luna universal qpcr master mix  (New England Biolabs)


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    Luna Universal qPCR Master Mix
    Description:
    Luna Universal qPCR Master Mix 2 500 rxns
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    m3003e
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    2 500 rxns
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    PCR Product Visualization Kits
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    New England Biolabs luna universal qpcr master mix
    Luna Universal qPCR Master Mix
    Luna Universal qPCR Master Mix 2 500 rxns
    https://www.bioz.com/result/luna universal qpcr master mix/product/New England Biolabs
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    luna universal qpcr master mix - by Bioz Stars, 2020-05
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    Images

    1) Product Images from "Nutrient Sensing, Nutrition, and Metabolism: Hepatic ZIP8 deficiency is associated with disrupted selenium homeostasis, liver pathology, and tumor formation"

    Article Title: Nutrient Sensing, Nutrition, and Metabolism: Hepatic ZIP8 deficiency is associated with disrupted selenium homeostasis, liver pathology, and tumor formation

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00165.2018

    Liver pathology showing significant nodular tumors in Slc39a8(+/neo) mice. A : wild-type (WT) mouse ( far left column ) was 20 mo of age; Slc39a8(+/neo) mice ( three right columns ) were 13, 18 and 21 mo of age, respectively. Macroscopic livers ( top row ) and microscopic hematoxylin-eosin (H E) staining ( bottom two rows ) is shown. Nodular tumors are visualized in parts or in the entire liver of Slc39a8(+/neo) mice. Out of 22 hypomorph mice, 10 were identified with similar nodular tumors. Scale bars: 500 μm ( middle row ); 100 μm ( bottom row ). B : hepatic Slc39a8 mRNA expression in WT ( n = 6) and Slc39a8(+/neo) mice ages 13–21 mo ( n = 7). C : typical immunohistochemistry (IHC) staining for α-fetoprotein (AFP) and proliferating cell nuclear antigen (PCNA) in WT and Slc39a8(+/neo) mice (20 mo of age) ( top ). Bottom : quantitative PCR analysis of Afp in WT and in nontumor portion and tumor of Slc39a8(+/neo) ( n = 3 for each of the three histograms). All data represent means ± SE, with 1.0 designated for WT mean expression. * P ≤ 0.05 by Student’s t -test. T, tumor.
    Figure Legend Snippet: Liver pathology showing significant nodular tumors in Slc39a8(+/neo) mice. A : wild-type (WT) mouse ( far left column ) was 20 mo of age; Slc39a8(+/neo) mice ( three right columns ) were 13, 18 and 21 mo of age, respectively. Macroscopic livers ( top row ) and microscopic hematoxylin-eosin (H E) staining ( bottom two rows ) is shown. Nodular tumors are visualized in parts or in the entire liver of Slc39a8(+/neo) mice. Out of 22 hypomorph mice, 10 were identified with similar nodular tumors. Scale bars: 500 μm ( middle row ); 100 μm ( bottom row ). B : hepatic Slc39a8 mRNA expression in WT ( n = 6) and Slc39a8(+/neo) mice ages 13–21 mo ( n = 7). C : typical immunohistochemistry (IHC) staining for α-fetoprotein (AFP) and proliferating cell nuclear antigen (PCNA) in WT and Slc39a8(+/neo) mice (20 mo of age) ( top ). Bottom : quantitative PCR analysis of Afp in WT and in nontumor portion and tumor of Slc39a8(+/neo) ( n = 3 for each of the three histograms). All data represent means ± SE, with 1.0 designated for WT mean expression. * P ≤ 0.05 by Student’s t -test. T, tumor.

    Techniques Used: Mouse Assay, Staining, Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction

    2) Product Images from "Analysis of Global Collection of Group A Streptococcus Genomes Reveals that the Majority Encode a Trio of M and M-Like Proteins"

    Article Title: Analysis of Global Collection of Group A Streptococcus Genomes Reveals that the Majority Encode a Trio of M and M-Like Proteins

    Journal: mSphere

    doi: 10.1128/mSphere.00806-19

    Expression analysis of Mga regulon genes. cDNA from 19 isolates grown to mid-log phase in rich medium were analyzed for the expression of Mga regulon genes. The isolates were selected to be representative of all possible Mga regulon configurations and emm cluster diversity where possible. Primers were designed to amplify all members of the gene family where possible ( mrp , enn , pgs , sph , and scpA ) and to amplify a subset where sequence diversity necessitates. The dot plot symbols represent the mean value of the four qPCR analyses for each isolate, and the error bars represent the standard errors for all isolates for each gene.
    Figure Legend Snippet: Expression analysis of Mga regulon genes. cDNA from 19 isolates grown to mid-log phase in rich medium were analyzed for the expression of Mga regulon genes. The isolates were selected to be representative of all possible Mga regulon configurations and emm cluster diversity where possible. Primers were designed to amplify all members of the gene family where possible ( mrp , enn , pgs , sph , and scpA ) and to amplify a subset where sequence diversity necessitates. The dot plot symbols represent the mean value of the four qPCR analyses for each isolate, and the error bars represent the standard errors for all isolates for each gene.

