expression of Tuba8 is again low, with the exception of the olfactory bulbs and cerebellum, where it is expressed at modest levels. (C) Expression of TUBA1A , TUBB2B , TUBB3 , and TUBA8 in the developing human brain at GW13 (frontal lobe) and GW22 (total fetal brain). Again, TUBA8 is expressed at low levels. Error bars indicate the SEM.
Figure Legend Snippet: Real-Time qPCR Analysis of Tuba8 Expression Levels in Mouse and Human Brain (A) Relative expression levels of Tuba1a , Tubb2b , Tubb3 , and Tuba8 in the developing mouse brain (E10.5 [head], E12.5, E14.5, E16.5, E18.5, P0, and P6). Tuba8 is expressed at very low levels at all developmental stages. (B) In the adult mouse brain, there are regional expression differences when comparing Tuba1a , Tubb2b , Tubb3 , and Tuba8 . The expression of Tuba8 is again low, with the exception of the olfactory bulbs and cerebellum, where it is expressed at modest levels. (C) Expression of TUBA1A , TUBB2B , TUBB3 , and TUBA8 in the developing human brain at GW13 (frontal lobe) and GW22 (total fetal brain). Again, TUBA8 is expressed at low levels. Error bars indicate the SEM.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

21) Product Images from "Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish"

Article Title: Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish

Journal: Scientific Reports

doi: 10.1038/s41598-019-56466-z

A zebrafish model of Farber disease. ( a ) Schematic of asah1a/b −/− zebrafish generation and propagation. ( b ) DNA fragment analysis demonstrating presence of altered alleles. The 30-bp difference in the asah1b spectra (rather than the noted 20-bp deletion) is due to presence of different polymorphisms across the introns of the WT and KO alleles (Fig. S2b , see Fig. S3 for sequencing of full cDNA). ( c ) asah1a/b −/− (DKO) and asah1b −/− (SKO) zebrafish at 3.5 months. ( d ) Length of DKO and SKO zebrafish at 3.5–4 months. Zebrafish length measurements: Student t-test, SEM, combined data from n = 13 SKO and n = 12 DKO fish, fish within each data set (n = 4–5 SKO, n = 4 DKO) are age-matched. ***p
Figure Legend Snippet: A zebrafish model of Farber disease. ( a ) Schematic of asah1a/b −/− zebrafish generation and propagation. ( b ) DNA fragment analysis demonstrating presence of altered alleles. The 30-bp difference in the asah1b spectra (rather than the noted 20-bp deletion) is due to presence of different polymorphisms across the introns of the WT and KO alleles (Fig. S2b , see Fig. S3 for sequencing of full cDNA). ( c ) asah1a/b −/− (DKO) and asah1b −/− (SKO) zebrafish at 3.5 months. ( d ) Length of DKO and SKO zebrafish at 3.5–4 months. Zebrafish length measurements: Student t-test, SEM, combined data from n = 13 SKO and n = 12 DKO fish, fish within each data set (n = 4–5 SKO, n = 4 DKO) are age-matched. ***p

Techniques Used: Sequencing, Fluorescence In Situ Hybridization

22) Product Images from "AXL is not essential for Zika virus infection in the mouse brain"

Article Title: AXL is not essential for Zika virus infection in the mouse brain

Journal: Emerging Microbes & Infections

doi: 10.1038/emi.2017.10

Zika virus infection in Axl knockout ( Axl −/− ) mice. ( A ) Generation of BalB/c- Axl knockout ( Axl −/− ) mice. A 26-bp deletion was introduced into exon 1 of Axl using CRISPR/Cas9, resulting in premature termination of translation (indicated). The deletion was confirmed by sequencing. ( B ) Confirmation of Axl knockout using fluorescence-activated cell sorting. Monocytes isolated from the spleens of WT and Axl −/− mice were stained with rat anti-AXL antibody. ( C ) Axl −/− mouse brains support Zika virus infection. Neonatal mice underwent intracerebral inoculation with Zika virus at 1200, 120 and 1.2 PFU/mouse. Zika virus titers in the brain were measured using real-time PCR. No significant difference ( P > 0.05) was observed between WT and Axl −/− mice. plaque-forming unit, PFU; wild type, WT.
Figure Legend Snippet: Zika virus infection in Axl knockout ( Axl −/− ) mice. ( A ) Generation of BalB/c- Axl knockout ( Axl −/− ) mice. A 26-bp deletion was introduced into exon 1 of Axl using CRISPR/Cas9, resulting in premature termination of translation (indicated). The deletion was confirmed by sequencing. ( B ) Confirmation of Axl knockout using fluorescence-activated cell sorting. Monocytes isolated from the spleens of WT and Axl −/− mice were stained with rat anti-AXL antibody. ( C ) Axl −/− mouse brains support Zika virus infection. Neonatal mice underwent intracerebral inoculation with Zika virus at 1200, 120 and 1.2 PFU/mouse. Zika virus titers in the brain were measured using real-time PCR. No significant difference ( P > 0.05) was observed between WT and Axl −/− mice. plaque-forming unit, PFU; wild type, WT.

Techniques Used: Infection, Knock-Out, Mouse Assay, CRISPR, Sequencing, Fluorescence, FACS, Isolation, Staining, Real-time Polymerase Chain Reaction

23) Product Images from "Pharmacological targeting of the ?-amyloid precursor protein intracellular domain"

Article Title: Pharmacological targeting of the ?-amyloid precursor protein intracellular domain

Journal: Scientific Reports

doi: 10.1038/srep04618

The photoactivatable CHF5074-BpB derivative retains the γ-secretase modulatory properties of the parent compound. (a) Chemical structure of the benzophenone-biotin (BpB) derivative of CHF5074, whose synthesis is described in Supplementary Methods . Similar to the parent compound, CHF5074-BpB reduced Aβ 40 and Aβ 42 secretion (b) and increased Aβ 38 production ( cf . data reported in Supplementary Fig. S1a and S1b ) (c). Aβ peptide release was evaluated by ELISAs performed on conditioned culture media from H4swe cells exposed to the indicated concentrations of CHF5074-BpB (0.1–100 μM) for 18 hours. Data are percentages of control ± s.e.m. from three independent experiments. (d) Representative results obtained from immunoblot analysis (anti-APP C-terminus and control anti-actin antibodies as in Fig. 2c ) of extracts from H4swe cells treated with vehicle or CHF5074-BpB (1–100 μM). Full-length blots were presented in Supplementary Figure S4 . (e) Cell viability, assayed with the lactate dehydrogenase (LDH) release assay, was not appreciably affected by CHF5074-BpB at any of the tested concentrations; data are presented as percentages of control ± s.e.m. from three independent experiments.
Figure Legend Snippet: The photoactivatable CHF5074-BpB derivative retains the γ-secretase modulatory properties of the parent compound. (a) Chemical structure of the benzophenone-biotin (BpB) derivative of CHF5074, whose synthesis is described in Supplementary Methods . Similar to the parent compound, CHF5074-BpB reduced Aβ 40 and Aβ 42 secretion (b) and increased Aβ 38 production ( cf . data reported in Supplementary Fig. S1a and S1b ) (c). Aβ peptide release was evaluated by ELISAs performed on conditioned culture media from H4swe cells exposed to the indicated concentrations of CHF5074-BpB (0.1–100 μM) for 18 hours. Data are percentages of control ± s.e.m. from three independent experiments. (d) Representative results obtained from immunoblot analysis (anti-APP C-terminus and control anti-actin antibodies as in Fig. 2c ) of extracts from H4swe cells treated with vehicle or CHF5074-BpB (1–100 μM). Full-length blots were presented in Supplementary Figure S4 . (e) Cell viability, assayed with the lactate dehydrogenase (LDH) release assay, was not appreciably affected by CHF5074-BpB at any of the tested concentrations; data are presented as percentages of control ± s.e.m. from three independent experiments.

Techniques Used: Lactate Dehydrogenase Assay

APP processing in CHF5074-treated H4swe cells. (a) Immunoblot analysis of whole extracts from H4swe cells treated for 18 hours with vehicle or different concentrations of CHF5074 (1–30 μM). CTFβ and FL-APP, whose levels did not appreciably change upon treatment with CHF5074, were detected with the 6E10 antibody; GAPDH, measured in the same blot with an anti-GAPDH antibody, was used as a loading control. (b) Immunoblot analysis as in (a) performed with the anti-APP N-terminal antibody in culture medium of H4swe cells treated for 18 hours with vehicle or different concentration of CHF5074 (1-30 μM). Similar sAPP fragment levels were detected in all samples regardless of CHF5074 treatment. sAPP fragments in the medium were normalized to the actin content detected in the corresponding releasing cells. Full-length blots were presented in Supplementary Figure S3a c) Immunoblot analysis, performed with the anti-APP C-terminal antibody of extracts from H4swe cells treated with vehicle, CHF5074 (1–30 μM) or DAPT (0.2 μM) for 18 hours. DAPT, but not CHF5074, increases CTFα and β levels (*, p
Figure Legend Snippet: APP processing in CHF5074-treated H4swe cells. (a) Immunoblot analysis of whole extracts from H4swe cells treated for 18 hours with vehicle or different concentrations of CHF5074 (1–30 μM). CTFβ and FL-APP, whose levels did not appreciably change upon treatment with CHF5074, were detected with the 6E10 antibody; GAPDH, measured in the same blot with an anti-GAPDH antibody, was used as a loading control. (b) Immunoblot analysis as in (a) performed with the anti-APP N-terminal antibody in culture medium of H4swe cells treated for 18 hours with vehicle or different concentration of CHF5074 (1-30 μM). Similar sAPP fragment levels were detected in all samples regardless of CHF5074 treatment. sAPP fragments in the medium were normalized to the actin content detected in the corresponding releasing cells. Full-length blots were presented in Supplementary Figure S3a c) Immunoblot analysis, performed with the anti-APP C-terminal antibody of extracts from H4swe cells treated with vehicle, CHF5074 (1–30 μM) or DAPT (0.2 μM) for 18 hours. DAPT, but not CHF5074, increases CTFα and β levels (*, p

