rna  (Qiagen)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96
    Name:
    RNase Free DNase Set
    Description:
    For DNase digestion during RNA purification Kit contents Qiagen RNase free DNase Set 50 preps For DNase Digestion During RNA Purification Silica gel Membrane Spin column Technology Efficiently Removes the Majority of the DNA Without DNase Treatment The Buffer is Also Well suited for Efficient DNase Digestion in Solution Includes 1500U RNase free DNase I RNase free Buffer RDD and RNase free Water
    Catalog Number:
    79254
    Price:
    108
    Category:
    RNase Free DNase Set
    Buy from Supplier


    Structured Review

    Qiagen rna
    RNase Free DNase Set
    For DNase digestion during RNA purification Kit contents Qiagen RNase free DNase Set 50 preps For DNase Digestion During RNA Purification Silica gel Membrane Spin column Technology Efficiently Removes the Majority of the DNA Without DNase Treatment The Buffer is Also Well suited for Efficient DNase Digestion in Solution Includes 1500U RNase free DNase I RNase free Buffer RDD and RNase free Water
    https://www.bioz.com/result/rna/product/Qiagen
    Average 96 stars, based on 30392 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-07
    96/100 stars

    Images

    1) Product Images from "Depleting Components of the THO Complex Causes Increased Telomere Length by Reducing the Expression of the Telomere-Associated Protein Rif1p"

    Article Title: Depleting Components of the THO Complex Causes Increased Telomere Length by Reducing the Expression of the Telomere-Associated Protein Rif1p

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033498

    THO2 and HPR1 function in the same pathway as RIF1 to regulate telomere length. ( A ) Yeast DNA from the indicated mutations was isolated and analyzed by Southern blotting assays using the Y′ element probe. ( B ) The decreased Tel1p level does not contribute to telomere lengthening in tho2 and hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for GCN1 and TEL1 RNA levels using real time RT-PCR (left panel). Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations determined from three independent experiments. Yeast DNA from indicated strains was analyzed for telomere length (right panel). ( C ) Overexpressing Rif1p suppresses the telomere lengthening in tho2 and hpr1 cells. Wild-type, tho2 , or hpr1 yeast cells carrying plasmids pRS426 or pRS426- RIF1 (o/e RIF1 ) were cultured at 30°C. Telomere lengths of these cells were then analyzed using Southern blotting assays (top panel). Immunoblotting analysis of the Rif1p level was also performed (bottom panel).
    Figure Legend Snippet: THO2 and HPR1 function in the same pathway as RIF1 to regulate telomere length. ( A ) Yeast DNA from the indicated mutations was isolated and analyzed by Southern blotting assays using the Y′ element probe. ( B ) The decreased Tel1p level does not contribute to telomere lengthening in tho2 and hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for GCN1 and TEL1 RNA levels using real time RT-PCR (left panel). Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations determined from three independent experiments. Yeast DNA from indicated strains was analyzed for telomere length (right panel). ( C ) Overexpressing Rif1p suppresses the telomere lengthening in tho2 and hpr1 cells. Wild-type, tho2 , or hpr1 yeast cells carrying plasmids pRS426 or pRS426- RIF1 (o/e RIF1 ) were cultured at 30°C. Telomere lengths of these cells were then analyzed using Southern blotting assays (top panel). Immunoblotting analysis of the Rif1p level was also performed (bottom panel).

    Techniques Used: Isolation, Southern Blot, Quantitative RT-PCR, Expressing, Cell Culture

    Overexpressing SUB2 cannot restore the Rif1p level and telomere length in tho2 and hpr1 cells. ( A ) Overexpressing SUB2 suppressed the growth defect of tho2 or hpr1 cells. Strains of the indicated genotypes derived from THO2 / tho2 , or HPR1 / hpr1 diploids carrying SUB2 or sub2-5 overexpressing plasmids were grown on YC plates at 30° or 37°C. ( B ) SUB2 or sub2-5 overexpression did not restore the Rif1p level in tho2 or hpr1 cells. Immunoblotting assays were carried out as described. ( C ) SUB2 overexpression did not restore the RIF1 level in tho2 or hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for the RIF1 RNA level using real time RT-PCR. Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations calculated using data from three independent experiments. ( D ) SUB2 or sub2-5 overexpression did not restore the telomere length in tho2 or hpr1 cells. Telomere length analyses were performed as previously described.
    Figure Legend Snippet: Overexpressing SUB2 cannot restore the Rif1p level and telomere length in tho2 and hpr1 cells. ( A ) Overexpressing SUB2 suppressed the growth defect of tho2 or hpr1 cells. Strains of the indicated genotypes derived from THO2 / tho2 , or HPR1 / hpr1 diploids carrying SUB2 or sub2-5 overexpressing plasmids were grown on YC plates at 30° or 37°C. ( B ) SUB2 or sub2-5 overexpression did not restore the Rif1p level in tho2 or hpr1 cells. Immunoblotting assays were carried out as described. ( C ) SUB2 overexpression did not restore the RIF1 level in tho2 or hpr1 cells. Total mRNA from the indicated strains was isolated and analyzed for the RIF1 RNA level using real time RT-PCR. Results were presented as relative levels normalized to the wild-type expression level. The bars were standard deviations calculated using data from three independent experiments. ( D ) SUB2 or sub2-5 overexpression did not restore the telomere length in tho2 or hpr1 cells. Telomere length analyses were performed as previously described.

    Techniques Used: Derivative Assay, Over Expression, Isolation, Quantitative RT-PCR, Expressing

    2) Product Images from "Extensive Natural Variation in Arabidopsis Seed Mucilage Structure"

    Article Title: Extensive Natural Variation in Arabidopsis Seed Mucilage Structure

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.00803

    Gene expression in siliques of accessions with altered Gal and Man content. (A,B) qRT-PCR analysis of RNA isolated from 7 DPA siliques. Data show means + SD of two biological replicates analyzed at least twice (only technical error shown for one Le-0 plant). Target gene transcript levels were first normalized to the geometric mean of two reference genes ( UBQ5 and elF4a ), and were then set as 1.0 for Col-0. Significant changes in gene expression relative to Col-0 ( t -test, P
    Figure Legend Snippet: Gene expression in siliques of accessions with altered Gal and Man content. (A,B) qRT-PCR analysis of RNA isolated from 7 DPA siliques. Data show means + SD of two biological replicates analyzed at least twice (only technical error shown for one Le-0 plant). Target gene transcript levels were first normalized to the geometric mean of two reference genes ( UBQ5 and elF4a ), and were then set as 1.0 for Col-0. Significant changes in gene expression relative to Col-0 ( t -test, P

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation

    3) 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

    4) Product Images from "Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation"

    Article Title: Loss of Ezh2 promotes a midbrain-to-forebrain identity switch by direct gene derepression and Wnt-dependent regulation

    Journal: BMC Biology

    doi: 10.1186/s12915-015-0210-9

    Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. ( a ) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. ( b ) qRT-PCR for Ezh2 , cell cycle regulators Cdkn2a and Cdkn2c , and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, * P ≤0.05, Student’s t -test. ( c ) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a , Cdkn2c , Wif1 , and Dkk2 . Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( d – e ) In situ hybridization for Cdkn2a ( d ) and Wif1 ( e ) mRNA illustrates increased gene expression in Ezh2 mutants. ( f ) Immunostaining for β-galactosidase + cells on the BAT- gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, ** P ≤0.01, Student’s t -test. Cartoon insert indicates area of analysis for f and g . ( g ) Immunostaining against CyclinD1 and qRT-PCR. ( h ) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( i ) H E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f , g ; Scale bars: d , e , 100 μm; f , g , 40 μm; i , 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain
    Figure Legend Snippet: Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. ( a ) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. ( b ) qRT-PCR for Ezh2 , cell cycle regulators Cdkn2a and Cdkn2c , and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, * P ≤0.05, Student’s t -test. ( c ) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a , Cdkn2c , Wif1 , and Dkk2 . Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( d – e ) In situ hybridization for Cdkn2a ( d ) and Wif1 ( e ) mRNA illustrates increased gene expression in Ezh2 mutants. ( f ) Immunostaining for β-galactosidase + cells on the BAT- gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, ** P ≤0.01, Student’s t -test. Cartoon insert indicates area of analysis for f and g . ( g ) Immunostaining against CyclinD1 and qRT-PCR. ( h ) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, *** P ≤0.001, ** P ≤0.01, Student’s t -test. ( i ) H E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f , g ; Scale bars: d , e , 100 μm; f , g , 40 μm; i , 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain

    Techniques Used: Microarray, Mutagenesis, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, In Situ Hybridization, Expressing, Immunostaining, Staining, Marker

    5) Product Images from "Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4"

    Article Title: Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

    Journal: Scientific Reports

    doi: 10.1038/srep30529

    Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.
    Figure Legend Snippet: Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.

    Techniques Used: Expressing, Transfection, Stable Transfection, Infection, Western Blot, Software, RNA Expression, Quantitative RT-PCR

    6) Product Images from "FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice"

    Article Title: FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105349

    Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.
    Figure Legend Snippet: Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.

    Techniques Used: Mouse Assay, Expressing, Marker

    7) Product Images from "A novel role for the 3′-5′ exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth"

    Article Title: A novel role for the 3′-5′ exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth

    Journal: RNA Biology

    doi: 10.1080/15476286.2016.1232238

    RNA-seq validation. (A) Comparison between the fold changes of the selected transcripts by RNA-seq (red dots) and qRT-PCR (gray bars). The parental control (left) and dis3L2 knockdown (right) values are presented for each transcript. n ≥ 4, error bars represent standard error. See Table 1 for p-values. (B) pyrexia ( pyx) and CG2678 show post-transcriptional changes in gene expression as the mature mRNA (dark gray) but not the pre-mRNA (light gray) show significant increases in expression. cyt-c-d and CG31808 show transcriptional changes as both the pre- (light gray) and mature (dark gray) mRNA increase in expression. The parental control (left) and dis3L2 knockdown (right) values are presented for each transcript for both the pre- and mature mRNA. n ≥ 4, error bars represent standard error. See Table 1 for p-values.
    Figure Legend Snippet: RNA-seq validation. (A) Comparison between the fold changes of the selected transcripts by RNA-seq (red dots) and qRT-PCR (gray bars). The parental control (left) and dis3L2 knockdown (right) values are presented for each transcript. n ≥ 4, error bars represent standard error. See Table 1 for p-values. (B) pyrexia ( pyx) and CG2678 show post-transcriptional changes in gene expression as the mature mRNA (dark gray) but not the pre-mRNA (light gray) show significant increases in expression. cyt-c-d and CG31808 show transcriptional changes as both the pre- (light gray) and mature (dark gray) mRNA increase in expression. The parental control (left) and dis3L2 knockdown (right) values are presented for each transcript for both the pre- and mature mRNA. n ≥ 4, error bars represent standard error. See Table 1 for p-values.