    Techniques Used: Expressing, Sequencing, Real-time Polymerase Chain Reaction

    3) Product Images from "TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism"

    Article Title: TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

    Journal: Open Biology

    doi: 10.1098/rsob.170134

    Timed induction of bmp4 affects expression of BMP antagonists. ( a ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of tg(hsp70:bmp4, cmlc2:GFP) embryos at 30 hpf after heat shock at 24 hpf as represented by the log2(fold change) of individual samples. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 0.170 and 0.049, respectively. ( b , c ) WMISH for fsta (30 hpf) in tg(hsp70:bmp4, cmlc2:GFP) after heat shock at 24 hpf ( b ) and control embryos ( c ). Strong upregulation after the heat shock is seen in the optic fissure and other expression domains, but not the ciliary marginal zone (arrow). WMISHs in ( b ) and ( c ) were stained in parallel for the same amount of time.
    Figure Legend Snippet: Timed induction of bmp4 affects expression of BMP antagonists. ( a ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of tg(hsp70:bmp4, cmlc2:GFP) embryos at 30 hpf after heat shock at 24 hpf as represented by the log2(fold change) of individual samples. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 0.170 and 0.049, respectively. ( b , c ) WMISH for fsta (30 hpf) in tg(hsp70:bmp4, cmlc2:GFP) after heat shock at 24 hpf ( b ) and control embryos ( c ). Strong upregulation after the heat shock is seen in the optic fissure and other expression domains, but not the ciliary marginal zone (arrow). WMISHs in ( b ) and ( c ) were stained in parallel for the same amount of time.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Staining

    BMP antagonists grem2b and fsta are expressed in the optic fissure. WMISHs were performed at 30 hpf and are shown in lateral view. ( a ) bmp4 is expressed in the dorsal optic cup (arrow). ( b ) Expression of vax2 in the ventral retina. ( c , d ) Expression of grem2b in the optic cup is restricted to the optic fissure (arrow). ( d ) Confocal view of grem2b expression (red) with DAPI counterstaining (blue). ( e , f ) fsta is expressed in the optic fissure (arrows), as well as the ciliary marginal zone (CMZ, arrowhead). ( f ) Confocal view of fsta expression (red) with DAPI counterstaining (blue). ( g ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of SIS3-treated embryos (30 hpf) as represented by the log2(fold change) of individual samples. Embryos were treated from 24 hpf onward, controls were treated with DMSO. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 4.3 × 10 −3 and 0.877, respectively. ( h ) Model of the proposed role of TGFβ and BMP antagonism during optic fissure fusion. TGFβ signalling domains in the optic fissure margins are shielded from BMP by induced BMP antagonists. ( i ) Scheme of a heat shock inducible BMP construct used to create the transgenic line tg(hsp70:bmp4, cmlc2:GFP) . GFP expressed under the cardiac cmlc2 promoter serves as transgenesis marker. Experimental procedure using heat shocks at different time points between 21 and 26 hpf to induce bmp4 expression, aiming at disrupting optic fissure fusion. Analysis of phenotypes was scheduled for 3 dpf.
    Figure Legend Snippet: BMP antagonists grem2b and fsta are expressed in the optic fissure. WMISHs were performed at 30 hpf and are shown in lateral view. ( a ) bmp4 is expressed in the dorsal optic cup (arrow). ( b ) Expression of vax2 in the ventral retina. ( c , d ) Expression of grem2b in the optic cup is restricted to the optic fissure (arrow). ( d ) Confocal view of grem2b expression (red) with DAPI counterstaining (blue). ( e , f ) fsta is expressed in the optic fissure (arrows), as well as the ciliary marginal zone (CMZ, arrowhead). ( f ) Confocal view of fsta expression (red) with DAPI counterstaining (blue). ( g ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of SIS3-treated embryos (30 hpf) as represented by the log2(fold change) of individual samples. Embryos were treated from 24 hpf onward, controls were treated with DMSO. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 4.3 × 10 −3 and 0.877, respectively. ( h ) Model of the proposed role of TGFβ and BMP antagonism during optic fissure fusion. TGFβ signalling domains in the optic fissure margins are shielded from BMP by induced BMP antagonists. ( i ) Scheme of a heat shock inducible BMP construct used to create the transgenic line tg(hsp70:bmp4, cmlc2:GFP) . GFP expressed under the cardiac cmlc2 promoter serves as transgenesis marker. Experimental procedure using heat shocks at different time points between 21 and 26 hpf to induce bmp4 expression, aiming at disrupting optic fissure fusion. Analysis of phenotypes was scheduled for 3 dpf.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Construct, Transgenic Assay, Marker