Techniques Used: Concentration Assay

Photoaffinity labeling analysis of specific APP fragments. (a) Schematic representation of APP processing products, including the binding sites of the antibodies utilized in this work and the APP fragments that were subjected to photolabeling analysis: the CHF5074-BpB-reactive CTFα and AICD-59 fragments ( black bars ) and the non-reactive Aβ 42 peptide ( white bar ). (b) Photoaffinity labeling profiles ( upper panels ) obtained after incubation with (+) or without (−) CHF5074-BpB (3 μM) of the synthetic AICD-59 (20 pmol, left panel ) and Aβ 42 (20 and 90 pmol, central panel ) peptides, and the empty glutathione S-transferase carrier ( GST ) and the recombinant GST-CTFα fusion polypeptide (20 pmol, right panel ). CHF5074-BpB reactive products were detected with IrDye 680-conjugated streptavidin. Labeling competition experiments were performed by preincubating 20 pmol of the AICD-59 peptide ( left panel ) or the GST-CTFα fusion polypeptide ( right panel ) in the presence (+) of unmodified CHF5074 (100 μM), followed by exposure to 3 μM CHF5074-BpB. Parallel immunoblot analyses performed with the anti-APP C-terminal antibody (AICD-59), the 6E10 antibody (Aβ 42 ) and with an antibody directed against GST (GST and GST-CTFα), served as controls to verify the input of the various (poly)peptides utilized for these experiments ( lower panels ). Cropped gel images are shown and the gels were run under the same experimental conditions.
Figure Legend Snippet: Photoaffinity labeling analysis of specific APP fragments. (a) Schematic representation of APP processing products, including the binding sites of the antibodies utilized in this work and the APP fragments that were subjected to photolabeling analysis: the CHF5074-BpB-reactive CTFα and AICD-59 fragments ( black bars ) and the non-reactive Aβ 42 peptide ( white bar ). (b) Photoaffinity labeling profiles ( upper panels ) obtained after incubation with (+) or without (−) CHF5074-BpB (3 μM) of the synthetic AICD-59 (20 pmol, left panel ) and Aβ 42 (20 and 90 pmol, central panel ) peptides, and the empty glutathione S-transferase carrier ( GST ) and the recombinant GST-CTFα fusion polypeptide (20 pmol, right panel ). CHF5074-BpB reactive products were detected with IrDye 680-conjugated streptavidin. Labeling competition experiments were performed by preincubating 20 pmol of the AICD-59 peptide ( left panel ) or the GST-CTFα fusion polypeptide ( right panel ) in the presence (+) of unmodified CHF5074 (100 μM), followed by exposure to 3 μM CHF5074-BpB. Parallel immunoblot analyses performed with the anti-APP C-terminal antibody (AICD-59), the 6E10 antibody (Aβ 42 ) and with an antibody directed against GST (GST and GST-CTFα), served as controls to verify the input of the various (poly)peptides utilized for these experiments ( lower panels ). Cropped gel images are shown and the gels were run under the same experimental conditions.

Techniques Used: Labeling, Binding Assay, Incubation, Recombinant

Photoaffinity labeling analysis of CHF5074-BpB-treated H4swe cells. (a) Photoaffinity labeling of FL-APP and CTFβ in membrane fractions and total lysates from H4swe cells incubated with CHF5074-BpB (3 μM) and exposed to ultraviolet light (350 nm, 30 minutes), prior to streptavidin precipitation ( IP ), gel-fractionation and immunoblot analysis with the 6E10 monoclonal antibody. Immunoreactivity of naïve membrane fractions (i.e., membranes not treated with CHF5074-BpB and not subjected to streptavidin precipitation) is shown on the right ( “start ”). Full-length blots were presented in Supplementary Figure S5a . (b) Reduced CHF5074-BpB labeling of APP-C-terminal fragments upon preincubation with unmodified CHF5074 (100 μM). Labeling was performed on membrane fractions, followed by streptavidin precipitation, gel-fractionation and immunoblot analysis with the anti-APP C-terminal antibody (see legend to panel a for further details). Specific labeling by CHF5074-BpB was detected over the background signals observed when omitting the photoprobe ( lane 2 ) or the membranes ( lane 3 ) from the reaction mixture. Full-length blots are presented in Supplementary Figure S5b . (c) Immunoblot analysis of CHF5074-BpB-treated membranes as in (a), performed with antibodies directed against the indicated γ-secretase subunits: nicastrin, Nct ; presenilin-1 N-terminal fragment, PS1-NTF ; presenilin-1 C-terminal fragment, PS1-CTF ; presenilin-2, PS2 ; presenilin enhancer-2, Pen-2 (see legend to panel a for further details). Lack of photolabeling was observed even when the membranes were exposed to the CHF5074-BpB photoprobe prior to detergent solubilization (“native conditions”). Cropped blot images (derived from ten different experiments) were shown.
Figure Legend Snippet: Photoaffinity labeling analysis of CHF5074-BpB-treated H4swe cells. (a) Photoaffinity labeling of FL-APP and CTFβ in membrane fractions and total lysates from H4swe cells incubated with CHF5074-BpB (3 μM) and exposed to ultraviolet light (350 nm, 30 minutes), prior to streptavidin precipitation ( IP ), gel-fractionation and immunoblot analysis with the 6E10 monoclonal antibody. Immunoreactivity of naïve membrane fractions (i.e., membranes not treated with CHF5074-BpB and not subjected to streptavidin precipitation) is shown on the right ( “start ”). Full-length blots were presented in Supplementary Figure S5a . (b) Reduced CHF5074-BpB labeling of APP-C-terminal fragments upon preincubation with unmodified CHF5074 (100 μM). Labeling was performed on membrane fractions, followed by streptavidin precipitation, gel-fractionation and immunoblot analysis with the anti-APP C-terminal antibody (see legend to panel a for further details). Specific labeling by CHF5074-BpB was detected over the background signals observed when omitting the photoprobe ( lane 2 ) or the membranes ( lane 3 ) from the reaction mixture. Full-length blots are presented in Supplementary Figure S5b . (c) Immunoblot analysis of CHF5074-BpB-treated membranes as in (a), performed with antibodies directed against the indicated γ-secretase subunits: nicastrin, Nct ; presenilin-1 N-terminal fragment, PS1-NTF ; presenilin-1 C-terminal fragment, PS1-CTF ; presenilin-2, PS2 ; presenilin enhancer-2, Pen-2 (see legend to panel a for further details). Lack of photolabeling was observed even when the membranes were exposed to the CHF5074-BpB photoprobe prior to detergent solubilization (“native conditions”). Cropped blot images (derived from ten different experiments) were shown.

Techniques Used: Labeling, Incubation, Fractionation, Derivative Assay

Effect of in vivo CHF5074 treatment on AICD occupancy, histone H3 acetylation and transcriptional activity of the KAI1 gene. (a) Chromatin immunoprecipitation assays performed on brain cortices from Tg2576 mice treated for 4 weeks with standard ( black bars ) or CHF5074-supplemented diet (375 ppm; ≈60 mg/kg/day; white bars ) using anti-APP C-terminus and anti-acetyl H3 as test antibodies and an anti-IgG as a negative control antibody. DNA extracted from immunoprecipitated chromatin was analyzed by qRT-PCR, setting C T values relative to untreated samples to 1.0 (see ‘Methods' for details). (b) KAI1 mRNA levels determined by qRT-PCR analysis of reverse-transcribed total RNA extracted from brain cortices of Tg2576 mice fed on standard ( black bars ) and CHF5074-supplemented ( white bars ) diet as above. Data are presented as relative values determined with the comparative C T method using β-actin as house-keeping gene reference; bars are the mean ± s.e.m. of three independent experiments (**, p
Figure Legend Snippet: Effect of in vivo CHF5074 treatment on AICD occupancy, histone H3 acetylation and transcriptional activity of the KAI1 gene. (a) Chromatin immunoprecipitation assays performed on brain cortices from Tg2576 mice treated for 4 weeks with standard ( black bars ) or CHF5074-supplemented diet (375 ppm; ≈60 mg/kg/day; white bars ) using anti-APP C-terminus and anti-acetyl H3 as test antibodies and an anti-IgG as a negative control antibody. DNA extracted from immunoprecipitated chromatin was analyzed by qRT-PCR, setting C T values relative to untreated samples to 1.0 (see ‘Methods' for details). (b) KAI1 mRNA levels determined by qRT-PCR analysis of reverse-transcribed total RNA extracted from brain cortices of Tg2576 mice fed on standard ( black bars ) and CHF5074-supplemented ( white bars ) diet as above. Data are presented as relative values determined with the comparative C T method using β-actin as house-keeping gene reference; bars are the mean ± s.e.m. of three independent experiments (**, p

Techniques Used: In Vivo, Activity Assay, Chromatin Immunoprecipitation, Mouse Assay, Negative Control, Immunoprecipitation, Quantitative RT-PCR

CHF5074 lowers AICD nuclear levels in H4swe cells. (a) Chemical structure of CHF5074. (b) Immunoblot analysis (anti-APP C-terminal antibody) of nuclear extracts from H4swe cells treated for 18 hours with vehicle or with different concentrations of CHF5074 (1–10 μM) as indicated. A single polypeptide band immunoreactive to the anti-APP C-terminal antibody was detected in nuclear templates. Synthetic AICD-59 polypeptide (7 kDa; shown on the right) was used as a molecular mass and electrophoretic mobility reference for these experiments (upper row). Polypeptide band from the same gel was detected with an anti-histone H3 antibody (lower row). Full-length blots were presented in Supplementary Figure S2 . Data from multiple experiments are presented as bar plots derived from densitometric quantification of anti-APP C-terminal antibody immunoreactive bands (“test”) normalized with respect to the immunoreactivity signals (“control”) produced by the anti-histone H3 antibody. The test/control ratio for vehicle-treated samples was arbitrarily set to 100%; bars are the mean ± s.e.m. of three independent experiments (*, p
Figure Legend Snippet: CHF5074 lowers AICD nuclear levels in H4swe cells. (a) Chemical structure of CHF5074. (b) Immunoblot analysis (anti-APP C-terminal antibody) of nuclear extracts from H4swe cells treated for 18 hours with vehicle or with different concentrations of CHF5074 (1–10 μM) as indicated. A single polypeptide band immunoreactive to the anti-APP C-terminal antibody was detected in nuclear templates. Synthetic AICD-59 polypeptide (7 kDa; shown on the right) was used as a molecular mass and electrophoretic mobility reference for these experiments (upper row). Polypeptide band from the same gel was detected with an anti-histone H3 antibody (lower row). Full-length blots were presented in Supplementary Figure S2 . Data from multiple experiments are presented as bar plots derived from densitometric quantification of anti-APP C-terminal antibody immunoreactive bands (“test”) normalized with respect to the immunoreactivity signals (“control”) produced by the anti-histone H3 antibody. The test/control ratio for vehicle-treated samples was arbitrarily set to 100%; bars are the mean ± s.e.m. of three independent experiments (*, p

Techniques Used: Derivative Assay, Produced

24) Product Images from "GDNF is not required for catecholaminergic neuron survival in vivo"

Article Title: GDNF is not required for catecholaminergic neuron survival in vivo