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

    8) Product Images from "Towards an understanding of the molecular basis of effective RNAi against a global insect pest, the whitefly Bemisia tabaci"

    Article Title: Towards an understanding of the molecular basis of effective RNAi against a global insect pest, the whitefly Bemisia tabaci

    Journal: Insect Biochemistry and Molecular Biology

    doi: 10.1016/j.ibmb.2017.07.005

    The dsRNase genes of Bemisia tabaci . (A) Neighbor-joining phylogenetic tree constructed using the protein sequence of the conserved DNA/RNA non-specific nuclease domain of insect dsRNase genes. The numbers at the branches indicate the %bootstrap support, based on the frequency of the clusters for 1000 bootstraps. The d sRNase sequences were: Bombyx mori 2 ( NP_001091744.1 ), Papilio machaon ( XP_014355571.1 ), Spodoptera littorallis (CAR92522.1), Spodoptera frugiperda (CAR92521.1), Aedes aegypti ( XP_001648469.1 ), Drosophila melanogaster 1 ( NM_140821.4 ), D. melanogaster 2 ( NP_649078.1 , CG3819), Tribolium castaneum 1 ( XP_973587.1 ), T. castaneum 2 ( XP_970494.1 ), Schistocerca gregaria 1 ( KJ135008 ), S. gregaria 2 ( KJ135009 ), S. gregaria 3 ( KJ135010 ), S. gregaria 4 ( KJ135011 ), Acyrthosiphon pisum (ACYPI008471), Myzus persicae (MYZPE13164_0_v1.0_000125730.4_pep) and Bemisia tabaci 1(KX390872), B. tabaci 2 (KX390873) and B. tabaci 3 (Unigene11878_BT_Q_SG_ZJU). (B) qRT-PCR analysis of the expression of BtdsRNase-1 (top) and BtdsRNase-2 (bottom) in dissected guts of B. tabaci , relative to the whole body (Wb). Mean ± s. e. from 3 replicates are shown.
    Figure Legend Snippet: The dsRNase genes of Bemisia tabaci . (A) Neighbor-joining phylogenetic tree constructed using the protein sequence of the conserved DNA/RNA non-specific nuclease domain of insect dsRNase genes. The numbers at the branches indicate the %bootstrap support, based on the frequency of the clusters for 1000 bootstraps. The d sRNase sequences were: Bombyx mori 2 ( NP_001091744.1 ), Papilio machaon ( XP_014355571.1 ), Spodoptera littorallis (CAR92522.1), Spodoptera frugiperda (CAR92521.1), Aedes aegypti ( XP_001648469.1 ), Drosophila melanogaster 1 ( NM_140821.4 ), D. melanogaster 2 ( NP_649078.1 , CG3819), Tribolium castaneum 1 ( XP_973587.1 ), T. castaneum 2 ( XP_970494.1 ), Schistocerca gregaria 1 ( KJ135008 ), S. gregaria 2 ( KJ135009 ), S. gregaria 3 ( KJ135010 ), S. gregaria 4 ( KJ135011 ), Acyrthosiphon pisum (ACYPI008471), Myzus persicae (MYZPE13164_0_v1.0_000125730.4_pep) and Bemisia tabaci 1(KX390872), B. tabaci 2 (KX390873) and B. tabaci 3 (Unigene11878_BT_Q_SG_ZJU). (B) qRT-PCR analysis of the expression of BtdsRNase-1 (top) and BtdsRNase-2 (bottom) in dissected guts of B. tabaci , relative to the whole body (Wb). Mean ± s. e. from 3 replicates are shown.

    Techniques Used: Construct, Sequencing, Quantitative RT-PCR, Expressing, Western Blot

    9) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC
    Figure Legend Snippet: Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC

    Techniques Used: RNA Sequencing Assay

    Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p
    Figure Legend Snippet: Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p

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

    10) Product Images from "Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis"

    Article Title: Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky437

    No overlap in DEGs between the different LOF methods upon depletion of SLC25A25-AS1 . ( A ) Expression levels of SLC25A25-AS1 after RNAi, LNA and CRISPRi-mediated depletion. qPCR revealed only a 25% reduction in SLC25A25-AS1 transcription after siRNA-mediated knockdown relative to negative control siRNA from Dharmacon (Control Dharm), and no significant difference relative to negative control siRNA from Ambion (Control Ambion). LNA-mediated knockdown of SLC25A25-AS1 was performed using LNA oligonucleotide sequence 2 (LNA 2), and showed 90% reduction. CRISPRi-mediated repression of SLC25A25-AS1 using two guide RNAs targeting the TSS of SLC25A25-AS1 relative to the negative (non-targeting) guide RNA 2 yielded 70–90% knockdown in clonal cells. Only one guide RNA (guide 9) was efficient in depleting SLC25A25-AS1 in non-clonal cells. Statistical significance by two-tailed Student's t -test: ** P
    Figure Legend Snippet: No overlap in DEGs between the different LOF methods upon depletion of SLC25A25-AS1 . ( A ) Expression levels of SLC25A25-AS1 after RNAi, LNA and CRISPRi-mediated depletion. qPCR revealed only a 25% reduction in SLC25A25-AS1 transcription after siRNA-mediated knockdown relative to negative control siRNA from Dharmacon (Control Dharm), and no significant difference relative to negative control siRNA from Ambion (Control Ambion). LNA-mediated knockdown of SLC25A25-AS1 was performed using LNA oligonucleotide sequence 2 (LNA 2), and showed 90% reduction. CRISPRi-mediated repression of SLC25A25-AS1 using two guide RNAs targeting the TSS of SLC25A25-AS1 relative to the negative (non-targeting) guide RNA 2 yielded 70–90% knockdown in clonal cells. Only one guide RNA (guide 9) was efficient in depleting SLC25A25-AS1 in non-clonal cells. Statistical significance by two-tailed Student's t -test: ** P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Sequencing, Two Tailed Test

    SLC25A25-AS1 is an archetypal lncRNA expressed in the nucleus. ( A ) Schematic representation of the genomic landscape surrounding SLC25A25-AS1 (annotated in RefSeq as loc100289019; chr9:128108581-128118693, hg38), including three transcriptional start sites ( 101 ) and a polyadenylation site ( 102 ) . SLC25A25-AS1 is not occupied by ribosomes ( 64 ), shows no protein coding potential (PhyloCSF, ( 46 )), and has clear hallmarks of active transcription in HeLa cells (H3K4me3 and H3K27ac data sets obtained from ENCODE via the UCSC browser). The arrows denote the direction of transcription, and green boxes represent the five exons. Note that all PhyloCSF scores at this locus are negative. ( B ) Expression of SLC25A25-AS1 in cytosol and nuclei of ENCODE cell lines ( www.ebi.ac.uk/gxa/home ), shown as reads per kilobase of exon per million reads mapped (RPKM). ( C ) Computational analysis of the mature SLC25A25-AS1 transcript using the CPC and CPAT tools reveals SLC25A25-AS1 has low coding potential. ( D ) Nuclear localization of SLC25A25-AS1 in HeLa cells was determined using single-molecule RNA FISH with exonic probes (green). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar represents 5 μm. ( E ) SLC25A25-AS1 is enriched in chromatin of HeLa cells. RNA distribution in the cytoplasm, nucleoplasm and chromatin was quantified by qPCR, and RPS18 and MALAT1 were used as positive controls for the cytoplasmic and chromatin fraction, respectively. Error bars represent the standard error of the mean (s.e.m) values of four independent experiments.
    Figure Legend Snippet: SLC25A25-AS1 is an archetypal lncRNA expressed in the nucleus. ( A ) Schematic representation of the genomic landscape surrounding SLC25A25-AS1 (annotated in RefSeq as loc100289019; chr9:128108581-128118693, hg38), including three transcriptional start sites ( 101 ) and a polyadenylation site ( 102 ) . SLC25A25-AS1 is not occupied by ribosomes ( 64 ), shows no protein coding potential (PhyloCSF, ( 46 )), and has clear hallmarks of active transcription in HeLa cells (H3K4me3 and H3K27ac data sets obtained from ENCODE via the UCSC browser). The arrows denote the direction of transcription, and green boxes represent the five exons. Note that all PhyloCSF scores at this locus are negative. ( B ) Expression of SLC25A25-AS1 in cytosol and nuclei of ENCODE cell lines ( www.ebi.ac.uk/gxa/home ), shown as reads per kilobase of exon per million reads mapped (RPKM). ( C ) Computational analysis of the mature SLC25A25-AS1 transcript using the CPC and CPAT tools reveals SLC25A25-AS1 has low coding potential. ( D ) Nuclear localization of SLC25A25-AS1 in HeLa cells was determined using single-molecule RNA FISH with exonic probes (green). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar represents 5 μm. ( E ) SLC25A25-AS1 is enriched in chromatin of HeLa cells. RNA distribution in the cytoplasm, nucleoplasm and chromatin was quantified by qPCR, and RPS18 and MALAT1 were used as positive controls for the cytoplasmic and chromatin fraction, respectively. Error bars represent the standard error of the mean (s.e.m) values of four independent experiments.

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Staining, Real-time Polymerase Chain Reaction

    11) Product Images from "Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells"

    Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04602-0

    OxPAPC elicits ATP release and inhibition of ATP release prevents induction of MTHFD2 . a−d Nucleoside measurement in HAEC exposed to medium (1% FCS) with (oxP) or without (Ct) oxPAPC for 24 h. Cell lysates were measured by mass spectrometry ( n = 6). (* p ≤ 0.05 Student’s t test). e−h Nucleoside measurement in supernatants of HAEC exposed to medium (1% FCS) with or without oxPAPC for 24 h. Supernatants were measured by mass spectrometry ( n = 4) (* p ≤ 0.05 Student’s t -test) i Scheme of flow of serine- and glycine-derived carbons which can be incorporated into the purine backbone. j , k HAEC were treated with 13 C 3 -serine ( j ) or 13 C 2 -glycine ( k ) and oxPAPC or control for 24 h and supernatants were measured by mass spectrometry ( n = 3). Relative fractions of extracellular AMP containing no ( m ), one ( m + 1), two ( m + 2) or three ( m + 3) heavy carbons are shown. l 24 h flux analysis with 13 C 3 -serine labeling in HAEC with or without siRNA mediated knockdown of MTHFD2 ( n = 3). m ATP measurement of supernatants of HAEC exposed to medium (1% FCS) with or without oxPAPC and flufenamic acid (FFA, 50 µM) for 8 h. ATP was measured by luminescence and normalized to intracellular RNA concentration ( n = 7). n , o qRT-PCR detection of MTHFD2 and PHGDH in HAEC exposed to medium (1% FCS) with or without oxPAPC and flufenamic acid (FFA, 50 µM) for 24 h ( n = 5). p , q Spheroid outgrowth assay ( p ) and quantification ( q ) of the cumulative sprout length of HUVEC treated with combinations of oxPAPC, flufenamic acid (FFA, 50 µM) and VEGF-A165 (10 ng ml −1 ) as indicated ( n = 6). Scale bar: 50 µM. Data are represented as mean ± SEM, * p ≤ 0.05 (oxP vs Ct), # p ≤ 0.05 (inhibitor present vs absent), $ p ≤ 0.05 (VEGFA vs Ct), (ANOVA with Bonferroni post-hoc test if not otherwise indicated)
    Figure Legend Snippet: OxPAPC elicits ATP release and inhibition of ATP release prevents induction of MTHFD2 . a−d Nucleoside measurement in HAEC exposed to medium (1% FCS) with (oxP) or without (Ct) oxPAPC for 24 h. Cell lysates were measured by mass spectrometry ( n = 6). (* p ≤ 0.05 Student’s t test). e−h Nucleoside measurement in supernatants of HAEC exposed to medium (1% FCS) with or without oxPAPC for 24 h. Supernatants were measured by mass spectrometry ( n = 4) (* p ≤ 0.05 Student’s t -test) i Scheme of flow of serine- and glycine-derived carbons which can be incorporated into the purine backbone. j , k HAEC were treated with 13 C 3 -serine ( j ) or 13 C 2 -glycine ( k ) and oxPAPC or control for 24 h and supernatants were measured by mass spectrometry ( n = 3). Relative fractions of extracellular AMP containing no ( m ), one ( m + 1), two ( m + 2) or three ( m + 3) heavy carbons are shown. l 24 h flux analysis with 13 C 3 -serine labeling in HAEC with or without siRNA mediated knockdown of MTHFD2 ( n = 3). m ATP measurement of supernatants of HAEC exposed to medium (1% FCS) with or without oxPAPC and flufenamic acid (FFA, 50 µM) for 8 h. ATP was measured by luminescence and normalized to intracellular RNA concentration ( n = 7). n , o qRT-PCR detection of MTHFD2 and PHGDH in HAEC exposed to medium (1% FCS) with or without oxPAPC and flufenamic acid (FFA, 50 µM) for 24 h ( n = 5). p , q Spheroid outgrowth assay ( p ) and quantification ( q ) of the cumulative sprout length of HUVEC treated with combinations of oxPAPC, flufenamic acid (FFA, 50 µM) and VEGF-A165 (10 ng ml −1 ) as indicated ( n = 6). Scale bar: 50 µM. Data are represented as mean ± SEM, * p ≤ 0.05 (oxP vs Ct), # p ≤ 0.05 (inhibitor present vs absent), $ p ≤ 0.05 (VEGFA vs Ct), (ANOVA with Bonferroni post-hoc test if not otherwise indicated)