Journal: Nature neuroscience

doi: 10.1038/nn.3941

Dopamine system analysis after striatal Gdnf reduction by AAV5- Cre injection in adult mice. ( a ) Experimental timeline for analysis, time points for AAV5 encoding for synapsin promoter driven Cre injection (AAV5-Cre), RNA (R); protein (P); histology (H); striatal dopamine level measurements (DA) and behavior by corridor test (C) and after amphetamine (Amphet) or apomorphine (Apo) injections are indicated. ( b–c ) Relative levels of Gdnf mRNA determined by qPCR in the intact and AAV5 -Cre injected striata. ( d–f ) Measurements of rotations (Rot) after amphetamine (Amphet) and apomorphine (Apo) injection at the indicated time-points after AAV5 -Cre injection. ( g ) Corridor test at the indicated timepoints after AAV5 -Cre injection. ( h ) Unbiased stereological cell counts of substanti nigra tyrosine hydroxylase (TH) immunoreactive neurons 7 months (210 days) after Gdnf reduction. ( i ) Striatal tissue dopamine (DA) levels 7 months after Gdnf reduction. §-experiments were independently performed in Lund University, Sweden and University of Helsinki, Finland; § H -experiment performed in Helsinki; § L- experiment performed in Lund. F-floxed ; C-control containing only wt alleles; n = number of animals analyzed in each experiment; n.a. = not applicable. Error bars are mean ± s.e.m.
Figure Legend Snippet: Dopamine system analysis after striatal Gdnf reduction by AAV5- Cre injection in adult mice. ( a ) Experimental timeline for analysis, time points for AAV5 encoding for synapsin promoter driven Cre injection (AAV5-Cre), RNA (R); protein (P); histology (H); striatal dopamine level measurements (DA) and behavior by corridor test (C) and after amphetamine (Amphet) or apomorphine (Apo) injections are indicated. ( b–c ) Relative levels of Gdnf mRNA determined by qPCR in the intact and AAV5 -Cre injected striata. ( d–f ) Measurements of rotations (Rot) after amphetamine (Amphet) and apomorphine (Apo) injection at the indicated time-points after AAV5 -Cre injection. ( g ) Corridor test at the indicated timepoints after AAV5 -Cre injection. ( h ) Unbiased stereological cell counts of substanti nigra tyrosine hydroxylase (TH) immunoreactive neurons 7 months (210 days) after Gdnf reduction. ( i ) Striatal tissue dopamine (DA) levels 7 months after Gdnf reduction. §-experiments were independently performed in Lund University, Sweden and University of Helsinki, Finland; § H -experiment performed in Helsinki; § L- experiment performed in Lund. F-floxed ; C-control containing only wt alleles; n = number of animals analyzed in each experiment; n.a. = not applicable. Error bars are mean ± s.e.m.

Techniques Used: Injection, Mouse Assay, Real-time Polymerase Chain Reaction

25) Product Images from "Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain"

Article Title: Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain

Journal: American Journal of Human Genetics

doi: 10.1016/j.ajhg.2010.03.019

Tuba8 Expression in the Developing Mouse Brain Coronal sections (14 μm) of the developing cortex (E14.5, E16.5, P0) showing our in situ hybridization results obtained with the probe employed by Sheridan and colleagues (A, D, G) and the short (B, E, H) and long (C, F, I) probe targeted to the unique 3′ UTR of Tuba8 . High levels of staining are observed in the developing mouse brain with the Sheridan probe (particularly in the cortical plate [CP] and to a lesser extent in the intermediate zone [IZ] and ventricular zone [VZ]), but none is apparent with probes that exclusively target the 3′ UTR. We observed no staining when employing control sense probes (data not shown). Scale bars represent 50 μm. MZ indicates the marginal zone.
Figure Legend Snippet: Tuba8 Expression in the Developing Mouse Brain Coronal sections (14 μm) of the developing cortex (E14.5, E16.5, P0) showing our in situ hybridization results obtained with the probe employed by Sheridan and colleagues (A, D, G) and the short (B, E, H) and long (C, F, I) probe targeted to the unique 3′ UTR of Tuba8 . High levels of staining are observed in the developing mouse brain with the Sheridan probe (particularly in the cortical plate [CP] and to a lesser extent in the intermediate zone [IZ] and ventricular zone [VZ]), but none is apparent with probes that exclusively target the 3′ UTR. We observed no staining when employing control sense probes (data not shown). Scale bars represent 50 μm. MZ indicates the marginal zone.

Techniques Used: Expressing, In Situ Hybridization, Staining

Real-Time qPCR Analysis of Tuba8 Expression Levels in Mouse and Human Brain (A) Relative expression levels of Tuba1a , Tubb2b , Tubb3 , and Tuba8 in the developing mouse brain (E10.5 [head], E12.5, E14.5, E16.5, E18.5, P0, and P6). Tuba8 is expressed at very low levels at all developmental stages. (B) In the adult mouse brain, there are regional expression differences when comparing Tuba1a , Tubb2b , Tubb3 , and Tuba8 . The expression of Tuba8 is again low, with the exception of the olfactory bulbs and cerebellum, where it is expressed at modest levels. (C) Expression of TUBA1A , TUBB2B , TUBB3 , and TUBA8 in the developing human brain at GW13 (frontal lobe) and GW22 (total fetal brain). Again, TUBA8 is expressed at low levels. Error bars indicate the SEM.
Figure Legend Snippet: Real-Time qPCR Analysis of Tuba8 Expression Levels in Mouse and Human Brain (A) Relative expression levels of Tuba1a , Tubb2b , Tubb3 , and Tuba8 in the developing mouse brain (E10.5 [head], E12.5, E14.5, E16.5, E18.5, P0, and P6). Tuba8 is expressed at very low levels at all developmental stages. (B) In the adult mouse brain, there are regional expression differences when comparing Tuba1a , Tubb2b , Tubb3 , and Tuba8 . The expression of Tuba8 is again low, with the exception of the olfactory bulbs and cerebellum, where it is expressed at modest levels. (C) Expression of TUBA1A , TUBB2B , TUBB3 , and TUBA8 in the developing human brain at GW13 (frontal lobe) and GW22 (total fetal brain). Again, TUBA8 is expressed at low levels. Error bars indicate the SEM.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

26) Product Images from "Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions"

Article Title: Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky929

The presence of pathological prefibrillar oligomerized forms of Tau protein repressed the expression of Tau-interacting genes. Relative gene expression was analyzed by RT-qPCR with respect to three reference genes ( Ppib, Rplp0, Hmbs ). Total RNA was collected from the CA1 region of 6 month old mice either wild-type (WT) or THY-Tau22 expressing Tau oligomers, not submitted (CTRL) or after being submitted to HS (HS). ( A ) and analyzed by laser scanning confocal microscopy. The nuclei were identified using DAPI staining. The scale bars represent 20 μm. ( B ) The labeling intensity of Tau-oligomers was quantified within the nuclei (based on DAPI detection) of CA1 cells. The data shown are the means ± s.d of 60 to 160 nuclei. *** P -values
Figure Legend Snippet: The presence of pathological prefibrillar oligomerized forms of Tau protein repressed the expression of Tau-interacting genes. Relative gene expression was analyzed by RT-qPCR with respect to three reference genes ( Ppib, Rplp0, Hmbs ). Total RNA was collected from the CA1 region of 6 month old mice either wild-type (WT) or THY-Tau22 expressing Tau oligomers, not submitted (CTRL) or after being submitted to HS (HS). ( A ) and analyzed by laser scanning confocal microscopy. The nuclei were identified using DAPI staining. The scale bars represent 20 μm. ( B ) The labeling intensity of Tau-oligomers was quantified within the nuclei (based on DAPI detection) of CA1 cells. The data shown are the means ± s.d of 60 to 160 nuclei. *** P -values

Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Confocal Microscopy, Staining, Labeling

27) Product Images from "Minocycline plus N-acteylcysteine induces remyelination, synergistically protects oligodendrocytes and modifies neuroinflammation in a rat model of mild traumatic brain injury"

Article Title: Minocycline plus N-acteylcysteine induces remyelination, synergistically protects oligodendrocytes and modifies neuroinflammation in a rat model of mild traumatic brain injury

Journal: Journal of Cerebral Blood Flow & Metabolism

doi: 10.1177/0271678X17718106

MINO plus NAC synergistically increases transcripts of inflammatory markers associated with remyelination 14 days after mCCI. qPCR analysis of cDNA isolated from the corpus callosum of shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC 2 and 14 days after surgery. MINO plus NAC synergistically increases CD40 (a) and CD86 (b) RNA expression 14 days after injury (post hoc, * p
Figure Legend Snippet: MINO plus NAC synergistically increases transcripts of inflammatory markers associated with remyelination 14 days after mCCI. qPCR analysis of cDNA isolated from the corpus callosum of shamCCI-saline, mCCI-saline, mCCI-MINO, mCCI-NAC, and mCCI-MINO plus NAC 2 and 14 days after surgery. MINO plus NAC synergistically increases CD40 (a) and CD86 (b) RNA expression 14 days after injury (post hoc, * p

Techniques Used: Real-time Polymerase Chain Reaction, Isolation, RNA Expression

28) Product Images from "Phytochemical analysis and antioxidant and anticancer activities of mastic gum resin from Pistacia atlantica subspecies kurdica"

Article Title: Phytochemical analysis and antioxidant and anticancer activities of mastic gum resin from Pistacia atlantica subspecies kurdica

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S170827

mRNA expression levels of Bcl-2 , Bax , and Cyt-c normalized to the transcription levels of β-actin and GAPDH . The qRT-PCR analysis was performed on COLO205 cells treated with 5.2 ± 0.8 μg/mL MGR. The experiment was done in triplicates, and data are expressed as mean ± SD. *Significant difference from control ( P
Figure Legend Snippet: mRNA expression levels of Bcl-2 , Bax , and Cyt-c normalized to the transcription levels of β-actin and GAPDH . The qRT-PCR analysis was performed on COLO205 cells treated with 5.2 ± 0.8 μg/mL MGR. The experiment was done in triplicates, and data are expressed as mean ± SD. *Significant difference from control ( P

Techniques Used: Expressing, Quantitative RT-PCR

29) Product Images from "The ZEB1 Transcription Factor Is a Novel Repressor of Adiposity in Female Mice"

Article Title: The ZEB1 Transcription Factor Is a Novel Repressor of Adiposity in Female Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008460

The mass of parametrial fat pads does not differ with genotype. The mass of parametrial fat pads for TCF8+/− (black bars) and WT controls (gray bars) ages 2–6 months for mice fed ( A ) a high fat diet or ( B ) regular chow was determined by weighing. Note differences in the y-axes. Data are represented as a ratio of parametrial fat weight to total body weight. n = 5–12.
Figure Legend Snippet: The mass of parametrial fat pads does not differ with genotype. The mass of parametrial fat pads for TCF8+/− (black bars) and WT controls (gray bars) ages 2–6 months for mice fed ( A ) a high fat diet or ( B ) regular chow was determined by weighing. Note differences in the y-axes. Data are represented as a ratio of parametrial fat weight to total body weight. n = 5–12.