    Techniques Used: Inhibition, Mass Spectrometry, Flow Cytometry, Derivative Assay, Labeling, Concentration Assay, Quantitative RT-PCR

    12) Product Images from "Genome-wide transcriptional profiling for elucidating the effects of brassinosteroids on Glycine max during early vegetative development"

    Article Title: Genome-wide transcriptional profiling for elucidating the effects of brassinosteroids on Glycine max during early vegetative development

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52599-3

    Validation and comparison of eight differently expressed genes selected from RNA-seq analysis by qRT-PCR. qRT-PCR data were normalized to the stable endogenous control (Cyclin gene). The fold changes are presented as mean with standard errors (SE) of three biological replications. RNA-seq results showed in gray and qRT-PCR results showed in black.
    Figure Legend Snippet: Validation and comparison of eight differently expressed genes selected from RNA-seq analysis by qRT-PCR. qRT-PCR data were normalized to the stable endogenous control (Cyclin gene). The fold changes are presented as mean with standard errors (SE) of three biological replications. RNA-seq results showed in gray and qRT-PCR results showed in black.

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR

    13) Product Images from "IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP"

    Article Title: IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP

    Journal: eLife

    doi: 10.7554/eLife.49500

    Effect of IFI16 knockdown (KD) on H3K9me3 and RNA Pol II deposition on KSHV lytic gene promoters. ( A ) IFI16 was KD in BCBL-1 cells using shRNA lentivirus for 72 hr and KD efficiency was assessed by q-RT PCR and successful induction of lytic KSHV ORF50 gene as a result of IFI16 KD was assessed by q-RT PCR of ORF50. ( B ) WB showing IFI16 KD compared to untreated or shC treated BCBL-1 cells. ( C ) ChIP was performed after lentivirus-mediated IFI16 KD in BCBL-1 cells or shC-BCBL-1 cells. Deposition of different histone H3 lysine tri-methylation marks (H3, H3K4me3, H3K9me3, H3K27me3, H3K36me3 and H3K79me3) and RNA Pol II on four different KSHV promoters (pORF73- La, pK8- IE, pvIRF2- E, and pORF63- L) representing the four different temporal KSHV gene classes were tested by q-PCR. ChIP efficiencies normalized to input chromatin are shown as relative to shC control. ( D - H ) TIME cells were electroporated with either siC or siIFI16. After 72 hr, cells were de novo infected with KSHV (100 DNA copies/cell) for 6 or 48 hr. IFI16 KD efficiencies were assessed by q-RT PCR of the IFI16 gene ( D ) and WB of IFI16 ( E ). ( F ) ChIP was performed after 48 hr of de novo infection of IFI16 KD TIME. ( G and H ) q-RT PCR (one step TaqMan) of KSHV latent ORF73 ( G ) and lytic ORF50 ( H ) mRNA expression normalized to cellular RNaseP after 6 and 48 hr of de novo infection of TIME cells previously treated with siIFI16 of siC for 72 hr. ( I ) Lytic cycle was induced in TRExBCBL1-RTA cells using doxycycline. At 0, 1, 2, 3 and 4 days post-induction, ChIP was performed. Deposition of different H3 lysine tri-methylation marks (H3, H3K4me3, H3K9me3 and H3K27me3) and RNA Pol II on the ORF63 promoters was tested by q-PCR. ChIP with control IgG was also performed. ChIP efficiencies are represented as % input. Data shown are averages of the results of at least three experiments ± SD. *=p
    Figure Legend Snippet: Effect of IFI16 knockdown (KD) on H3K9me3 and RNA Pol II deposition on KSHV lytic gene promoters. ( A ) IFI16 was KD in BCBL-1 cells using shRNA lentivirus for 72 hr and KD efficiency was assessed by q-RT PCR and successful induction of lytic KSHV ORF50 gene as a result of IFI16 KD was assessed by q-RT PCR of ORF50. ( B ) WB showing IFI16 KD compared to untreated or shC treated BCBL-1 cells. ( C ) ChIP was performed after lentivirus-mediated IFI16 KD in BCBL-1 cells or shC-BCBL-1 cells. Deposition of different histone H3 lysine tri-methylation marks (H3, H3K4me3, H3K9me3, H3K27me3, H3K36me3 and H3K79me3) and RNA Pol II on four different KSHV promoters (pORF73- La, pK8- IE, pvIRF2- E, and pORF63- L) representing the four different temporal KSHV gene classes were tested by q-PCR. ChIP efficiencies normalized to input chromatin are shown as relative to shC control. ( D - H ) TIME cells were electroporated with either siC or siIFI16. After 72 hr, cells were de novo infected with KSHV (100 DNA copies/cell) for 6 or 48 hr. IFI16 KD efficiencies were assessed by q-RT PCR of the IFI16 gene ( D ) and WB of IFI16 ( E ). ( F ) ChIP was performed after 48 hr of de novo infection of IFI16 KD TIME. ( G and H ) q-RT PCR (one step TaqMan) of KSHV latent ORF73 ( G ) and lytic ORF50 ( H ) mRNA expression normalized to cellular RNaseP after 6 and 48 hr of de novo infection of TIME cells previously treated with siIFI16 of siC for 72 hr. ( I ) Lytic cycle was induced in TRExBCBL1-RTA cells using doxycycline. At 0, 1, 2, 3 and 4 days post-induction, ChIP was performed. Deposition of different H3 lysine tri-methylation marks (H3, H3K4me3, H3K9me3 and H3K27me3) and RNA Pol II on the ORF63 promoters was tested by q-PCR. ChIP with control IgG was also performed. ChIP efficiencies are represented as % input. Data shown are averages of the results of at least three experiments ± SD. *=p

    Techniques Used: shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Methylation, Polymerase Chain Reaction, Infection, Expressing

    14) Product Images from "Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro"

    Article Title: Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.4400

    LMP1 of Epstein-Barr virus promotes miR-155 expression in CNE-2 cells. CNE-2 cells were separated into three groups: Non-transfected CNE-2 cells (Blank), CNE-2 cells transfected with empty pcDNA3.1 as negative control (Con) and LMP1-pcDNA3.1-transfected CNE-2 cells (LMP1). (A) Relative mRNA expression levels of LMP1 in the three groups of CNE-2 cells, compared with the levels of GAPDH. (B) Percentage of LMP1 expression at the protein level in the three CNE-2 cell groups, as revealed by western blot analysis. (C) Relative mRNA levels of LMP1 vs. GAPDH in the three groups of CNE-2 cells upon a number of serial passages, as determined by reverse transcription-quantitative polymerase chain reaction. (D) Overexpression of LMP1 protein in CNE-2 cells following a number of serial passages. (E) Relative miR-155 levels in the three groups of CNE-2 cells, compared with the levels of U6 snRNA. (F) Relative miR-155 levels in CNE-2 cells transfected with various concentrations of LMP1-pcDNA3.1, compared with the levels of U6 snRNA. *P
    Figure Legend Snippet: LMP1 of Epstein-Barr virus promotes miR-155 expression in CNE-2 cells. CNE-2 cells were separated into three groups: Non-transfected CNE-2 cells (Blank), CNE-2 cells transfected with empty pcDNA3.1 as negative control (Con) and LMP1-pcDNA3.1-transfected CNE-2 cells (LMP1). (A) Relative mRNA expression levels of LMP1 in the three groups of CNE-2 cells, compared with the levels of GAPDH. (B) Percentage of LMP1 expression at the protein level in the three CNE-2 cell groups, as revealed by western blot analysis. (C) Relative mRNA levels of LMP1 vs. GAPDH in the three groups of CNE-2 cells upon a number of serial passages, as determined by reverse transcription-quantitative polymerase chain reaction. (D) Overexpression of LMP1 protein in CNE-2 cells following a number of serial passages. (E) Relative miR-155 levels in the three groups of CNE-2 cells, compared with the levels of U6 snRNA. (F) Relative miR-155 levels in CNE-2 cells transfected with various concentrations of LMP1-pcDNA3.1, compared with the levels of U6 snRNA. *P

    Techniques Used: Expressing, Transfection, Negative Control, Western Blot, Real-time Polymerase Chain Reaction, Over Expression

    15) Product Images from "Characterization of macroautophagic flux in vivo using a leupeptin-based assay"

    Article Title: Characterization of macroautophagic flux in vivo using a leupeptin-based assay

    Journal: Autophagy

    doi: 10.4161/auto.7.6.15100

    Analysis of basal macroautophagic flux in beclin 1 +/+ and beclin 1 +/− mice using the leupeptin assay. Mice were injected with PBS or 40 mg/kg leupeptin and sacrificed 0–180 min later (n = 3–4 mice per time point). (A) Western blot
    Figure Legend Snippet: Analysis of basal macroautophagic flux in beclin 1 +/+ and beclin 1 +/− mice using the leupeptin assay. Mice were injected with PBS or 40 mg/kg leupeptin and sacrificed 0–180 min later (n = 3–4 mice per time point). (A) Western blot

    Techniques Used: Mouse Assay, Injection, Western Blot

    16) Product Images from "Preclinical development of HIvax: Human survivin highly immunogenic vaccines"

    Article Title: Preclinical development of HIvax: Human survivin highly immunogenic vaccines

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.1080/21645515.2015.1050572

    HIvax1 and HIvax2 induce activation of CD8 + and CD4 + T cells, respectively, in a donor with haplotype HLA-A 01:01, 11:01; HLA-B 08:01, 42:01; HLA-DRB1 03:01. Human dendritic cells (DC) were infected with HIvax1 or HIvax2 and co-cultured for 2 wks with
    Figure Legend Snippet: HIvax1 and HIvax2 induce activation of CD8 + and CD4 + T cells, respectively, in a donor with haplotype HLA-A 01:01, 11:01; HLA-B 08:01, 42:01; HLA-DRB1 03:01. Human dendritic cells (DC) were infected with HIvax1 or HIvax2 and co-cultured for 2 wks with

    Techniques Used: Activation Assay, Infection, Cell Culture

    Survivin epitopes have been included in HIvax1 e HIvax2. ( A ) HIvax 1 and 2 diagrams. Epitopes from Hsurv 5–7 were included in HIvax1, separated by AAY spacers and targeted to the ER by including an IgK signal sequence. This strategy facilitates
    Figure Legend Snippet: Survivin epitopes have been included in HIvax1 e HIvax2. ( A ) HIvax 1 and 2 diagrams. Epitopes from Hsurv 5–7 were included in HIvax1, separated by AAY spacers and targeted to the ER by including an IgK signal sequence. This strategy facilitates

    Techniques Used: Sequencing

    Cytolytic potential of HIvax-activated (T)cells against malignant mesothelioma cells overexpressing survivin. ( A ) ELISPOT analysis of released granzyme B was performed after stimulation by DC infected with FP-ctrl or HIvax1/HIvax2 simultaneously (HIvax).
    Figure Legend Snippet: Cytolytic potential of HIvax-activated (T)cells against malignant mesothelioma cells overexpressing survivin. ( A ) ELISPOT analysis of released granzyme B was performed after stimulation by DC infected with FP-ctrl or HIvax1/HIvax2 simultaneously (HIvax).

    Techniques Used: Enzyme-linked Immunospot, Infection

    17) Product Images from "Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza"

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04792-7

    The transfection efficiency and antiviral activity of TAT-P1 with DIG-3 could be increased by P1 peptide. a Transfection efficiency of TAT-P1/pLuc increased by additional ATPase inhibitor (bafilomycin A1) or P1 peptide. Before TAT-P1/pLuc (2.0 μg/0.5 μg) complex was added to 293T cells for transfection, the indicated concentrations of bafilomycin A1 (A1, nM), P1 peptide (μg per ml), or PA1 peptide (μg per ml) were added to cell culture media. Mock indicates cells treated with A1 or TAT-P1 without DNA. Data were presented as mean ± SD of three independent experiments. b Transfection efficiency of TAT-P1/pCMV-Luc increased by P1 in mouse lungs. c Representative In Vivo Imaging System image showed increased luciferase expression by P1 in mouse lungs. TAT-P1/pCMV-Luc (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) or PA1 (20 μg) were inoculated to mouse lungs at 48 and 24 h before measuring bioluminescence signal or taking bioluminescence image. Mock indicates mouse lungs inoculated with TAT-P1 + P1 without DNA. d The RNA expression of DI-PA increased by P1 in mouse lungs. TAT-P1/DI-PA (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) were inoculated to mouse lungs at 48 and 24 h before detecting RNA expression. Data were presented as mean ± SD of ≥3 mice. * Indicates P
    Figure Legend Snippet: The transfection efficiency and antiviral activity of TAT-P1 with DIG-3 could be increased by P1 peptide. a Transfection efficiency of TAT-P1/pLuc increased by additional ATPase inhibitor (bafilomycin A1) or P1 peptide. Before TAT-P1/pLuc (2.0 μg/0.5 μg) complex was added to 293T cells for transfection, the indicated concentrations of bafilomycin A1 (A1, nM), P1 peptide (μg per ml), or PA1 peptide (μg per ml) were added to cell culture media. Mock indicates cells treated with A1 or TAT-P1 without DNA. Data were presented as mean ± SD of three independent experiments. b Transfection efficiency of TAT-P1/pCMV-Luc increased by P1 in mouse lungs. c Representative In Vivo Imaging System image showed increased luciferase expression by P1 in mouse lungs. TAT-P1/pCMV-Luc (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) or PA1 (20 μg) were inoculated to mouse lungs at 48 and 24 h before measuring bioluminescence signal or taking bioluminescence image. Mock indicates mouse lungs inoculated with TAT-P1 + P1 without DNA. d The RNA expression of DI-PA increased by P1 in mouse lungs. TAT-P1/DI-PA (20 μg/5 μg) with additional P1 (20 μg, 10 μg, or 0 μg) were inoculated to mouse lungs at 48 and 24 h before detecting RNA expression. Data were presented as mean ± SD of ≥3 mice. * Indicates P

    Techniques Used: Transfection, Activity Assay, Cell Culture, In Vivo Imaging, Luciferase, Expressing, RNA Expression, Mouse Assay

    Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P
    Figure Legend Snippet: Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Techniques Used: Activity Assay, Plasmid Preparation, RNA Expression, Transfection, Quantitative RT-PCR, Negative Control, Infection, Plaque Assay

    18) Product Images from "Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells"

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.01.013

    BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.
    Figure Legend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Techniques Used: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker

    19) Product Images from "Prostaglandin E2 affects in vitro maturation of bovine oocytes"

    Article Title: Prostaglandin E2 affects in vitro maturation of bovine oocytes

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/s12958-020-00598-9

    The effect of PGE 2 (10 μM) treatment of oocyte maturation medium on apoptosis in COCs. Panel (A) depicts representative fluorescent images of bovine COCs used to TUNEL labeling: (a) negative control, (b) positive control, (c) control COCs, and (d) COCs matured in the presence of PGE 2 . White arrows indicate TUNEL stained apoptotic nuclei (green) in contrast to DAPI stained nuclei (blue). Bars = 100 μm. Panel (B) depicts quantitative analysis of PGE 2 effect on apoptosis in COCs. The data are presented as a percentage of TUNEL positive apoptotic cells within all detected DAPI positive cells and expressed as mean ± SEM of three independent repeats (25 COCs per treatment group). Different letters indicate significant differences ( P
    Figure Legend Snippet: The effect of PGE 2 (10 μM) treatment of oocyte maturation medium on apoptosis in COCs. Panel (A) depicts representative fluorescent images of bovine COCs used to TUNEL labeling: (a) negative control, (b) positive control, (c) control COCs, and (d) COCs matured in the presence of PGE 2 . White arrows indicate TUNEL stained apoptotic nuclei (green) in contrast to DAPI stained nuclei (blue). Bars = 100 μm. Panel (B) depicts quantitative analysis of PGE 2 effect on apoptosis in COCs. The data are presented as a percentage of TUNEL positive apoptotic cells within all detected DAPI positive cells and expressed as mean ± SEM of three independent repeats (25 COCs per treatment group). Different letters indicate significant differences ( P

    Techniques Used: TUNEL Assay, Labeling, Negative Control, Positive Control, Staining

    20) Product Images from "Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses"

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01588

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Figure Legend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Techniques Used: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    21) Product Images from "A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division"

    Article Title: A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14978-7

    Gain-of-function and rescue studies of linc00899 and its effect on TPPP expression. a Schematic diagram of ectopic overexpression of linc00899. Linc00899 and TPPP expression were analysed by qPCR after lentiviral overexpression using lincXpress vector encoding linc00899 cDNA in HeLa (left) and RPE1 cells (right). The expression was normalised to the scrambled linc00899 vector (negative control). n = 3–4 biological replicates, * P
    Figure Legend Snippet: Gain-of-function and rescue studies of linc00899 and its effect on TPPP expression. a Schematic diagram of ectopic overexpression of linc00899. Linc00899 and TPPP expression were analysed by qPCR after lentiviral overexpression using lincXpress vector encoding linc00899 cDNA in HeLa (left) and RPE1 cells (right). The expression was normalised to the scrambled linc00899 vector (negative control). n = 3–4 biological replicates, * P

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Plasmid Preparation, Negative Control

    22) Product Images from "Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells"

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

    Journal: Immunity

    doi: 10.1016/j.immuni.2018.01.013

    BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.
    Figure Legend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Techniques Used: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker

    23) Product Images from "MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae"

    Article Title: MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1458171

    MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.
    Figure Legend Snippet: MoSNT2 is associated with the MoTor signaling pathway. (A) Vegetative growth of M. oryzae on CM agar medium supplemented with or without 1 μg/ml rapamycin (rapa.). (B) Inhibition rate of rapamycin on the mycelial growth. (C) Expression profiles of MoSNT2 and MoTOR in the wild-type Guy11 strain at different developmental processes. (D) Linear correlation between qRT-PCR-measured expression levels of MoSNT2 and MoTOR . (E) qRT-PCR analysis of MoSNT2 expression levels in the Guy11 strain in response to rapamycin. The Guy11 strain grown in liquid CM for 48 h was transferred into fresh liquid CM in the presence or absence of 1 μg/ml rapamycin for 6 h before total RNA extraction.

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, RNA Extraction

    24) Product Images from "Gain-of-function mutation in Gli3 causes ventricular septal defects"

    Article Title: Gain-of-function mutation in Gli3 causes ventricular septal defects

    Journal: bioRxiv

    doi: 10.1101/2020.02.10.942144

    Reduced proliferation rate in ciliated regions of Gli3 Δ699/Δ699 embryonic hearts. (A, B) Immunofluorescence on mouse embryonic heart sections obtained from WT (n=12), Gli3 Δ699/Δ699 (n=8) and Gli3 XtJ/XtJ (n=12) embryos at E12.5. Proliferating cells are marked by pH3 (A) or KI67 (B). Cell nuclei are marked by DAPI. Scale bars represent a length of 100 µm in the overview and 15 µm in the insets. LV, left ventricle; RV, right ventricle.
    Figure Legend Snippet: Reduced proliferation rate in ciliated regions of Gli3 Δ699/Δ699 embryonic hearts. (A, B) Immunofluorescence on mouse embryonic heart sections obtained from WT (n=12), Gli3 Δ699/Δ699 (n=8) and Gli3 XtJ/XtJ (n=12) embryos at E12.5. Proliferating cells are marked by pH3 (A) or KI67 (B). Cell nuclei are marked by DAPI. Scale bars represent a length of 100 µm in the overview and 15 µm in the insets. LV, left ventricle; RV, right ventricle.

    Techniques Used: Immunofluorescence

    PDGFRα signalling activators rescue proliferation rate in Gli3 Δ699/Δ699 MEFs. (A-C) Immunofluorescence on and ciliary length quantifications in MEFs obtained from WT and Gli3 Δ699/Δ699 (n = 4, respectively) embryos at E12.5. (A) The ciliary axoneme is stained in green by acetylated α-tubulin, the basal body is stained in blue by γ-tubulin. The amount of PDGFRα is significantly reduced in cilia of Gli3 Δ699/Δ699 MEFs. The scale bar represents a length of 0.5 µm. (B) Ciliary length measurement. (C) Proliferating cells are marked by pH3. Cell nuclei are marked by DAPI.
    Figure Legend Snippet: PDGFRα signalling activators rescue proliferation rate in Gli3 Δ699/Δ699 MEFs. (A-C) Immunofluorescence on and ciliary length quantifications in MEFs obtained from WT and Gli3 Δ699/Δ699 (n = 4, respectively) embryos at E12.5. (A) The ciliary axoneme is stained in green by acetylated α-tubulin, the basal body is stained in blue by γ-tubulin. The amount of PDGFRα is significantly reduced in cilia of Gli3 Δ699/Δ699 MEFs. The scale bar represents a length of 0.5 µm. (B) Ciliary length measurement. (C) Proliferating cells are marked by pH3. Cell nuclei are marked by DAPI.

    Techniques Used: Immunofluorescence, Staining

    Gli3 Δ699/Δ699 mutant embryos display VSDs and a decreased HH signalling. (A) HE staining of heart sections from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ embryos at E14.5. Asterisk indicates VSD. The scale bar represents a length of 500 µm. (B, C) Quantification of Gli1 (B) and Ptch1 (C) expression via qRT-PCR analysis. Lysates were obtained from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 3, respectively) mouse embryonic hearts at E12.5. (D) Western blot analysis of GLI2 with lysates obtained from Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 3, respectively) mouse embryonic hearts at E12.5. Actin serves as loading control.
    Figure Legend Snippet: Gli3 Δ699/Δ699 mutant embryos display VSDs and a decreased HH signalling. (A) HE staining of heart sections from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ embryos at E14.5. Asterisk indicates VSD. The scale bar represents a length of 500 µm. (B, C) Quantification of Gli1 (B) and Ptch1 (C) expression via qRT-PCR analysis. Lysates were obtained from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 3, respectively) mouse embryonic hearts at E12.5. (D) Western blot analysis of GLI2 with lysates obtained from Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 3, respectively) mouse embryonic hearts at E12.5. Actin serves as loading control.