Techniques Used: Mouse Assay

ZEB1 mRNA expression increases concomitantly with weight in WT female mice. Mice were fed regular chow ad libitum or a diet restricted to 75% of the calories of the ad libitum group (calorie restricted). ( A ) Body weights (g) were recorded as indicated for mice that were fed ad libitum (gray line) or calorie restricted (black line). n = 3–7 mice/group. ( B ) Corresponding ZEB1 mRNA expression in parametrial fat was determined by quantitative SYBR real time PCR. ZEB1 mRNA was expressed relative to β-actin mRNA, ad libitum (gray bars), calorie restricted (black bars). n = 3–7 mice/group ( C ) Western blot confirming that ZEB1 protein expression increases in response to increased body weight in mice fed ad libitum . GAPDH was used as a loading control. Individual lanes are labeled as months of age.
Figure Legend Snippet: ZEB1 mRNA expression increases concomitantly with weight in WT female mice. Mice were fed regular chow ad libitum or a diet restricted to 75% of the calories of the ad libitum group (calorie restricted). ( A ) Body weights (g) were recorded as indicated for mice that were fed ad libitum (gray line) or calorie restricted (black line). n = 3–7 mice/group. ( B ) Corresponding ZEB1 mRNA expression in parametrial fat was determined by quantitative SYBR real time PCR. ZEB1 mRNA was expressed relative to β-actin mRNA, ad libitum (gray bars), calorie restricted (black bars). n = 3–7 mice/group ( C ) Western blot confirming that ZEB1 protein expression increases in response to increased body weight in mice fed ad libitum . GAPDH was used as a loading control. Individual lanes are labeled as months of age.

Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Labeling

30) Product Images from "Role of the TGF‐β pathway in dedifferentiation of human mature adipocytes"

Article Title: Role of the TGF‐β pathway in dedifferentiation of human mature adipocytes

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12250

Expression levels of (A) TGF‐β1, (B) TGF‐β2, (C) TGF‐β3, (D) COL1A1, (E) COL1A2, and (F) COL6A3 in whole adipose tissue, SVF, and DFAT cells (day 12) ( n = 5 donors). Values are mean ± SEM.
Figure Legend Snippet: Expression levels of (A) TGF‐β1, (B) TGF‐β2, (C) TGF‐β3, (D) COL1A1, (E) COL1A2, and (F) COL6A3 in whole adipose tissue, SVF, and DFAT cells (day 12) ( n = 5 donors). Values are mean ± SEM.

Techniques Used: Expressing

31) Product Images from "Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines"

Article Title: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

Journal: BMC Research Notes

doi: 10.1186/s13104-017-2959-0

Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p
Figure Legend Snippet: Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Synthesized, Expressing, Construct, Activated Clotting Time Assay

Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method
Figure Legend Snippet: Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method

Techniques Used: Isolation, Purification

32) Product Images from "Sox2 Is Essential for Oligodendroglial Proliferation and Differentiation during Postnatal Brain Myelination and CNS Remyelination"

Article Title: Sox2 Is Essential for Oligodendroglial Proliferation and Differentiation during Postnatal Brain Myelination and CNS Remyelination

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1291-17.2018

Sox2 is required for OL regeneration and remyelination in cuprizone-induced demyelinated corpus callosum. A , Transgenic mice (male, 2–3 months old at onset of cuprizone diet) and experimental designs for B1–H . The 0.25% curprizone diet was used in the design. B1 , B2 , Representative confocal images of Sox2, PDGFRα, and EYFP triple immunostaining in corpus callosum. Arrowheads point to PDGFRα + Sox2 + EYFP − cells (left) and PDGFRα + Sox2 − EYFP + cells (right). B2 , Right, Boxed area represents higher-magnification images. B3 , Density of Sox2 + cells (cell no./mm 2 ) in corpus callosum ( n = 3 each group). C ). D , Densities of marker-positive cell as indicated ( n = 3 each group). E , Representative confocal images of APC and TCF7l2 double immunostaining. Arrowheads point to APC + TCF7l2 + ). F , Quantification of APC + TCF7l2 + cells ( n = 3, Sox2 WT + cuprizone; n = 3, Sox2 cKO + cuprizone; n = 4 Sox2 WT + normal diet). G , Confocal images showing MBP and SMI312 in the posterior corpus callosum (coronal forebrain sections). H , Quantification of MBP and SMI312 immunoreactive signals in H ( n = 3 each group). B3 , D , F , H , * p
Figure Legend Snippet: Sox2 is required for OL regeneration and remyelination in cuprizone-induced demyelinated corpus callosum. A , Transgenic mice (male, 2–3 months old at onset of cuprizone diet) and experimental designs for B1–H . The 0.25% curprizone diet was used in the design. B1 , B2 , Representative confocal images of Sox2, PDGFRα, and EYFP triple immunostaining in corpus callosum. Arrowheads point to PDGFRα + Sox2 + EYFP − cells (left) and PDGFRα + Sox2 − EYFP + cells (right). B2 , Right, Boxed area represents higher-magnification images. B3 , Density of Sox2 + cells (cell no./mm 2 ) in corpus callosum ( n = 3 each group). C ). D , Densities of marker-positive cell as indicated ( n = 3 each group). E , Representative confocal images of APC and TCF7l2 double immunostaining. Arrowheads point to APC + TCF7l2 + ). F , Quantification of APC + TCF7l2 + cells ( n = 3, Sox2 WT + cuprizone; n = 3, Sox2 cKO + cuprizone; n = 4 Sox2 WT + normal diet). G , Confocal images showing MBP and SMI312 in the posterior corpus callosum (coronal forebrain sections). H , Quantification of MBP and SMI312 immunoreactive signals in H ( n = 3 each group). B3 , D , F , H , * p

Techniques Used: Transgenic Assay, Mouse Assay, Triple Immunostaining, Marker, Double Immunostaining

Oligodendrocyte number appears normal in the spinal cord of P14 Cnp-Cre Sox2 fl/fl mice. A , Low-power confocal images showing the distribution of CC1 + differentiated OLs in the spinal cord. B , Quantification of Olig2 + CC1 + differentiated OLs in the spinal cord ( n = 4 Cnp-Cre Sox2 fl/fl , n = 6 Sox2 fl/fl ). C , Representative images of Western blotting of MBP, Sox2, and the internal loading control β-actin in the spinal cord. D , qRT-PCR quantification of mRNA levels of Sox2, Mbp, proteolipid protein (Plp), myelin-associated protein (Mag), myelin-associated oligodendrocyte basic protein (Mobp), and Sox10 ( n = 4 Cnp-Cre Sox2 fl/fl n = 8 Sox2 fl/fl ). E , Representative confocal images and quantification showing Sox2 is completely deleted in CC1 + OLs in the spinal cord of Cnp-Cre Sox2 fl/fl mice ( n = 4 Cnp-Cre Sox2 fl/fl n = 3 Sox2 fl/fl ). Arrowheads point to Sox2 + CC1 + OLs. ** p
Figure Legend Snippet: Oligodendrocyte number appears normal in the spinal cord of P14 Cnp-Cre Sox2 fl/fl mice. A , Low-power confocal images showing the distribution of CC1 + differentiated OLs in the spinal cord. B , Quantification of Olig2 + CC1 + differentiated OLs in the spinal cord ( n = 4 Cnp-Cre Sox2 fl/fl , n = 6 Sox2 fl/fl ). C , Representative images of Western blotting of MBP, Sox2, and the internal loading control β-actin in the spinal cord. D , qRT-PCR quantification of mRNA levels of Sox2, Mbp, proteolipid protein (Plp), myelin-associated protein (Mag), myelin-associated oligodendrocyte basic protein (Mobp), and Sox10 ( n = 4 Cnp-Cre Sox2 fl/fl n = 8 Sox2 fl/fl ). E , Representative confocal images and quantification showing Sox2 is completely deleted in CC1 + OLs in the spinal cord of Cnp-Cre Sox2 fl/fl mice ( n = 4 Cnp-Cre Sox2 fl/fl n = 3 Sox2 fl/fl ). Arrowheads point to Sox2 + CC1 + OLs. ** p

Techniques Used: Mouse Assay, Western Blot, Quantitative RT-PCR, Plasmid Purification

Cnp-Cre, Sox2 fl/fl mice display brain hypomyelination on postnatal day 14. A , B , Grayscale images of MBP immunostaining showing decreased density of myelin fibers in the corpus callosum at the cingular cortical areas ( A ) and in the subcortical white matter tracts ( B ). C , Single and merged channels of confocal images of MBP and SMI312 in the subcortical white matter and the overlaying cortex. Most SMI312 + axons (green) lack MBP + myelin (red) in the Cnp-Cre, Sox2 fl/fl mice (lower right), in sharp contrast to extensive colabeling of MBP and SMI312 in the Sox2 fl/fl littermate controls (top right). D , Quantification of relative densities of MBP and SMI312 immunoreactive signals ( n = 4 Cnp-Cre, Sox2 fl/fl , n = 5 Sox2 fl/fl mice). ** p
Figure Legend Snippet: Cnp-Cre, Sox2 fl/fl mice display brain hypomyelination on postnatal day 14. A , B , Grayscale images of MBP immunostaining showing decreased density of myelin fibers in the corpus callosum at the cingular cortical areas ( A ) and in the subcortical white matter tracts ( B ). C , Single and merged channels of confocal images of MBP and SMI312 in the subcortical white matter and the overlaying cortex. Most SMI312 + axons (green) lack MBP + myelin (red) in the Cnp-Cre, Sox2 fl/fl mice (lower right), in sharp contrast to extensive colabeling of MBP and SMI312 in the Sox2 fl/fl littermate controls (top right). D , Quantification of relative densities of MBP and SMI312 immunoreactive signals ( n = 4 Cnp-Cre, Sox2 fl/fl , n = 5 Sox2 fl/fl mice). ** p

Techniques Used: Mouse Assay, Immunostaining

Oligodendrocyte differentiation is inhibited in the brain of Cnp-Cre Sox2 fl/fl mice. A , Fate-mapping of Cnp-Cre -expressing cells in the subcortical white matter of P7 Cnp-Cre, Rosa-EYFP mice. Many PDGFRα + OPCs are EYFP − (arrowheads). B , Many EYFP + fate-mapped cells are O4 + differentiation-committed, late OPCs, and/or immature oligodendrocytes (arrowheads) in the subcortical white matter of P7 Cnp-Cre, Rosa-EYFP mice. C , Triple immunostaining of CC1, PDGFRα, and Sox2 in the corpus callosum. Arrows and arrowheads point to representative CC1 + OLs and PDGFRα + OPCs, respectively. In the Sox2 cKO corpus callosum (bottom), many OPCs retain Sox2 expression, whereas no OLs have detectable Sox2. D , Quantification of Olig2 + CC1 + OLs and Olig2 + PDGFRα + OPCs in the subcortical white matter tract. Olig2 is a pan-oligodendroglial marker expressed in both OPCs and OLs. E , F , Density (cell no./mm 2 ) ( E ) and representative images ( F ) of TCF7l2-expressing, newly differentiated OLs in the subcortical white matter tracts ( F , dotted areas). C–F , Animals are P14 old. D , E , n = 4 Cnp-Cre Sox2 fl/fl and n = 6 Sox2 fl/fl . ** p
Figure Legend Snippet: Oligodendrocyte differentiation is inhibited in the brain of Cnp-Cre Sox2 fl/fl mice. A , Fate-mapping of Cnp-Cre -expressing cells in the subcortical white matter of P7 Cnp-Cre, Rosa-EYFP mice. Many PDGFRα + OPCs are EYFP − (arrowheads). B , Many EYFP + fate-mapped cells are O4 + differentiation-committed, late OPCs, and/or immature oligodendrocytes (arrowheads) in the subcortical white matter of P7 Cnp-Cre, Rosa-EYFP mice. C , Triple immunostaining of CC1, PDGFRα, and Sox2 in the corpus callosum. Arrows and arrowheads point to representative CC1 + OLs and PDGFRα + OPCs, respectively. In the Sox2 cKO corpus callosum (bottom), many OPCs retain Sox2 expression, whereas no OLs have detectable Sox2. D , Quantification of Olig2 + CC1 + OLs and Olig2 + PDGFRα + OPCs in the subcortical white matter tract. Olig2 is a pan-oligodendroglial marker expressed in both OPCs and OLs. E , F , Density (cell no./mm 2 ) ( E ) and representative images ( F ) of TCF7l2-expressing, newly differentiated OLs in the subcortical white matter tracts ( F , dotted areas). C–F , Animals are P14 old. D , E , n = 4 Cnp-Cre Sox2 fl/fl and n = 6 Sox2 fl/fl . ** p