    Techniques Used: Mutagenesis, Staining, Expressing, Quantitative RT-PCR, Western Blot

    Reduced PDGFRα signalling and ciliary length in Gli3 Δ699/Δ699 hearts. (A) Immunofluorescence on heart sections obtained from WT (n=12), Gli3 Δ699/Δ699 (n=8) and Gli3 XtJ/XtJ (n=12) embryos at E12.5. The ciliary axoneme is stained in green by acetylated α-tubulin, the basal body is stained in blue by γ-tubulin. The scale bar represents a length of 0.5 µm. (B) Ciliary length measurement on mouse embryonic heart sections obtained from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 4, respectively) embryos at E12.5.
    Figure Legend Snippet: Reduced PDGFRα signalling and ciliary length in Gli3 Δ699/Δ699 hearts. (A) Immunofluorescence on heart sections obtained from WT (n=12), Gli3 Δ699/Δ699 (n=8) and Gli3 XtJ/XtJ (n=12) embryos at E12.5. The ciliary axoneme is stained in green by acetylated α-tubulin, the basal body is stained in blue by γ-tubulin. The scale bar represents a length of 0.5 µm. (B) Ciliary length measurement on mouse embryonic heart sections obtained from WT, Gli3 Δ699/Δ699 and Gli3 XtJ/XtJ (n = 4, respectively) embryos at E12.5.

    Techniques Used: Immunofluorescence, Staining

    25) Product Images from "Dedifferentiation and neuronal repression define Familial Alzheimer’s Disease"

    Article Title: Dedifferentiation and neuronal repression define Familial Alzheimer’s Disease

    Journal: bioRxiv

    doi: 10.1101/531202

    Dedifferentiation in PSEN1 M146L hiPSC-derived neurons is caused by changes in histone methylation leading to modulation of chromatin accessibility A Differentially methylated histone regions in PSEN1 M146L hiPSC-derived neurons relative to NDC as measured by ChIP-Seq for H3K4Me3 or H3K27Me3. ( n = 1) B-C Homer TF motif enrichment of all regions with a significant increase or decrease in B H3K4Me3 or C H3K27Me3 (O/S/N = OCT4-SOX2-Nanog). D-E Metascape functional enrichment of all regions with a significant increase or decrease in D H3K4Me3 or E H3K27Me3. F DEGs with a directionally corresponding increase or decrease in H3K4Me3 or H3K27Me3. G Enrichr enrichment of DEGs with significant change in H3K4Me3 and H3K27Me3 using the ENCODE/ChEA Consensus TF database. H ChIP-Seq read profiles around the summits of differential H3K4Me3 or H3K27Me3 methylation for DEGs with significant change in H3K4Me3 or H3K27Me3. I Metascape enrichment of DEGs with significant change in H3K4Me3 or H3K27Me3. J log 2 fold change in chromatin accessibility and RNA expression in genes with (left) increased gene expression and corresponding change in H3K trimethylation state or (right) decreased gene expression and corresponding change in H3K trimethylation state. K Metascape enrichment of genes with differential H3K trimethylation, chromatin accessibility, and gene expression. L-N ATAC-Seq, H3K4Me3, and H3K27Me3 read profiles around the promoter regions of L genes with increased expression, M genes with decreased expression, or N miRNAs with decreased expression. O Heatmap of log 2 fold change in RNA expression (RNA), chromatin accessibility within the promoter (ATAC), H3K4Me3 status, or H3K27Me3 (projected in negative log 2 space to correspond to expression) status for endotype-associated or previously identified AD-associated genes.
    Figure Legend Snippet: Dedifferentiation in PSEN1 M146L hiPSC-derived neurons is caused by changes in histone methylation leading to modulation of chromatin accessibility A Differentially methylated histone regions in PSEN1 M146L hiPSC-derived neurons relative to NDC as measured by ChIP-Seq for H3K4Me3 or H3K27Me3. ( n = 1) B-C Homer TF motif enrichment of all regions with a significant increase or decrease in B H3K4Me3 or C H3K27Me3 (O/S/N = OCT4-SOX2-Nanog). D-E Metascape functional enrichment of all regions with a significant increase or decrease in D H3K4Me3 or E H3K27Me3. F DEGs with a directionally corresponding increase or decrease in H3K4Me3 or H3K27Me3. G Enrichr enrichment of DEGs with significant change in H3K4Me3 and H3K27Me3 using the ENCODE/ChEA Consensus TF database. H ChIP-Seq read profiles around the summits of differential H3K4Me3 or H3K27Me3 methylation for DEGs with significant change in H3K4Me3 or H3K27Me3. I Metascape enrichment of DEGs with significant change in H3K4Me3 or H3K27Me3. J log 2 fold change in chromatin accessibility and RNA expression in genes with (left) increased gene expression and corresponding change in H3K trimethylation state or (right) decreased gene expression and corresponding change in H3K trimethylation state. K Metascape enrichment of genes with differential H3K trimethylation, chromatin accessibility, and gene expression. L-N ATAC-Seq, H3K4Me3, and H3K27Me3 read profiles around the promoter regions of L genes with increased expression, M genes with decreased expression, or N miRNAs with decreased expression. O Heatmap of log 2 fold change in RNA expression (RNA), chromatin accessibility within the promoter (ATAC), H3K4Me3 status, or H3K27Me3 (projected in negative log 2 space to correspond to expression) status for endotype-associated or previously identified AD-associated genes.

    Techniques Used: Derivative Assay, Methylation, Chromatin Immunoprecipitation, Functional Assay, RNA Expression, Expressing

    Modulation of chromatin accessibility drives differential gene expression and dedifferentiation in PSEN1 M146L hiPSC-derived neurons A Differentially accessible regions of chromatin in PSEN1 M146L hiPSC-derived neurons relative to NDC as measured by ATAC-Seq; top, all differentially accessible regions; bottom, differentially expressed genes with differentially accessible regions of chromatin. Colors maintained A-C. ( n = 2) B MA Plot of differential chromatin accessibility occurring within the promotor region or outside the promotor region. C Violin plot of RNA log 2 fold-change for genes with differential accessibility and gene expression. D Homer TF motif enrichment of all regions with increased accessibility (top, purple) or decreased accessibility (green, bottom). E TF motifs with a significant (adj. p-value
    Figure Legend Snippet: Modulation of chromatin accessibility drives differential gene expression and dedifferentiation in PSEN1 M146L hiPSC-derived neurons A Differentially accessible regions of chromatin in PSEN1 M146L hiPSC-derived neurons relative to NDC as measured by ATAC-Seq; top, all differentially accessible regions; bottom, differentially expressed genes with differentially accessible regions of chromatin. Colors maintained A-C. ( n = 2) B MA Plot of differential chromatin accessibility occurring within the promotor region or outside the promotor region. C Violin plot of RNA log 2 fold-change for genes with differential accessibility and gene expression. D Homer TF motif enrichment of all regions with increased accessibility (top, purple) or decreased accessibility (green, bottom). E TF motifs with a significant (adj. p-value

    Techniques Used: Expressing, Derivative Assay

    26) Product Images from "HeT-A_pi1, a piRNA Target Sequence in the Drosophila Telomeric Retrotransposon HeT-A, Is Extremely Conserved across Copies and Species"

    Article Title: HeT-A_pi1, a piRNA Target Sequence in the Drosophila Telomeric Retrotransposon HeT-A, Is Extremely Conserved across Copies and Species

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037405

    Alignment of 3′UTR transcripts obtained from testes and ovaries of D. melanogaster Oregon R. Nucleotide polymorphisms are indicated. piRNA target HeT-A_pi1 is labelled with a red rectangle.
    Figure Legend Snippet: Alignment of 3′UTR transcripts obtained from testes and ovaries of D. melanogaster Oregon R. Nucleotide polymorphisms are indicated. piRNA target HeT-A_pi1 is labelled with a red rectangle.

    Techniques Used:

    27) 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

    28) Product Images from "Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing"

    Article Title: Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069973

    Percentage of fungal OTUs shared between nucleic acid type (DNA; RNA) in different sampling times (t1 = 47 days; t2 = 104 days).
    Figure Legend Snippet: Percentage of fungal OTUs shared between nucleic acid type (DNA; RNA) in different sampling times (t1 = 47 days; t2 = 104 days).

    Techniques Used: Sampling

    NMDS analysis based on relative abundance of fungal OTUs based on environmental DNA and RNA both at sampling times t1-47 days and t2-104 days of plant age.
    Figure Legend Snippet: NMDS analysis based on relative abundance of fungal OTUs based on environmental DNA and RNA both at sampling times t1-47 days and t2-104 days of plant age.

    Techniques Used: Sampling

    Relative abundance of different fungal phyla recovered from environmental DNA and RNA in soils with two maize cultivars (M = Monumental; D = DKC 3420) and their respective genetically modified lines (M-GM = event MON810; D-GM = DKC 3421YG) at two different sampling times (t1 = 47 days; t2 = 104 days) of plant age.
    Figure Legend Snippet: Relative abundance of different fungal phyla recovered from environmental DNA and RNA in soils with two maize cultivars (M = Monumental; D = DKC 3420) and their respective genetically modified lines (M-GM = event MON810; D-GM = DKC 3421YG) at two different sampling times (t1 = 47 days; t2 = 104 days) of plant age.

    Techniques Used: Genetically Modified, Sampling

    Non-metric multidimensional scaling (NMDS) analysis of presence and absence of fungal OTUs based on Jaccard-index of similarity with 95% confidence intervals shared between sampling times (t1 = 47 days; t2 = 104 days) and nucleic acid type (DNA, RNA).
    Figure Legend Snippet: Non-metric multidimensional scaling (NMDS) analysis of presence and absence of fungal OTUs based on Jaccard-index of similarity with 95% confidence intervals shared between sampling times (t1 = 47 days; t2 = 104 days) and nucleic acid type (DNA, RNA).

    Techniques Used: Sampling

    29) Product Images from "Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells"

    Article Title: Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084072

    Differential expression profiles of cNCCs and tNCCs in P0-Cre/Floxed-EGFP mouse embryos. (A) Scatter plot of Craniofacial EGFP + cells (Cp) and Trunk EGFP + cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP + cells (blue) and Trunk EGFP + cells (red), compared with those in the EGFP + cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP − cells, and Tn; trunk EGFP − cells.
    Figure Legend Snippet: Differential expression profiles of cNCCs and tNCCs in P0-Cre/Floxed-EGFP mouse embryos. (A) Scatter plot of Craniofacial EGFP + cells (Cp) and Trunk EGFP + cells (Tp) as assessed by microarray analysis (3D-Gene; Toray Industries). (B) Most up-regulated genes in Craniofacial EGFP + cells (blue) and Trunk EGFP + cells (red), compared with those in the EGFP + cells of trunk and craniofacial regions, respectively. (C) Biplot of principal component analysis of the eight samples revealed three sample groups. Black dots indicate all genes and red dots indicate known stem cell genes selected from GO annotations. Cp, Tp, Cn; craniofacial EGFP − cells, and Tn; trunk EGFP − cells.