Techniques Used: Mouse Assay, Expressing, Triple Immunostaining, Marker

Oligodendrocyte differentiation and myelination are severely affected in the Sox2 cKO brain at later stages of postnatal development. A , Accelerating Rotarod test showing that Sox10-Sox2 cKO ( Sox10-Cre, Sox2 fl/fl ) mice have severely impaired motor performance function evidenced by significant less retention time on the rotating rod ( n = 8 Sox2 fl/fl , n = 4 Sox10-Sox2 cKO, P28). B , Double immunostaining of Sox2 and PDGFRα in the corpus callosum of Sox10-Sox2 cKO and control mice. Sox2 is expressed in OPCs of the control mice (arrowheads, left) and absent from OPCs in the Sox10-Sox2 cKO mice (arrowheads, right). C , Grayscale images showing decreased MBP + myelin fibers in the corpus callosum and overlaying cingular cortex and quantification of MBP and SMI312 densities ( n = 4 Sox10-Sox2 cKO, n = 5 Sox2 fl/fl ). D , Western blotting of Sox2, another SoxB1 family member Sox3, myelin-related genes MBP, CNP, and MAG (left), and quantifications (right). The protein levels of MBP and CNP are reduced by > 50% in P21 Sox10-Sox2 cKO brains, compared with non-Cre controls. E , qRT-PCR of forebrain mRNA levels of Sox2, MBP, and myelinating OL-specific Plp-Exon3b ( n = 4 Sox10-Sox2 cKO, n = 6 non-Cre controls, P21). F , Immunostaining of mature OL marker CC1 in the P21 corpus callosum. Arrowheads point to representative CC1 + OLs. G , Quantification of PDGFRα + OPCs and CC1 + OLs in forebrain subcortical white matter tracts ( n = 4 Sox10-Sox2 cKO, n = 5 Sox10-Sox2 hetero-cKO, and n = 7 non-Cre controls, P21). H , Low-magnification images showing similar distribution but decreased density of Olig1-expressing oligodendroglial lineage cells in the P21 spinal cord of Sox10-Sox2 cKO (right) compared with Sox2 fl/fl control (left). I , Quantification of PDGFRα + OPCs and CC1 + OLs in the P21 spinal cord ( n = 6 Sox2 fl/fl , n = 4 Sox10-Sox2 cKO). J , K , Corticospinal tract cross semithin sections of toluidine blue staining ( J ) and ultrathin sections of transmission electron microscopy ( K ) from a P28 Sox10-Sox2 mouse and a littermate Sox2 fl/fl control. The density of myelinated axons is substantially decreased at low ( J ) and high ( K ) magnification images. Two-tailed Student's t test in A , C , D , E , I , one-way ANOCA with Tukey's post hoc test in G : * p
Figure Legend Snippet: Oligodendrocyte differentiation and myelination are severely affected in the Sox2 cKO brain at later stages of postnatal development. A , Accelerating Rotarod test showing that Sox10-Sox2 cKO ( Sox10-Cre, Sox2 fl/fl ) mice have severely impaired motor performance function evidenced by significant less retention time on the rotating rod ( n = 8 Sox2 fl/fl , n = 4 Sox10-Sox2 cKO, P28). B , Double immunostaining of Sox2 and PDGFRα in the corpus callosum of Sox10-Sox2 cKO and control mice. Sox2 is expressed in OPCs of the control mice (arrowheads, left) and absent from OPCs in the Sox10-Sox2 cKO mice (arrowheads, right). C , Grayscale images showing decreased MBP + myelin fibers in the corpus callosum and overlaying cingular cortex and quantification of MBP and SMI312 densities ( n = 4 Sox10-Sox2 cKO, n = 5 Sox2 fl/fl ). D , Western blotting of Sox2, another SoxB1 family member Sox3, myelin-related genes MBP, CNP, and MAG (left), and quantifications (right). The protein levels of MBP and CNP are reduced by > 50% in P21 Sox10-Sox2 cKO brains, compared with non-Cre controls. E , qRT-PCR of forebrain mRNA levels of Sox2, MBP, and myelinating OL-specific Plp-Exon3b ( n = 4 Sox10-Sox2 cKO, n = 6 non-Cre controls, P21). F , Immunostaining of mature OL marker CC1 in the P21 corpus callosum. Arrowheads point to representative CC1 + OLs. G , Quantification of PDGFRα + OPCs and CC1 + OLs in forebrain subcortical white matter tracts ( n = 4 Sox10-Sox2 cKO, n = 5 Sox10-Sox2 hetero-cKO, and n = 7 non-Cre controls, P21). H , Low-magnification images showing similar distribution but decreased density of Olig1-expressing oligodendroglial lineage cells in the P21 spinal cord of Sox10-Sox2 cKO (right) compared with Sox2 fl/fl control (left). I , Quantification of PDGFRα + OPCs and CC1 + OLs in the P21 spinal cord ( n = 6 Sox2 fl/fl , n = 4 Sox10-Sox2 cKO). J , K , Corticospinal tract cross semithin sections of toluidine blue staining ( J ) and ultrathin sections of transmission electron microscopy ( K ) from a P28 Sox10-Sox2 mouse and a littermate Sox2 fl/fl control. The density of myelinated axons is substantially decreased at low ( J ) and high ( K ) magnification images. Two-tailed Student's t test in A , C , D , E , I , one-way ANOCA with Tukey's post hoc test in G : * p

Techniques Used: Mouse Assay, Double Immunostaining, Western Blot, Quantitative RT-PCR, Plasmid Purification, Immunostaining, Marker, Expressing, Staining, Transmission Assay, Electron Microscopy, Two Tailed Test

Sox2 regulates OPC proliferation but not survival. A , Confocal images of EdU (2 h of pulse labeling before death on P14) and Sox10 double immunostaining. B , Quantification (cell no./mm 2 ) of EdU + Sox10 + and Ki67 + Sox10 + proliferative OPCs in the P14 forebrain subcortical white matter. C , Representative confocal images of Sox10 and active caspase 3 double immunostaining in the P14 subcortical white matter (dotted areas). D , Quantification of Caspase3 + Sox10 + and TUNEL + Sox10 + oligodendroglial lineage cells in the P14 subcortical white matter. A–D , Animals are P14 old; tamoxifen treatment on P6 and P7. N = 3 in each group. * p
Figure Legend Snippet: Sox2 regulates OPC proliferation but not survival. A , Confocal images of EdU (2 h of pulse labeling before death on P14) and Sox10 double immunostaining. B , Quantification (cell no./mm 2 ) of EdU + Sox10 + and Ki67 + Sox10 + proliferative OPCs in the P14 forebrain subcortical white matter. C , Representative confocal images of Sox10 and active caspase 3 double immunostaining in the P14 subcortical white matter (dotted areas). D , Quantification of Caspase3 + Sox10 + and TUNEL + Sox10 + oligodendroglial lineage cells in the P14 subcortical white matter. A–D , Animals are P14 old; tamoxifen treatment on P6 and P7. N = 3 in each group. * p

Techniques Used: Labeling, Double Immunostaining, TUNEL Assay

Sox2 regulates OPC proliferation and OL regeneration in the spinal cord in response to inflammatory insults. A , Transgenic mice (female, 2–3 months old) and experimental designs for B1–F . EAE was induced at day 0 by immunizing study mice with MOG peptide 35-55 ). Seven days after the last tamoxifen injection, lumbar spinal cord was harvested for analysis. B1–B3 , Double immunostaining of Sox2 and NG2 in the lumbar spinal cords of each group. Right, Boxed areas represent higher power. B2 , B3 , Demyelination lesions with extensive inflammatory infiltrations indicated by dense DAPI nuclear counterstaining. B1 , B2 , Arrowheads point to Sox2 + NG2 + OPCs. NG2 + OPCs in Sox2 cKO have no Sox2 expression ( B3 ) but distribute within the demyelination lesions in a similar pattern to that in Sox2 WT mice ( B2 ). C1–C3 , Confocal images of NG2 and Sox10 double immunostaining ( C1 , C2 ) and quantification of Sox10 + NG2 + OPCs ( C3 ). Arrowheads point to NG2 + Sox10 + OPCs. Right, Boxed areas represent higher-magnification images. D1–E , Representative confocal images of Ki67 and Olig2 ( D1 , D2 ) and quantifications ( E ). F , Density of APC + TCF7l2 + newly regenerated OLs. N = 3 in each group. * p
Figure Legend Snippet: Sox2 regulates OPC proliferation and OL regeneration in the spinal cord in response to inflammatory insults. A , Transgenic mice (female, 2–3 months old) and experimental designs for B1–F . EAE was induced at day 0 by immunizing study mice with MOG peptide 35-55 ). Seven days after the last tamoxifen injection, lumbar spinal cord was harvested for analysis. B1–B3 , Double immunostaining of Sox2 and NG2 in the lumbar spinal cords of each group. Right, Boxed areas represent higher power. B2 , B3 , Demyelination lesions with extensive inflammatory infiltrations indicated by dense DAPI nuclear counterstaining. B1 , B2 , Arrowheads point to Sox2 + NG2 + OPCs. NG2 + OPCs in Sox2 cKO have no Sox2 expression ( B3 ) but distribute within the demyelination lesions in a similar pattern to that in Sox2 WT mice ( B2 ). C1–C3 , Confocal images of NG2 and Sox10 double immunostaining ( C1 , C2 ) and quantification of Sox10 + NG2 + OPCs ( C3 ). Arrowheads point to NG2 + Sox10 + OPCs. Right, Boxed areas represent higher-magnification images. D1–E , Representative confocal images of Ki67 and Olig2 ( D1 , D2 ) and quantifications ( E ). F , Density of APC + TCF7l2 + newly regenerated OLs. N = 3 in each group. * p

Techniques Used: Transgenic Assay, Mouse Assay, Injection, Double Immunostaining, Expressing

33) Product Images from "Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain"

Article Title: Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain

Journal: American Journal of Human Genetics

doi: 10.1016/j.ajhg.2010.03.019

Tuba8 Expression in the Developing Mouse Brain Coronal sections (14 μm) of the developing cortex (E14.5, E16.5, P0) showing our in situ hybridization results obtained with the probe employed by Sheridan and colleagues (A, D, G) and the short (B, E, H) and long (C, F, I) probe targeted to the unique 3′ UTR of Tuba8 . High levels of staining are observed in the developing mouse brain with the Sheridan probe (particularly in the cortical plate [CP] and to a lesser extent in the intermediate zone [IZ] and ventricular zone [VZ]), but none is apparent with probes that exclusively target the 3′ UTR. We observed no staining when employing control sense probes (data not shown). Scale bars represent 50 μm. MZ indicates the marginal zone.
Figure Legend Snippet: Tuba8 Expression in the Developing Mouse Brain Coronal sections (14 μm) of the developing cortex (E14.5, E16.5, P0) showing our in situ hybridization results obtained with the probe employed by Sheridan and colleagues (A, D, G) and the short (B, E, H) and long (C, F, I) probe targeted to the unique 3′ UTR of Tuba8 . High levels of staining are observed in the developing mouse brain with the Sheridan probe (particularly in the cortical plate [CP] and to a lesser extent in the intermediate zone [IZ] and ventricular zone [VZ]), but none is apparent with probes that exclusively target the 3′ UTR. We observed no staining when employing control sense probes (data not shown). Scale bars represent 50 μm. MZ indicates the marginal zone.

Techniques Used: Expressing, In Situ Hybridization, Staining

Real-Time qPCR Analysis of Tuba8 Expression Levels in Mouse and Human Brain (A) Relative expression levels of Tuba1a , Tubb2b , Tubb3 , and Tuba8 in the developing mouse brain (E10.5 [head], E12.5, E14.5, E16.5, E18.5, P0, and P6). Tuba8 is expressed at very low levels at all developmental stages. (B) In the adult mouse brain, there are regional expression differences when comparing Tuba1a , Tubb2b , Tubb3 , and Tuba8 . The expression of Tuba8 is again low, with the exception of the olfactory bulbs and cerebellum, where it is expressed at modest levels. (C) Expression of TUBA1A , TUBB2B , TUBB3 , and TUBA8 in the developing human brain at GW13 (frontal lobe) and GW22 (total fetal brain). Again, TUBA8 is expressed at low levels. Error bars indicate the SEM.
Figure Legend Snippet: Real-Time qPCR Analysis of Tuba8 Expression Levels in Mouse and Human Brain (A) Relative expression levels of Tuba1a , Tubb2b , Tubb3 , and Tuba8 in the developing mouse brain (E10.5 [head], E12.5, E14.5, E16.5, E18.5, P0, and P6). Tuba8 is expressed at very low levels at all developmental stages. (B) In the adult mouse brain, there are regional expression differences when comparing Tuba1a , Tubb2b , Tubb3 , and Tuba8 . The expression of Tuba8 is again low, with the exception of the olfactory bulbs and cerebellum, where it is expressed at modest levels. (C) Expression of TUBA1A , TUBB2B , TUBB3 , and TUBA8 in the developing human brain at GW13 (frontal lobe) and GW22 (total fetal brain). Again, TUBA8 is expressed at low levels. Error bars indicate the SEM.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

34) Product Images from "PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2"

Article Title: PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Journal: Prion

doi: 10.4161/pri.26416

Figure 6. Expression of PrP in PSP and FTLD (fFTLD-tau and FTLD-TDP-43) frontal cortex. ( A ) mRNA average expression levels of PrP in the frontal cortex in control (Con), PSP and FTLD cases determined by TaqMan PCR assays. Values for PrP mRNA are
Figure Legend Snippet: Figure 6. Expression of PrP in PSP and FTLD (fFTLD-tau and FTLD-TDP-43) frontal cortex. ( A ) mRNA average expression levels of PrP in the frontal cortex in control (Con), PSP and FTLD cases determined by TaqMan PCR assays. Values for PrP mRNA are

Techniques Used: Expressing, Polymerase Chain Reaction

35) Product Images from "Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish"

Article Title: Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish

Journal: Scientific Reports

doi: 10.1038/s41598-019-56466-z

A zebrafish model of Farber disease. ( a ) Schematic of asah1a/b −/− zebrafish generation and propagation. ( b ) DNA fragment analysis demonstrating presence of altered alleles. The 30-bp difference in the asah1b spectra (rather than the noted 20-bp deletion) is due to presence of different polymorphisms across the introns of the WT and KO alleles (Fig. S2b , see Fig. S3 for sequencing of full cDNA). ( c ) asah1a/b −/− (DKO) and asah1b −/− (SKO) zebrafish at 3.5 months. ( d ) Length of DKO and SKO zebrafish at 3.5–4 months. Zebrafish length measurements: Student t-test, SEM, combined data from n = 13 SKO and n = 12 DKO fish, fish within each data set (n = 4–5 SKO, n = 4 DKO) are age-matched. ***p
Figure Legend Snippet: A zebrafish model of Farber disease. ( a ) Schematic of asah1a/b −/− zebrafish generation and propagation. ( b ) DNA fragment analysis demonstrating presence of altered alleles. The 30-bp difference in the asah1b spectra (rather than the noted 20-bp deletion) is due to presence of different polymorphisms across the introns of the WT and KO alleles (Fig. S2b , see Fig. S3 for sequencing of full cDNA). ( c ) asah1a/b −/− (DKO) and asah1b −/− (SKO) zebrafish at 3.5 months. ( d ) Length of DKO and SKO zebrafish at 3.5–4 months. Zebrafish length measurements: Student t-test, SEM, combined data from n = 13 SKO and n = 12 DKO fish, fish within each data set (n = 4–5 SKO, n = 4 DKO) are age-matched. ***p

Techniques Used: Sequencing, Fluorescence In Situ Hybridization

36) Product Images from "Egr-1 Regulates Expression of the Glial Scar Component Phosphacan in Astrocytes after Experimental Stroke"

Article Title: Egr-1 Regulates Expression of the Glial Scar Component Phosphacan in Astrocytes after Experimental Stroke

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2008.070648

Phosphacan accumulation is impaired in the glial scar of Egr-1-deficient mice. A and B: Measurement of GFAP ( A ) and phosphacan ( B ) RNA expression levels 10 days after permanent occlusion of the MCA. RNA samples were isolated from wild-type (+/+) and Egr-1-deficient (−/−) mice and from the infarcted, left brain (L) and contralateral, right brain (R) hemispheres. Results were normalized to β-actin RNA expression levels setting arbitrarily the value of one wild-type sample from the right hemisphere as 100. Statistical analysis was performed with the Mann-Whitney rank sum test. Wild-type and Egr-1-deficient mice show comparable GFAP induction. Egr-1 −/− mice show a statistically significant reduction of phosphacan levels in the infarcted hemisphere compared with the contralateral site ( P = 0.001). The expression levels of phosphacan are also statistically significant different between the infarcted hemispheres of Egr-1 −/− mice and their wild-type littermates ( P ≤ 0.001). In contrast, there is no statistically significant change of phosphacan expression between both hemispheres ( P = 0.163) in wild-type mice and between noninfarcted hemispheres of WT and KO mice ( P = 0.380). C: Western blot analysis of brain tissue isolated from the glial scar 10 days after occlusion of the MCA and control (c) tissue from the contralateral brain hemisphere. Quantitative image analysis demonstrates that GFAP up-regulation after stroke is comparable in Egr-1 −/− and wild-type brain tissue, ie, 4.4 times in wild-type samples (SD, 0.6) and 4.7 times in Egr-1 −/− mice (SD, 1.8). The levels of the two phosphacan isoforms detected by the KAF13 antibody, namely the secreted form of phosphacan (Pcan) as well as the transmembrane isoform receptor protein tyrosine phosphatase β (RPTPβs) are up-regulated in the glial scar of wild types by 1.49-fold (SD, 0.14), but not in the glial scar of Egr-1-deficient animals (0.89-fold; SD, 0.26). β-Tubulin serves as loading control. Protein sizes are indicated in kDa.
Figure Legend Snippet: Phosphacan accumulation is impaired in the glial scar of Egr-1-deficient mice. A and B: Measurement of GFAP ( A ) and phosphacan ( B ) RNA expression levels 10 days after permanent occlusion of the MCA. RNA samples were isolated from wild-type (+/+) and Egr-1-deficient (−/−) mice and from the infarcted, left brain (L) and contralateral, right brain (R) hemispheres. Results were normalized to β-actin RNA expression levels setting arbitrarily the value of one wild-type sample from the right hemisphere as 100. Statistical analysis was performed with the Mann-Whitney rank sum test. Wild-type and Egr-1-deficient mice show comparable GFAP induction. Egr-1 −/− mice show a statistically significant reduction of phosphacan levels in the infarcted hemisphere compared with the contralateral site ( P = 0.001). The expression levels of phosphacan are also statistically significant different between the infarcted hemispheres of Egr-1 −/− mice and their wild-type littermates ( P ≤ 0.001). In contrast, there is no statistically significant change of phosphacan expression between both hemispheres ( P = 0.163) in wild-type mice and between noninfarcted hemispheres of WT and KO mice ( P = 0.380). C: Western blot analysis of brain tissue isolated from the glial scar 10 days after occlusion of the MCA and control (c) tissue from the contralateral brain hemisphere. Quantitative image analysis demonstrates that GFAP up-regulation after stroke is comparable in Egr-1 −/− and wild-type brain tissue, ie, 4.4 times in wild-type samples (SD, 0.6) and 4.7 times in Egr-1 −/− mice (SD, 1.8). The levels of the two phosphacan isoforms detected by the KAF13 antibody, namely the secreted form of phosphacan (Pcan) as well as the transmembrane isoform receptor protein tyrosine phosphatase β (RPTPβs) are up-regulated in the glial scar of wild types by 1.49-fold (SD, 0.14), but not in the glial scar of Egr-1-deficient animals (0.89-fold; SD, 0.26). β-Tubulin serves as loading control. Protein sizes are indicated in kDa.