    Techniques Used: Expressing, Microarray

    30) Product Images from "GmBZL3 acts as a major BR signaling regulator through crosstalk with multiple pathways in Glycine max"

    Article Title: GmBZL3 acts as a major BR signaling regulator through crosstalk with multiple pathways in Glycine max

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-019-1677-2

    Expression patterns of GmBZL3 target genes in response to a BR inhibitor in combination with or without epibrassinolide. a Heatmap representation of expression patterns of different GmBZL3 targets in soybean Williams 82 under following conditions (Pcz: 5 μM Pcz for 10 days. Pcz-BL: 5 μM Pcz with 10 nM BL for 10 days. Pcz-BL-1 h: 5 μM Pcz for 10 days then with 1 μM BL for 1 h. Pcz-BL-8 h: 5 μM Pcz for 10 days then with 1 μM BL for 8 h). The expression data values were median-centered and normalized for each gene before transforming to the color scale (log2-transformed ratios). The color bar at the bottom shows the range of expression values from highest expression level (red) to lowest expression level (green). 0 is the median expression level (Black). b qRT-PCR analysis of six GmBZL3 target genes was performed using total RNA isolated from Wm82 seedlings under control. Pcz. Pcz-BL. Pcz-BL-1 h and Pcz-BL-8 h treatments. Relative gene expression levels (fold change, log2) are shown following normalization with actin (Glyma.18G290800) transcript values. Error bars represent the standard error of the mean. The y-axis represents the relative gene expression level in different samples. Three independent experiments were performed. A representative result is shown. The star (*) indicates statistically significant differences among the means ( p
    Figure Legend Snippet: Expression patterns of GmBZL3 target genes in response to a BR inhibitor in combination with or without epibrassinolide. a Heatmap representation of expression patterns of different GmBZL3 targets in soybean Williams 82 under following conditions (Pcz: 5 μM Pcz for 10 days. Pcz-BL: 5 μM Pcz with 10 nM BL for 10 days. Pcz-BL-1 h: 5 μM Pcz for 10 days then with 1 μM BL for 1 h. Pcz-BL-8 h: 5 μM Pcz for 10 days then with 1 μM BL for 8 h). The expression data values were median-centered and normalized for each gene before transforming to the color scale (log2-transformed ratios). The color bar at the bottom shows the range of expression values from highest expression level (red) to lowest expression level (green). 0 is the median expression level (Black). b qRT-PCR analysis of six GmBZL3 target genes was performed using total RNA isolated from Wm82 seedlings under control. Pcz. Pcz-BL. Pcz-BL-1 h and Pcz-BL-8 h treatments. Relative gene expression levels (fold change, log2) are shown following normalization with actin (Glyma.18G290800) transcript values. Error bars represent the standard error of the mean. The y-axis represents the relative gene expression level in different samples. Three independent experiments were performed. A representative result is shown. The star (*) indicates statistically significant differences among the means ( p

    Techniques Used: Expressing, Transformation Assay, Quantitative RT-PCR, Isolation

    31) Product Images from "RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response."

    Article Title: RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response.

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.15044.1

    Analysis of protein levels of genes with increased transcription in hypoxia, IOX2 and VH032. HIF targets were increased in hypoxia, VH298 and FG-4592. 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 were introduced to ( A ) HeLa or HFF for 24 hours and ( B ) HeLa for indicated time. Protein levels were analysed by immunoblotting using antibodies against indicated proteins, with β-Actin as loading control. The blots shown are representative of three independent experiments. * indicates longer exposure.
    Figure Legend Snippet: Analysis of protein levels of genes with increased transcription in hypoxia, IOX2 and VH032. HIF targets were increased in hypoxia, VH298 and FG-4592. 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 were introduced to ( A ) HeLa or HFF for 24 hours and ( B ) HeLa for indicated time. Protein levels were analysed by immunoblotting using antibodies against indicated proteins, with β-Actin as loading control. The blots shown are representative of three independent experiments. * indicates longer exposure.

    Techniques Used:

    Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.
    Figure Legend Snippet: Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test

    Differential gene expression analysis of RNA-seq results. ( A ) Heatmap of Pearson correlations among RNA-seq samples that have been normalised to their total counts. ( B ) Multidimensional scaling plot of RNA-seq data. The distance between two samples reflects the leading logFC of the corresponding samples. The leading logFC is the average (root mean square) of the 2000 largest absolute logFCs for genes between those two samples. ( C–E ) Heatmaps of log2 counts per million (logcpm) across all the samples using the top 100 most differentially expressed (DE) genes in ( C ) Hypoxia, ( D ) VH032, and ( E ) IOX2. The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to a sample and each row corresponds to a specific gene. ( F ) Heatmaps of Pearson correlations between replicates of the same conditions. Each data had been normalised to their total counts. ( G ) Each dot represents a differentially expressed gene comparing the condition stated in the heading legend to DMSO vehicle control. Blue dots represent genes with increased expression (logFC > 0.58; to the right) or decreased expression (logFC
    Figure Legend Snippet: Differential gene expression analysis of RNA-seq results. ( A ) Heatmap of Pearson correlations among RNA-seq samples that have been normalised to their total counts. ( B ) Multidimensional scaling plot of RNA-seq data. The distance between two samples reflects the leading logFC of the corresponding samples. The leading logFC is the average (root mean square) of the 2000 largest absolute logFCs for genes between those two samples. ( C–E ) Heatmaps of log2 counts per million (logcpm) across all the samples using the top 100 most differentially expressed (DE) genes in ( C ) Hypoxia, ( D ) VH032, and ( E ) IOX2. The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to a sample and each row corresponds to a specific gene. ( F ) Heatmaps of Pearson correlations between replicates of the same conditions. Each data had been normalised to their total counts. ( G ) Each dot represents a differentially expressed gene comparing the condition stated in the heading legend to DMSO vehicle control. Blue dots represent genes with increased expression (logFC > 0.58; to the right) or decreased expression (logFC

    Techniques Used: Expressing, RNA Sequencing Assay

    RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.
    Figure Legend Snippet: RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test, Chromatin Immunoprecipitation, Binding Assay

    Transcription factor enrichment analysis. Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in ( A ) VH032, ( B ) IOX2 and ( C ) hypoxia, or ( D ) downregulated in hypoxia. The graph represents the adjusted p-value (-log10 false discovery rate (FDR)) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.
    Figure Legend Snippet: Transcription factor enrichment analysis. Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in ( A ) VH032, ( B ) IOX2 and ( C ) hypoxia, or ( D ) downregulated in hypoxia. The graph represents the adjusted p-value (-log10 false discovery rate (FDR)) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay

    32) Product Images from "RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response."

    Article Title: RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response.

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.15044.1

    Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.
    Figure Legend Snippet: Validation of genes with increased transcript level in hypoxia, IOX2 and VH032. ( A ) Bar plot showing log2FC according to data obtained from RNA seq analysis of known HIF target genes in hypoxia, IOX2 and VH032. ( B ) HeLa and ( C ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test

    Differential gene expression analysis of RNA-seq results. ( A ) Heatmap of Pearson correlations among RNA-seq samples that have been normalised to their total counts. ( B ) Multidimensional scaling plot of RNA-seq data. The distance between two samples reflects the leading logFC of the corresponding samples. The leading logFC is the average (root mean square) of the 2000 largest absolute logFCs for genes between those two samples. ( C–E ) Heatmaps of log2 counts per million (logcpm) across all the samples using the top 100 most differentially expressed (DE) genes in ( C ) Hypoxia, ( D ) VH032, and ( E ) IOX2. The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to a sample and each row corresponds to a specific gene. ( F ) Heatmaps of Pearson correlations between replicates of the same conditions. Each data had been normalised to their total counts. ( G ) Each dot represents a differentially expressed gene comparing the condition stated in the heading legend to DMSO vehicle control. Blue dots represent genes with increased expression (logFC > 0.58; to the right) or decreased expression (logFC
    Figure Legend Snippet: Differential gene expression analysis of RNA-seq results. ( A ) Heatmap of Pearson correlations among RNA-seq samples that have been normalised to their total counts. ( B ) Multidimensional scaling plot of RNA-seq data. The distance between two samples reflects the leading logFC of the corresponding samples. The leading logFC is the average (root mean square) of the 2000 largest absolute logFCs for genes between those two samples. ( C–E ) Heatmaps of log2 counts per million (logcpm) across all the samples using the top 100 most differentially expressed (DE) genes in ( C ) Hypoxia, ( D ) VH032, and ( E ) IOX2. The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to a sample and each row corresponds to a specific gene. ( F ) Heatmaps of Pearson correlations between replicates of the same conditions. Each data had been normalised to their total counts. ( G ) Each dot represents a differentially expressed gene comparing the condition stated in the heading legend to DMSO vehicle control. Blue dots represent genes with increased expression (logFC > 0.58; to the right) or decreased expression (logFC

    Techniques Used: Expressing, RNA Sequencing Assay

    RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.
    Figure Legend Snippet: RNA seq validation of genes solely upregulated in hypoxia and IOX2, but not VH032. ( A ) HeLa and ( B ) HFF cells were treated with 0.05% DMSO (vehicle control), 1% O 2 (hypoxia), 100 µM VH298 and 50 µM FG-4592 for 16 h prior to mRNA extraction. The graphs show relative mRNA transcripts normalised to actin mRNA levels. The mean + SEM were determined from three independent experiments. Two-tailed student t-test analysis was performed * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns: P > 0.05. ( C ) Table showing log2FC according to data obtained from RNA-seq analysis of known HIF target genes in hypoxia and IOX2, but not VH032. ( D ) Gene set enrichment analysis (GSEA) MsigDB showing significant enrichment of gene set signatures for genes upregulated in hypoxia and IOX2, but not found in VH032 at 5% false discovery rate (FDR). ( E ) Transcription factor enrichment analysis using TFEA.ChIP showing binding site enrichment for genes upregulated in hypoxia and IOX2, but not B032. The graph represents the adjusted p value (-log10 FDR) and the log-odds ratio (Log2.OR) for the association of ChIP datasets.

    Techniques Used: RNA Sequencing Assay, Two Tailed Test, Chromatin Immunoprecipitation, Binding Assay

    33) Product Images from "Sex differences in gene regulation in the dorsal root ganglion after nerve injury"

    Article Title: Sex differences in gene regulation in the dorsal root ganglion after nerve injury

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-5512-9

    Comparison of differential gene expression between CCI and naïve groups in male and female rats. a Schematic diagram of experiment. Male and female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR
    Figure Legend Snippet: Comparison of differential gene expression between CCI and naïve groups in male and female rats. a Schematic diagram of experiment. Male and female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    Gene ontology in differentially expressed genes that are upregulated after CCI. a Schematic diagram of experiment. Male and female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR
    Figure Legend Snippet: Gene ontology in differentially expressed genes that are upregulated after CCI. a Schematic diagram of experiment. Male and female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR

    Techniques Used: RNA Sequencing Assay

    Schematic diagram of experimental procedures. Male and female rats were randomly assigned to the naïve group or receive CCI. Total RNA was isolated from the L4-L6 DRG of naïve rats and on day 14 after CCI to the sciatic nerve. Libraries were constructed after poly(A) selection and sequenced. RNA-seq was performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG), defined as genes expressed after CCI versus naïve with a |log 2 fold change (FC)| > 0.5 and an FDR
    Figure Legend Snippet: Schematic diagram of experimental procedures. Male and female rats were randomly assigned to the naïve group or receive CCI. Total RNA was isolated from the L4-L6 DRG of naïve rats and on day 14 after CCI to the sciatic nerve. Libraries were constructed after poly(A) selection and sequenced. RNA-seq was performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG), defined as genes expressed after CCI versus naïve with a |log 2 fold change (FC)| > 0.5 and an FDR