Techniques Used: Mouse Assay, RNA Expression, Isolation, MANN-WHITNEY, Expressing, Western Blot

37) Product Images from "Gene Expression in the Spinal Cord in Female Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced with Myelin Basic Protein"

Article Title: Gene Expression in the Spinal Cord in Female Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced with Myelin Basic Protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048555

RT- PCR validation of 5 genes expressed differentially in the spinal cords of Lewis rats with MBP induced EAE. Tissue samples were snap frozen in liquid nitrogen and stored at −80°C prior to total RNA preparation using the QIAGEN RNeasy Lipid tissue kit. RNA quality analysis was carried out on the BioRadExperion automated electrophoresis system. All preparations used in both assays had RNA quality indicator (RQI) values of > 9.5. For RT-PCR, total RNA was reverse transcribed and amplified as described in the methods. Analysis of selected genes up or down regulated at the peak of disease in EAE. Bars represent the average fold change between expression in the spinal cord level at peak of disease compared to normal healthy animals (+/− SEMs, Microarray n = 4, RT-PCR n = 8). Dark columns represent fold change derived from the microarray data. Similar amplification patterns were obtained from RT-PCR amplification of the same total RNA samples and a second set of 4 animals samples at an identical time point.
Figure Legend Snippet: RT- PCR validation of 5 genes expressed differentially in the spinal cords of Lewis rats with MBP induced EAE. Tissue samples were snap frozen in liquid nitrogen and stored at −80°C prior to total RNA preparation using the QIAGEN RNeasy Lipid tissue kit. RNA quality analysis was carried out on the BioRadExperion automated electrophoresis system. All preparations used in both assays had RNA quality indicator (RQI) values of > 9.5. For RT-PCR, total RNA was reverse transcribed and amplified as described in the methods. Analysis of selected genes up or down regulated at the peak of disease in EAE. Bars represent the average fold change between expression in the spinal cord level at peak of disease compared to normal healthy animals (+/− SEMs, Microarray n = 4, RT-PCR n = 8). Dark columns represent fold change derived from the microarray data. Similar amplification patterns were obtained from RT-PCR amplification of the same total RNA samples and a second set of 4 animals samples at an identical time point.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Amplification, Expressing, Microarray, Derivative Assay

38) Product Images from "Elevated autophagy gene expression in adipose tissue of obese humans: A potential non-cell-cycle-dependent function of E2F1"

Article Title: Elevated autophagy gene expression in adipose tissue of obese humans: A potential non-cell-cycle-dependent function of E2F1

Journal: Autophagy

doi: 10.1080/15548627.2015.1094597

Attenuated basal and stimulated autophagy in e2f1 −/− MEF-derived adipocytes. ( A ) Differentiation of primary mouse embryonic fibroblasts (MEFs) from WT or e2f1 knockout mice into adipocyte-like cells. Confluent MEFs were exposed to differentiation medium for 14 d. At the end of differentiation period adipocyte-like cells were stained with Oil red O, and representative images were obtained at 40X magnification by light microscopy. Scale bar: 20 µm. ( B ) Quantitative real-time PCR analysis of 5 adipogenic genes in fibroblasts or adipocyte-like cells from WT and e2f1 −/− . Each transcript expression was normalized to Rn18s and Hprt/Hprt1 (hypoxanthine guanine phosphoribosyl transferase) mRNA levels. ( C ) Quantitative real-time PCR analysis of E2f1 mRNA levels in WT versus e2f1 −/− . ( D ) e2f1 −/− and WT adipocyte-like cells were treated, in full (serum-containing) medium, with TNF (10 ng/ml) + IL1B (10 ng/ml) for 24h and subjected to western blot analysis. Shown are representative blots of n=3 independent experiments, with their densitometry analysis ( E ). ACTB was used as a loading control. ***, P
Figure Legend Snippet: Attenuated basal and stimulated autophagy in e2f1 −/− MEF-derived adipocytes. ( A ) Differentiation of primary mouse embryonic fibroblasts (MEFs) from WT or e2f1 knockout mice into adipocyte-like cells. Confluent MEFs were exposed to differentiation medium for 14 d. At the end of differentiation period adipocyte-like cells were stained with Oil red O, and representative images were obtained at 40X magnification by light microscopy. Scale bar: 20 µm. ( B ) Quantitative real-time PCR analysis of 5 adipogenic genes in fibroblasts or adipocyte-like cells from WT and e2f1 −/− . Each transcript expression was normalized to Rn18s and Hprt/Hprt1 (hypoxanthine guanine phosphoribosyl transferase) mRNA levels. ( C ) Quantitative real-time PCR analysis of E2f1 mRNA levels in WT versus e2f1 −/− . ( D ) e2f1 −/− and WT adipocyte-like cells were treated, in full (serum-containing) medium, with TNF (10 ng/ml) + IL1B (10 ng/ml) for 24h and subjected to western blot analysis. Shown are representative blots of n=3 independent experiments, with their densitometry analysis ( E ). ACTB was used as a loading control. ***, P

Techniques Used: Derivative Assay, Knock-Out, Mouse Assay, Staining, Light Microscopy, Real-time Polymerase Chain Reaction, Expressing, Western Blot

39) Product Images from "Anti-CD11b antibody treatment suppresses the osteoclast generation, inflammatory cell infiltration, and autoantibody production in arthritis-prone FcγRIIB-deficient mice"

Article Title: Anti-CD11b antibody treatment suppresses the osteoclast generation, inflammatory cell infiltration, and autoantibody production in arthritis-prone FcγRIIB-deficient mice

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-018-1523-1

Comparisons of mRNA expression levels of receptor activator of NF-κB (RANK), RANK ligand (RANKL), osteoprotogerin (OPG), RANKL/OPG ratio, monocyte chemotactic protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), CX3CL1, TNFα, IL-6, IL-17, and IL-10 in the ankle joints analyzed by qRT-PCR among untreated, normal rat IgG-treated, and monoclonal antibody (5C6)-treated FcγRIIB-deficient mouse strain (KO1) mice at 10 months of age. Values in each of the three groups of mice were evaluated as fold change compared with the expression levels in B6 mice (broken line). Data are shown as mean + SEM of six mice from each group. Statistical significance is shown (analysis of variance * P
Figure Legend Snippet: Comparisons of mRNA expression levels of receptor activator of NF-κB (RANK), RANK ligand (RANKL), osteoprotogerin (OPG), RANKL/OPG ratio, monocyte chemotactic protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), CX3CL1, TNFα, IL-6, IL-17, and IL-10 in the ankle joints analyzed by qRT-PCR among untreated, normal rat IgG-treated, and monoclonal antibody (5C6)-treated FcγRIIB-deficient mouse strain (KO1) mice at 10 months of age. Values in each of the three groups of mice were evaluated as fold change compared with the expression levels in B6 mice (broken line). Data are shown as mean + SEM of six mice from each group. Statistical significance is shown (analysis of variance * P

Techniques Used: Expressing, Activation Assay, Quantitative RT-PCR, Mouse Assay

Comparisons of mRNA expression levels of B-cell-activating factor of the tumor-necrosis-factor family (BAFF), IL-1β, B cell stimulating factor-3 (BSF-3), IL-6, IL-10, IL-17, interferon-γ (IFN-γ), and TNFα in spleen ( a ) and peripheral leukocytes ( b ) analyzed by qRT-PCR among untreated, normal rat IgG-treated, and monoclonal antibody (5C6)-treated FcγRIIB-deficient mouse strain (KO1) mice at 10 months of age. Values of each of the three groups of mice were evaluated as fold change compared with the expression levels in B6 mice (broken line). Data are shown as mean + SEM of six mice from each group. Statistical significance is shown (analysis of variance * P
Figure Legend Snippet: Comparisons of mRNA expression levels of B-cell-activating factor of the tumor-necrosis-factor family (BAFF), IL-1β, B cell stimulating factor-3 (BSF-3), IL-6, IL-10, IL-17, interferon-γ (IFN-γ), and TNFα in spleen ( a ) and peripheral leukocytes ( b ) analyzed by qRT-PCR among untreated, normal rat IgG-treated, and monoclonal antibody (5C6)-treated FcγRIIB-deficient mouse strain (KO1) mice at 10 months of age. Values of each of the three groups of mice were evaluated as fold change compared with the expression levels in B6 mice (broken line). Data are shown as mean + SEM of six mice from each group. Statistical significance is shown (analysis of variance * P

Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

40) Product Images from "Gene Expression in the Spinal Cord in Female Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced with Myelin Basic Protein"

Article Title: Gene Expression in the Spinal Cord in Female Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced with Myelin Basic Protein

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048555

RT- PCR validation of 5 genes expressed differentially in the spinal cords of Lewis rats with MBP induced EAE. Tissue samples were snap frozen in liquid nitrogen and stored at −80°C prior to total RNA preparation using the QIAGEN RNeasy Lipid tissue kit. RNA quality analysis was carried out on the BioRadExperion automated electrophoresis system. All preparations used in both assays had RNA quality indicator (RQI) values of > 9.5. For RT-PCR, total RNA was reverse transcribed and amplified as described in the methods. Analysis of selected genes up or down regulated at the peak of disease in EAE. Bars represent the average fold change between expression in the spinal cord level at peak of disease compared to normal healthy animals (+/− SEMs, Microarray n = 4, RT-PCR n = 8). Dark columns represent fold change derived from the microarray data. Similar amplification patterns were obtained from RT-PCR amplification of the same total RNA samples and a second set of 4 animals samples at an identical time point.
Figure Legend Snippet: RT- PCR validation of 5 genes expressed differentially in the spinal cords of Lewis rats with MBP induced EAE. Tissue samples were snap frozen in liquid nitrogen and stored at −80°C prior to total RNA preparation using the QIAGEN RNeasy Lipid tissue kit. RNA quality analysis was carried out on the BioRadExperion automated electrophoresis system. All preparations used in both assays had RNA quality indicator (RQI) values of > 9.5. For RT-PCR, total RNA was reverse transcribed and amplified as described in the methods. Analysis of selected genes up or down regulated at the peak of disease in EAE. Bars represent the average fold change between expression in the spinal cord level at peak of disease compared to normal healthy animals (+/− SEMs, Microarray n = 4, RT-PCR n = 8). Dark columns represent fold change derived from the microarray data. Similar amplification patterns were obtained from RT-PCR amplification of the same total RNA samples and a second set of 4 animals samples at an identical time point.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Amplification, Expressing, Microarray, Derivative Assay

Related Articles

Real-time Polymerase Chain Reaction:

Article Title: OSBP-Related Proteins (ORPs) in Human Adipose Depots and Cultured Adipocytes: Evidence for Impacts on the Adipocyte Phenotype
Article Snippet: .. Quantitative RT-PCR (qPCR) Analyses Total RNA (RNeasy Lipid Tissue Mini kit, QIAGEN, Germantown, MD) was extracted from s.c. and visceral adipose tissues of 4 patients. .. The RNAs were reverse transcribed by using the VILO kit (Invitrogen, Carlsbad, CA) and subjected, by using primer sets specific for 13 major ORP transcripts , to qPCR analysis using SYBR Green I Master Mix (Roche Diagnostics) and a Roche LightCycler 480 II instrument.

Isolation:

Article Title: Immortalization and characterization of lineage-restricted neuronal progenitor cells derived from the porcine olfactory bulb
Article Snippet: .. Total RNA was independently isolated from 90% confluent monolayers of early-subpassaged OBGF400 and of PK15 (non-neuronal) cells in three 75 cm2 flasks (Midwest Scientific) by using the RNeasy Mini Kit (Qiagen) and from approximately 10 to 20 mg of minced OB tissue obtained from 1 to 3 day-old pigs by using the RNeasy Lipid Tissue Kit (Qiagen) following manufacturer's recommendations. .. Methods for DNase I treatment as well as subsequent RNA purification, RNA concentration determination and cDNA synthesis are described above. rRT-PCR was conducted for selected genes ( ) using the TaqMan™ Universal PCR Master Mix (Applied Biosystems) standard protocol as described above.

RNA Extraction:

Article Title: Gene Expression in the Spinal Cord in Female Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced with Myelin Basic Protein
Article Snippet: .. RNA Extraction Total RNA was extracted from tissues using QIAGEN RNeasy® Lipid tissue Midi kits as per the manufactures instructions and treated with DNase1 (QIAGEN) to remove all traces of genomic DNA prior to storage at −80°C. .. The RNA quality analysis was carried out using the BioRadExperion automated gel electrophoresis system (BioRad Laboratories Inc.).

Cell Culture:

Article Title: Egr-1 Regulates Expression of the Glial Scar Component Phosphacan in Astrocytes after Experimental Stroke
Article Snippet: .. Total RNA was extracted and prepared from cultured cells as well as brain hemispheres (ipsilateral and contralateral) 10 days after permanent occlusion of the MCA using the RNA lipid tissue mini kit (Qiagen, Hilden, Germany). .. Residual genomic DNA was removed by the RNase-Free DNase Set (Qiagen) and RNA was reverse-transcribed into cDNA as follows: 1 μg of RNA was mixed with 100 ng of oligo(dT)15 and incubated for 5 minutes at 65°C.

Quantitative RT-PCR:

Article Title: Phytochemical analysis and antioxidant and anticancer activities of mastic gum resin from Pistacia atlantica subspecies kurdica
Article Snippet: .. qRT-PCR assay RNeasy® lipid tissue mini kit (Qiagen NV, Venlo, the Netherlands) was used to extract total RNA from COLO205 cells prior to conducting qRT-PCR analysis. .. The nanophotometer (Implen GmbH, München, Germany) was used to quantify the RNA before aliquoting and storing at −80°C.

Article Title: OSBP-Related Proteins (ORPs) in Human Adipose Depots and Cultured Adipocytes: Evidence for Impacts on the Adipocyte Phenotype
Article Snippet: .. Quantitative RT-PCR (qPCR) Analyses Total RNA (RNeasy Lipid Tissue Mini kit, QIAGEN, Germantown, MD) was extracted from s.c. and visceral adipose tissues of 4 patients. .. The RNAs were reverse transcribed by using the VILO kit (Invitrogen, Carlsbad, CA) and subjected, by using primer sets specific for 13 major ORP transcripts , to qPCR analysis using SYBR Green I Master Mix (Roche Diagnostics) and a Roche LightCycler 480 II instrument.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Qiagen qrt pcr assay rneasy lipid tissue mini kit
    mRNA expression levels of Bcl-2 , Bax , and Cyt-c normalized to the transcription levels of β-actin and GAPDH . The <t>qRT-PCR</t> analysis was performed on COLO205 cells treated with 5.2 ± 0.8 μg/mL MGR. The experiment was done in triplicates, and data are expressed as mean ± SD. *Significant difference from control ( P
    Qrt Pcr Assay Rneasy Lipid Tissue Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr assay rneasy lipid tissue mini kit/product/Qiagen
    Average 99 stars, based on 126 article reviews
    Price from $9.99 to $1999.99
    qrt pcr assay rneasy lipid tissue mini kit - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    mRNA expression levels of Bcl-2 , Bax , and Cyt-c normalized to the transcription levels of β-actin and GAPDH . The qRT-PCR analysis was performed on COLO205 cells treated with 5.2 ± 0.8 μg/mL MGR. The experiment was done in triplicates, and data are expressed as mean ± SD. *Significant difference from control ( P

    Journal: OncoTargets and therapy

    Article Title: Phytochemical analysis and antioxidant and anticancer activities of mastic gum resin from Pistacia atlantica subspecies kurdica

    doi: 10.2147/OTT.S170827

    Figure Lengend Snippet: mRNA expression levels of Bcl-2 , Bax , and Cyt-c normalized to the transcription levels of β-actin and GAPDH . The qRT-PCR analysis was performed on COLO205 cells treated with 5.2 ± 0.8 μg/mL MGR. The experiment was done in triplicates, and data are expressed as mean ± SD. *Significant difference from control ( P

    Article Snippet: qRT-PCR assay RNeasy® lipid tissue mini kit (Qiagen NV, Venlo, the Netherlands) was used to extract total RNA from COLO205 cells prior to conducting qRT-PCR analysis.

    Techniques: Expressing, Quantitative RT-PCR

    Phosphacan accumulation is impaired in the glial scar of Egr-1-deficient mice. A and B: Measurement of GFAP ( A ) and phosphacan ( B ) RNA expression levels 10 days after permanent occlusion of the MCA. RNA samples were isolated from wild-type (+/+) and Egr-1-deficient (−/−) mice and from the infarcted, left brain (L) and contralateral, right brain (R) hemispheres. Results were normalized to β-actin RNA expression levels setting arbitrarily the value of one wild-type sample from the right hemisphere as 100. Statistical analysis was performed with the Mann-Whitney rank sum test. Wild-type and Egr-1-deficient mice show comparable GFAP induction. Egr-1 −/− mice show a statistically significant reduction of phosphacan levels in the infarcted hemisphere compared with the contralateral site ( P = 0.001). The expression levels of phosphacan are also statistically significant different between the infarcted hemispheres of Egr-1 −/− mice and their wild-type littermates ( P ≤ 0.001). In contrast, there is no statistically significant change of phosphacan expression between both hemispheres ( P = 0.163) in wild-type mice and between noninfarcted hemispheres of WT and KO mice ( P = 0.380). C: Western blot analysis of brain tissue isolated from the glial scar 10 days after occlusion of the MCA and control (c) tissue from the contralateral brain hemisphere. Quantitative image analysis demonstrates that GFAP up-regulation after stroke is comparable in Egr-1 −/− and wild-type brain tissue, ie, 4.4 times in wild-type samples (SD, 0.6) and 4.7 times in Egr-1 −/− mice (SD, 1.8). The levels of the two phosphacan isoforms detected by the KAF13 antibody, namely the secreted form of phosphacan (Pcan) as well as the transmembrane isoform receptor protein tyrosine phosphatase β (RPTPβs) are up-regulated in the glial scar of wild types by 1.49-fold (SD, 0.14), but not in the glial scar of Egr-1-deficient animals (0.89-fold; SD, 0.26). β-Tubulin serves as loading control. Protein sizes are indicated in kDa.

    Journal: The American Journal of Pathology

    Article Title: Egr-1 Regulates Expression of the Glial Scar Component Phosphacan in Astrocytes after Experimental Stroke

    doi: 10.2353/ajpath.2008.070648

    Figure Lengend Snippet: Phosphacan accumulation is impaired in the glial scar of Egr-1-deficient mice. A and B: Measurement of GFAP ( A ) and phosphacan ( B ) RNA expression levels 10 days after permanent occlusion of the MCA. RNA samples were isolated from wild-type (+/+) and Egr-1-deficient (−/−) mice and from the infarcted, left brain (L) and contralateral, right brain (R) hemispheres. Results were normalized to β-actin RNA expression levels setting arbitrarily the value of one wild-type sample from the right hemisphere as 100. Statistical analysis was performed with the Mann-Whitney rank sum test. Wild-type and Egr-1-deficient mice show comparable GFAP induction. Egr-1 −/− mice show a statistically significant reduction of phosphacan levels in the infarcted hemisphere compared with the contralateral site ( P = 0.001). The expression levels of phosphacan are also statistically significant different between the infarcted hemispheres of Egr-1 −/− mice and their wild-type littermates ( P ≤ 0.001). In contrast, there is no statistically significant change of phosphacan expression between both hemispheres ( P = 0.163) in wild-type mice and between noninfarcted hemispheres of WT and KO mice ( P = 0.380). C: Western blot analysis of brain tissue isolated from the glial scar 10 days after occlusion of the MCA and control (c) tissue from the contralateral brain hemisphere. Quantitative image analysis demonstrates that GFAP up-regulation after stroke is comparable in Egr-1 −/− and wild-type brain tissue, ie, 4.4 times in wild-type samples (SD, 0.6) and 4.7 times in Egr-1 −/− mice (SD, 1.8). The levels of the two phosphacan isoforms detected by the KAF13 antibody, namely the secreted form of phosphacan (Pcan) as well as the transmembrane isoform receptor protein tyrosine phosphatase β (RPTPβs) are up-regulated in the glial scar of wild types by 1.49-fold (SD, 0.14), but not in the glial scar of Egr-1-deficient animals (0.89-fold; SD, 0.26). β-Tubulin serves as loading control. Protein sizes are indicated in kDa.

    Article Snippet: Total RNA was extracted and prepared from cultured cells as well as brain hemispheres (ipsilateral and contralateral) 10 days after permanent occlusion of the MCA using the RNA lipid tissue mini kit (Qiagen, Hilden, Germany).

    Techniques: Mouse Assay, RNA Expression, Isolation, MANN-WHITNEY, Expressing, Western Blot

    RT- PCR validation of 5 genes expressed differentially in the spinal cords of Lewis rats with MBP induced EAE. Tissue samples were snap frozen in liquid nitrogen and stored at −80°C prior to total RNA preparation using the QIAGEN RNeasy Lipid tissue kit. RNA quality analysis was carried out on the BioRadExperion automated electrophoresis system. All preparations used in both assays had RNA quality indicator (RQI) values of > 9.5. For RT-PCR, total RNA was reverse transcribed and amplified as described in the methods. Analysis of selected genes up or down regulated at the peak of disease in EAE. Bars represent the average fold change between expression in the spinal cord level at peak of disease compared to normal healthy animals (+/− SEMs, Microarray n = 4, RT-PCR n = 8). Dark columns represent fold change derived from the microarray data. Similar amplification patterns were obtained from RT-PCR amplification of the same total RNA samples and a second set of 4 animals samples at an identical time point.

    Journal: PLoS ONE

    Article Title: Gene Expression in the Spinal Cord in Female Lewis Rats with Experimental Autoimmune Encephalomyelitis Induced with Myelin Basic Protein

    doi: 10.1371/journal.pone.0048555

    Figure Lengend Snippet: RT- PCR validation of 5 genes expressed differentially in the spinal cords of Lewis rats with MBP induced EAE. Tissue samples were snap frozen in liquid nitrogen and stored at −80°C prior to total RNA preparation using the QIAGEN RNeasy Lipid tissue kit. RNA quality analysis was carried out on the BioRadExperion automated electrophoresis system. All preparations used in both assays had RNA quality indicator (RQI) values of > 9.5. For RT-PCR, total RNA was reverse transcribed and amplified as described in the methods. Analysis of selected genes up or down regulated at the peak of disease in EAE. Bars represent the average fold change between expression in the spinal cord level at peak of disease compared to normal healthy animals (+/− SEMs, Microarray n = 4, RT-PCR n = 8). Dark columns represent fold change derived from the microarray data. Similar amplification patterns were obtained from RT-PCR amplification of the same total RNA samples and a second set of 4 animals samples at an identical time point.

    Article Snippet: Tissue samples were snap frozen in liquid nitrogen and stored at −80°C prior to total RNA preparation using the QIAGEN RNeasy Lipid tissue kit.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Amplification, Expressing, Microarray, Derivative Assay