    Techniques Used: Isolation, Construct, Selection, RNA Sequencing Assay

    Sex differences of gene expression in DRGs from naïve rats. a Schematic diagram of experiment. RNA-seq performed on ipsilateral L4-L6 DRGs from naïve male and female rats. Differentially expressed genes (DEG) in one sex are identified by a |log 2 fold change (FC)| > 0.5 and an FDR
    Figure Legend Snippet: Sex differences of gene expression in DRGs from naïve rats. a Schematic diagram of experiment. RNA-seq performed on ipsilateral L4-L6 DRGs from naïve male and female rats. Differentially expressed genes (DEG) in one sex are identified by a |log 2 fold change (FC)| > 0.5 and an FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    Differential gene expression between CCI and naïve groups in female rats. a Schematic diagram of experiment. Female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) are defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR
    Figure Legend Snippet: Differential gene expression between CCI and naïve groups in female rats. a Schematic diagram of experiment. Female rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) are defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an FDR

    Techniques Used: Expressing, RNA Sequencing Assay

    Differential gene expression between CCI and naïve groups in male rats. a Schematic diagram of experiment. Male rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) are defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an adjusted p -value
    Figure Legend Snippet: Differential gene expression between CCI and naïve groups in male rats. a Schematic diagram of experiment. Male rats were randomly assigned to the naïve group or receive CCI. RNA-seq performed on ipsilateral L4-L6 DRGs from each animal. Differentially expressed genes (DEG) are defined as genes expressed after CCI versus naïve with a |log 2 FC| > 0.5 and an adjusted p -value

    Techniques Used: Expressing, RNA Sequencing Assay

    34) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    35) Product Images from "Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer"

    Article Title: Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

    Journal: Epigenetics

    doi: 10.1080/15592294.2017.1300729

    Relationship between methylation and expression of LCT14 in CRC. (A) Schematic diagram of the LCT14 genomic locus on human chromosome 5 (coordinates: 24,487,209–27,038,689) with indicated positions of the annotated genes CDH10, LOC105374693 , and CDH9 and of the intact intergenic LINE1 (L1) that drives transcription of LCT14. At the bottom is an enlargement of the region including the LINE-1 (L1PA2; chr5:25,378,639–25,384,665) from which LCT14 originates with the regions (black bars) tested by bisulfite or hydroxymethylated DNA (hMeDIP) and chromatin (ChIP) immunoprecipitations and, below these, the LCT14 transcript (chr5: 25,384,485–25,384,958) and the region amplified for expression studies. All coordinates are from hg19 annotations; scale is in kb. (B) Expression of LCT14 measured by real-time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray) and tumor (light gray) tissues from 4 colorectal cancer patients (left panel) and of 5 colorectal cancer cell lines (right panel). (C) Methylation levels measured by bisulfite sequencing in the paired normal and tumor tissues of the 4 patients (left panel) and cell lines (right panel) described in B.
    Figure Legend Snippet: Relationship between methylation and expression of LCT14 in CRC. (A) Schematic diagram of the LCT14 genomic locus on human chromosome 5 (coordinates: 24,487,209–27,038,689) with indicated positions of the annotated genes CDH10, LOC105374693 , and CDH9 and of the intact intergenic LINE1 (L1) that drives transcription of LCT14. At the bottom is an enlargement of the region including the LINE-1 (L1PA2; chr5:25,378,639–25,384,665) from which LCT14 originates with the regions (black bars) tested by bisulfite or hydroxymethylated DNA (hMeDIP) and chromatin (ChIP) immunoprecipitations and, below these, the LCT14 transcript (chr5: 25,384,485–25,384,958) and the region amplified for expression studies. All coordinates are from hg19 annotations; scale is in kb. (B) Expression of LCT14 measured by real-time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray) and tumor (light gray) tissues from 4 colorectal cancer patients (left panel) and of 5 colorectal cancer cell lines (right panel). (C) Methylation levels measured by bisulfite sequencing in the paired normal and tumor tissues of the 4 patients (left panel) and cell lines (right panel) described in B.

    Techniques Used: Methylation, Expressing, Chromatin Immunoprecipitation, Amplification, Quantitative RT-PCR, Methylation Sequencing

    36) Product Images from "Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators"

    Article Title: Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators

    Journal: Nature

    doi: 10.1038/nature25179

    MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).
    Figure Legend Snippet: MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Western Blot, Knock-Out, Clone Assay, Derivative Assay, Fluorescence In Situ Hybridization, Quantitation Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Northern Blot, Sequencing, Marker

    HUSH/MORC2 binding at L1s decreases active host gene expression. a. Heatmaps showing MPP8 and H3K9me3 ChIP signal enrichment, centered on MPP8 and MORC2 summits and separated by L1 presence or absence. b. Expression change of genes with intronic full-length L1s that are bound or unbound by MORC2 or MPP8 (RNA-seq reads from KO K562 clones compared to Ctrl). Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. p-value, two-sided Mann-Whitney-Wilcoxon test. c. Genome browser tracks: HUSH/MORC2 loss causing H3K9me3 decrease at the target L1 and expression increase at both the target L1 and its host gene, independently repeated once with similar results. d. Deleting the target intronic L1 from CYP3A5 in K562 increases CYP3A5 expression, by RT-qPCR normalized to wild-type sample. n = 2 biological replicates × 3 technical replicates (center value as median). Gel image confirms L1 deletion; two experiments repeated independently with similar results. e. RT-qPCR for CYP3A5 expression in K562 clones, normalized to Ctrl. n = 2 biological replicates × 3 technical replicates (center value as median). f. Model: HUSH/MORC2 bind young full-length L1s within transcriptionally active genes, and promote H3K9me3 deposition at target L1s to silence L1 transcription. This pathway not only inhibits L1 retrotransposition, but also decreases host gene expression.
    Figure Legend Snippet: HUSH/MORC2 binding at L1s decreases active host gene expression. a. Heatmaps showing MPP8 and H3K9me3 ChIP signal enrichment, centered on MPP8 and MORC2 summits and separated by L1 presence or absence. b. Expression change of genes with intronic full-length L1s that are bound or unbound by MORC2 or MPP8 (RNA-seq reads from KO K562 clones compared to Ctrl). Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. p-value, two-sided Mann-Whitney-Wilcoxon test. c. Genome browser tracks: HUSH/MORC2 loss causing H3K9me3 decrease at the target L1 and expression increase at both the target L1 and its host gene, independently repeated once with similar results. d. Deleting the target intronic L1 from CYP3A5 in K562 increases CYP3A5 expression, by RT-qPCR normalized to wild-type sample. n = 2 biological replicates × 3 technical replicates (center value as median). Gel image confirms L1 deletion; two experiments repeated independently with similar results. e. RT-qPCR for CYP3A5 expression in K562 clones, normalized to Ctrl. n = 2 biological replicates × 3 technical replicates (center value as median). f. Model: HUSH/MORC2 bind young full-length L1s within transcriptionally active genes, and promote H3K9me3 deposition at target L1s to silence L1 transcription. This pathway not only inhibits L1 retrotransposition, but also decreases host gene expression.

    Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay, Clone Assay, MANN-WHITNEY, Quantitative RT-PCR

    HUSH/MORC2 preferentially bind intronic L1s within actively transcribed genes. a. Genes that contain MPP8 or MORC2 bound intronic L1s are expressed at significantly higher levels in Ctrl K562 cells, compared to genes that contain intronic full-length L1s unbound by MPP8 or MORC2. p-value, two-sided Mann-Whitney-Wilcoxon test. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. b. The promoters of genes that contain MPP8 or MORC2 bound intronic full-length L1s are marked by transcriptionally permissive H3K27ac in wild-type K562 cells. H3K27ac ChIP-seq data are taken from K562 epigenome pilot study, accession number PRJEB8620. TSS, transcription start site. c. Genes selectively occupied by MORC2/MPP8 either in K562 or in hESC cells exhibit higher gene expression in the corresponding cell line (p-values = 4.3 × 10 −107 for MPP8 binding; p-values = 5.0 × 10 −92 for MORC2 binding, Kruskal-Wallis test). Boxplots defined as in panel a. RNA-seq datasets for hESC are from SRA entries SRR2043329 and SRR2043330. d. ChIP-qPCR assays quantifying HUSH/MORC2 binding to an inducible L1 transgene in K562 cells before or after its transcriptional induction via Dox. Transcriptional induction increases binding of MORC2 and MPP8 to the L1 transgene. n = 2 biological replicates × 3 technical replicates (center value as median).
    Figure Legend Snippet: HUSH/MORC2 preferentially bind intronic L1s within actively transcribed genes. a. Genes that contain MPP8 or MORC2 bound intronic L1s are expressed at significantly higher levels in Ctrl K562 cells, compared to genes that contain intronic full-length L1s unbound by MPP8 or MORC2. p-value, two-sided Mann-Whitney-Wilcoxon test. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. b. The promoters of genes that contain MPP8 or MORC2 bound intronic full-length L1s are marked by transcriptionally permissive H3K27ac in wild-type K562 cells. H3K27ac ChIP-seq data are taken from K562 epigenome pilot study, accession number PRJEB8620. TSS, transcription start site. c. Genes selectively occupied by MORC2/MPP8 either in K562 or in hESC cells exhibit higher gene expression in the corresponding cell line (p-values = 4.3 × 10 −107 for MPP8 binding; p-values = 5.0 × 10 −92 for MORC2 binding, Kruskal-Wallis test). Boxplots defined as in panel a. RNA-seq datasets for hESC are from SRA entries SRR2043329 and SRR2043330. d. ChIP-qPCR assays quantifying HUSH/MORC2 binding to an inducible L1 transgene in K562 cells before or after its transcriptional induction via Dox. Transcriptional induction increases binding of MORC2 and MPP8 to the L1 transgene. n = 2 biological replicates × 3 technical replicates (center value as median).

    Techniques Used: MANN-WHITNEY, Chromatin Immunoprecipitation, Expressing, Binding Assay, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    37) Product Images from "Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators"

    Article Title: Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators

    Journal: Nature

    doi: 10.1038/nature25179

    MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).
    Figure Legend Snippet: MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Western Blot, Knock-Out, Clone Assay, Derivative Assay, Fluorescence In Situ Hybridization, Quantitation Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Northern Blot, Sequencing, Marker

    38) Product Images from "A nested parallel experiment demonstrates differences in intensity-dependence between RNA-seq and microarrays"

    Article Title: A nested parallel experiment demonstrates differences in intensity-dependence between RNA-seq and microarrays

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv636

    Comparison of the log fold change estimates measured with qPCR compared to estimates from the microarray or RNA-Seq, for 13 selected low-intensity genes that disagreed between RNA-Seq and microarray.
    Figure Legend Snippet: Comparison of the log fold change estimates measured with qPCR compared to estimates from the microarray or RNA-Seq, for 13 selected low-intensity genes that disagreed between RNA-Seq and microarray.

    Techniques Used: Real-time Polymerase Chain Reaction, Microarray, RNA Sequencing Assay

    Boxplots comparing the normalized and centered expression values of microarrays, RNA-Seq and qPCR of the six genes for which microarray and qPCR most disagreed. This shows that in many cases, microarray measurements were very consistent between biological, technical and chip replicates. This suggests that the problem is not variation at low-intensity microarray measurements, but rather bias.
    Figure Legend Snippet: Boxplots comparing the normalized and centered expression values of microarrays, RNA-Seq and qPCR of the six genes for which microarray and qPCR most disagreed. This shows that in many cases, microarray measurements were very consistent between biological, technical and chip replicates. This suggests that the problem is not variation at low-intensity microarray measurements, but rather bias.

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Microarray, Chromatin Immunoprecipitation

    39) Product Images from "Global identification and analysis of long non-coding RNAs in diploid strawberry Fragaria vesca during flower and fruit development"

    Article Title: Global identification and analysis of long non-coding RNAs in diploid strawberry Fragaria vesca during flower and fruit development

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2014-2

    Validation of anther/pollen specific expression of lncRNAs by qRT-PCR. a Gel image of RT-PCR products of ten randomly selected anther/pollen specific lncRNAs. b to g The expression of six lncRNAs quantified by qRT-PCR (black bar and Y-axis on the left). Error bar indicates standard deviation (SD) of two biological replicates with three technical replicates each. The relative FPKM of the same six lncRNAs based on RNA-seq data was also shown (red line and Y-axis on the right). Gene11892 was used as the internal control for both RNA-seq (red line) and qRT-PCR (black bar). RNAs were from anthers at stage7/8, stage9, stage10, stage11, and stage12 as well as mature pollen
    Figure Legend Snippet: Validation of anther/pollen specific expression of lncRNAs by qRT-PCR. a Gel image of RT-PCR products of ten randomly selected anther/pollen specific lncRNAs. b to g The expression of six lncRNAs quantified by qRT-PCR (black bar and Y-axis on the left). Error bar indicates standard deviation (SD) of two biological replicates with three technical replicates each. The relative FPKM of the same six lncRNAs based on RNA-seq data was also shown (red line and Y-axis on the right). Gene11892 was used as the internal control for both RNA-seq (red line) and qRT-PCR (black bar). RNAs were from anthers at stage7/8, stage9, stage10, stage11, and stage12 as well as mature pollen

    Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, RNA Sequencing Assay

    40) Product Images from "Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway"

    Article Title: Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway

    Journal: Scientific Reports

    doi: 10.1038/srep35783

    Transcriptome profiling of bladder cancer compared to normal bladder tissues pointed out the PPAR family. ( a ) Heat map of the differentially expressed genes in three bladder cancer tissues compared with three normal bladder tissues. Red color indicated upregulated genes and green color indicated downregulated genes. ( b ) GO-map network analysis by GCBI platform revealed fatty acid biosynthesis and glycerolipid metabolism were linked with bladder cancer via PPAR and ErbB signalling pathways, as well as a close correlation between bladder cancer and cell cycle. ( c ) Semiquantitative RT-PCR analysis for alterations of PPAR family ( PPARα , PPARβ and PPAR γ) using pooled total RNA isolated from the three bladder cancer tissues versus three normal bladder tissues. The expression of the GAPDH mRNA was used as a loading control. ( d ) ELISA analysis revealed the relative PPARγ DNA-binding activity in the BCa tissues was significantly decreased comparing with the normal bladder tissues (n = 3). *p
    Figure Legend Snippet: Transcriptome profiling of bladder cancer compared to normal bladder tissues pointed out the PPAR family. ( a ) Heat map of the differentially expressed genes in three bladder cancer tissues compared with three normal bladder tissues. Red color indicated upregulated genes and green color indicated downregulated genes. ( b ) GO-map network analysis by GCBI platform revealed fatty acid biosynthesis and glycerolipid metabolism were linked with bladder cancer via PPAR and ErbB signalling pathways, as well as a close correlation between bladder cancer and cell cycle. ( c ) Semiquantitative RT-PCR analysis for alterations of PPAR family ( PPARα , PPARβ and PPAR γ) using pooled total RNA isolated from the three bladder cancer tissues versus three normal bladder tissues. The expression of the GAPDH mRNA was used as a loading control. ( d ) ELISA analysis revealed the relative PPARγ DNA-binding activity in the BCa tissues was significantly decreased comparing with the normal bladder tissues (n = 3). *p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, BIA-KA

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts
    Article Snippet: .. qPCR Confluent monocultures of MCF-7s and NBFs were trypsinized and ∼20×104 cells were used for mRNA isolation (RNEasy with RNAse-Free DNase set, Qiagen). qPCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) on a LightCycler 480 (Roche, Indianapolis, IN). ..

    Isolation:

    Article Title: Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts
    Article Snippet: .. qPCR Confluent monocultures of MCF-7s and NBFs were trypsinized and ∼20×104 cells were used for mRNA isolation (RNEasy with RNAse-Free DNase set, Qiagen). qPCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) on a LightCycler 480 (Roche, Indianapolis, IN). ..

    Purification:

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza
    Article Snippet: .. Extracted RNA were treated with DNase I (QIAGEN, Cat# 79254, USA) according to the manufacturer’s protocol and purified by RNeasy Mini Kit (QIAGEN, Cat# 74106, USA) to exclude plasmid DNA contamination. .. Real-time RT-qPCR was performed as we described previously .

    SYBR Green Assay:

    Article Title: Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts
    Article Snippet: .. qPCR Confluent monocultures of MCF-7s and NBFs were trypsinized and ∼20×104 cells were used for mRNA isolation (RNEasy with RNAse-Free DNase set, Qiagen). qPCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) on a LightCycler 480 (Roche, Indianapolis, IN). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts
    Article Snippet: .. qPCR Confluent monocultures of MCF-7s and NBFs were trypsinized and ∼20×104 cells were used for mRNA isolation (RNEasy with RNAse-Free DNase set, Qiagen). qPCR was performed using the QuantiFast SYBR Green RT-PCR kit (Qiagen) on a LightCycler 480 (Roche, Indianapolis, IN). ..

    Lysis:

    Article Title: Isolation and identification of cell-specific microRNAs targeting a messenger RNA using a biotinylated anti-sense oligonucleotide capture affinity technique
    Article Snippet: .. Cell lysis was performed in lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton, 0.1 % sodium deoxycholate) using a FastPrep cell disrupter 4–5 times at speed 5.5 for 30 s. DNA removal was performed by adding RNase-free DNase (Qiagen) and incubating at 37°C for 15 min. .. This reaction was stopped with a final concentration of 20 mM EDTA.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells
    Article Snippet: .. Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat79254) was performed, followed by elution with 10μl of RNase-free water. .. Total RNA from 1K cells was reverse transcribed by SuperScript II (Invitrogen, Cat#18064-014) with oligo-dT and LNA-containing TSO primers in a final reaction volume of 10μl using the condition: 42°C for 90min, 10 cycles of 50°C 2min to 42°C 2min, 70°C for 15min and hold at 4°C. cDNA was pre-amplified by PCR using KAPA HiFi HotStart ReadyMix (KAPABIOSYSTEMS Cat#KK2602) with IS PCR for 12 cycles in 25μl.

    Plasmid Preparation:

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza
    Article Snippet: .. Extracted RNA were treated with DNase I (QIAGEN, Cat# 79254, USA) according to the manufacturer’s protocol and purified by RNeasy Mini Kit (QIAGEN, Cat# 74106, USA) to exclude plasmid DNA contamination. .. Real-time RT-qPCR was performed as we described previously .

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Qiagen rnase free dnase set
    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including <t>DNase</t> digestion). Optional steps are indicated by dotted arrows. Note that <t>RNase</t> digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Rnase Free Dnase Set, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase free dnase set/product/Qiagen
    Average 99 stars, based on 2160 article reviews
    Price from $9.99 to $1999.99
    rnase free dnase set - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Journal: Frontiers in Microbiology

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    doi: 10.3389/fmicb.2016.01588

    Figure Lengend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

    Techniques: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Journal: Immunity

    Article Title: Transformation of accessible chromatin and 3D nucleome underlies lineage commitment of early T cells

    doi: 10.1016/j.immuni.2018.01.013

    Figure Lengend Snippet: BCL11B binding is associated with an increase in chromatin interaction (A) Expression of Bcl11b from HSPC to DP from RNA-Seq analysis. (B) UCSC genome browser image showing the distribution of ChIP-Seq read density across the genomic region enclosing the Id2 locus (in red) for BCL11B binding, an active histone modification H3K27ac (two independent experiments), and a repressive histone modification H3K27me3, all in DP cells. Top track: distribution of DNase-Seq read density; Yellow and pink rectangles: BCL11B binding sites enriched with H3K27ac and H3K27me3, respectively; K.Z.: a representative BCL11B Chip-Seq data from Dr. Zhao’s lab, NHLBI (two independent experiments); E.V.R.: a representative BCL11B ChIP-Seq data from Prof. Rothenberg’s lab, Cal Tech (two independent experiments). (C) Gene Ontology enrichment analysis for genes with promoters bound by BCL11B and marked by repressive histone modification H3K27me3 in DP cells. (D) Observed versus expected number of genes, sorted based on the status of BCL11B binding and H3K27me3 marker at promoters and expression change by Bcl11b deletion in DP cells. Blue and red arrow heads: gene set repressed and activated by BCL11B, respectively. (E) Empirical cumulative distribution of the fold change of the number of TAD PETs from DN2 to DP cells for TADs sorted into four equal size groups based on the BCL11B coverage, defined by the percentage of genomic region bound by BCL11B in DP cells. P -value by K.-S. test. (F) WashU genome browser showing the distribution of BCL11B ChIP-Seq reads in DPs and the distribution of intra-TAD PETs in DN2 and DP cells for a 360K bps genomic region in chromosome 11. Red rectangle: TAD enriched with BCL11B binding and showing an increase in intra-TAD PETs; Green lines: TAD boundaries.

    Article Snippet: Total RNA was extracted and on-column digestion with DNase (QIAGEN, Cat#79254) was performed, followed by elution with 10μl of RNase-free water.

    Techniques: Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Modification, Marker

    Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Journal: Nature Communications

    Article Title: Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza

    doi: 10.1038/s41467-018-04792-7

    Figure Lengend Snippet: Construction and antiviral activity of defective interfering genes (DIG). a The plasmid construction of DI-PB2, DI-PB1, and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1, and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes. b , c DI RNA expression in 293T and A549 cells. The plasmids of DI-PB2, DI-PB1, and DI-PA were co-transfected into cells with the indicated concentrations. At 24 h post transfection, DI RNAs were extracted from cells and digested by DNase I for RT-qPCR. Empty vector was used as a negative control for RT-qPCR. d Anti-A(H7N7) virus activity of individual plasmid of DI-PB2, DI-PB1, and DI-PA or three combined plasmid DIG (DIG-3, 0.6 μg per well). e , f Dose-dependent anti-A(H7N7) virus activity of DIG-3 in 293T and A549 cells. g Anti-A(H5N1) virus activity of DIG-3. Empty vector phw2000 and plasmids with DIG were individually transfected to cells. At 24 h post transfection, cells were infected with A(H7N7) or A(H5N1) virus at MOI = 0.005 and cell supernatants were collected at 40 h post infection. Viral titers in the supernatants were detected by plaque assay. Data were presented as mean ± SD of three independent experiments. * Indicates P

    Article Snippet: Extracted RNA were treated with DNase I (QIAGEN, Cat# 79254, USA) according to the manufacturer’s protocol and purified by RNeasy Mini Kit (QIAGEN, Cat# 74106, USA) to exclude plasmid DNA contamination.

    Techniques: Activity Assay, Plasmid Preparation, RNA Expression, Transfection, Quantitative RT-PCR, Negative Control, Infection, Plaque Assay

    Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

    Journal: Science Advances

    Article Title: Matrix-bound nanovesicles within ECM bioscaffolds

    doi: 10.1126/sciadv.1600502

    Figure Lengend Snippet: Enzymatic digestion of decellularized ECM scaffolds releases small RNA molecules. ( A ) Nucleic acid extracted from untreated UBM (no digest) and pepsin-, proteinase K–, or collagenase-treated UBM was exposed to RNase A, DNase I, or no-nuclease treatment (control). ( B ) Electropherogram depicting the small RNA pattern of nucleic acid in fluorescence units (FU) before (top panel) and after (bottom panel) DNase I treatment. ( C ) Electropherogram depicting small RNA pattern from the indicated samples in FU. ( D ) A subset of nucleic molecules in biologic scaffolds is protected from nuclease degradation.

    Article Snippet: RNase-free DNase was obtained from Qiagen.

    Techniques: Fluorescence