    Loss of TGFβ2 ligand results in coloboma. ( a , b ) Frontal sections (E18.5, H E) of ( b ) TGFβ2 KO and ( a ) wild-type mouse embryos from mixed genetic background. Note the persisting optic fissure (boxed in b ), scale bars 200 µm. ( c , d ) Frontal sections (E16.5, H E) of ( d ) TGFβ2 KO and ( c ) wild-type mouse embryos from a sole genetic background, mild coloboma phenotype boxed in ( d ), Scale bars 200 µm. ( e ) Expression analysis of gremlin and follistatin, decrease in TGFβ2 KO (TGFβ2/GDNF KO) as represented by the log2(fold change). Error bars represent the 95% confidence interval. Corrected p -values of control gene expression compared to KO for Grem1 and Fst, 5.5 × 10 −3 and 1.2 × 10 −3 , respectively. ( f ) Expression analysis of gremlin and follistatin by quantitative PCR, decrease in TGFβ2 KO (TGFβ2 −/−, GDNF +/− ) compared to controls (TGFβ2 +/+ , GDNF +/− ) as represented by the log2(fold change) of individual samples; n = 3, horizontal bars represent the arithmetic mean. p -values for Grem1 and Fst, 7.8 × 10 −3 and 0.029, respectively. ( g ) Selected terms enriched in the set of downregulated genes in TGFβ2/GDNF KO microarray based on gProfiler analysis. BP, biological process; CC, cellular component; MF, molecular function. ( h ) Expression analysis of several ECM-related genes. Error bars represent the 95% confidence interval. ( i ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in TGFβ2 KO (sole genetic background) compared to wild-type as represented by the log2(fold change) of individual samples; n = 5, one KO sample was excluded as an outlier. Horizontal bars represent the arithmetic mean. p -Values for Grem1 and Fst, 0.312 and 0.027, respectively.
    Figure Legend Snippet: Loss of TGFβ2 ligand results in coloboma. ( a , b ) Frontal sections (E18.5, H E) of ( b ) TGFβ2 KO and ( a ) wild-type mouse embryos from mixed genetic background. Note the persisting optic fissure (boxed in b ), scale bars 200 µm. ( c , d ) Frontal sections (E16.5, H E) of ( d ) TGFβ2 KO and ( c ) wild-type mouse embryos from a sole genetic background, mild coloboma phenotype boxed in ( d ), Scale bars 200 µm. ( e ) Expression analysis of gremlin and follistatin, decrease in TGFβ2 KO (TGFβ2/GDNF KO) as represented by the log2(fold change). Error bars represent the 95% confidence interval. Corrected p -values of control gene expression compared to KO for Grem1 and Fst, 5.5 × 10 −3 and 1.2 × 10 −3 , respectively. ( f ) Expression analysis of gremlin and follistatin by quantitative PCR, decrease in TGFβ2 KO (TGFβ2 −/−, GDNF +/− ) compared to controls (TGFβ2 +/+ , GDNF +/− ) as represented by the log2(fold change) of individual samples; n = 3, horizontal bars represent the arithmetic mean. p -values for Grem1 and Fst, 7.8 × 10 −3 and 0.029, respectively. ( g ) Selected terms enriched in the set of downregulated genes in TGFβ2/GDNF KO microarray based on gProfiler analysis. BP, biological process; CC, cellular component; MF, molecular function. ( h ) Expression analysis of several ECM-related genes. Error bars represent the 95% confidence interval. ( i ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in TGFβ2 KO (sole genetic background) compared to wild-type as represented by the log2(fold change) of individual samples; n = 5, one KO sample was excluded as an outlier. Horizontal bars represent the arithmetic mean. p -Values for Grem1 and Fst, 0.312 and 0.027, respectively.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Microarray

    4) Product Images from "CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding"

    Article Title: CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0288-7

    Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments
    Figure Legend Snippet: Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments

    Techniques Used: Polymerase Chain Reaction, Diagnostic Assay, Activation Assay, Real-time Polymerase Chain Reaction, Positive Control, In Vitro, Negative Control, DNA Extraction

    5) Product Images from "Enhanced vascular activity of a new chimeric promoter containing the full CaMV 35S promoter and the plant XYLOGEN PROTEIN 1 promoter"

    Article Title: Enhanced vascular activity of a new chimeric promoter containing the full CaMV 35S promoter and the plant XYLOGEN PROTEIN 1 promoter

    Journal: 3 Biotech

    doi: 10.1007/s13205-018-1379-8

    Quantitative PCR analysis of β -glucuronidase (GUS) transcriptional expression in vascular and leaf tissues: In lines Z4, uidA is regulated by the promoter of Arabidopsis thaliana XYLOGEN PROTEIN 1; In lines Z5, uidA is regulated by the chimeric promoter 35S-Px. WT, wild-type tobacco; V, vascular tissues of 50- to 60-day-old plants; L, leaves with main vein removed. Experiments were performed in triplicate (biological replicates) and data represent mean relative expression ± standard deviation. Transcription expression between vascular and leaf tissues (paired-samples t -test): For Z4 and Z5lines, *** P = 0.001
    Figure Legend Snippet: Quantitative PCR analysis of β -glucuronidase (GUS) transcriptional expression in vascular and leaf tissues: In lines Z4, uidA is regulated by the promoter of Arabidopsis thaliana XYLOGEN PROTEIN 1; In lines Z5, uidA is regulated by the chimeric promoter 35S-Px. WT, wild-type tobacco; V, vascular tissues of 50- to 60-day-old plants; L, leaves with main vein removed. Experiments were performed in triplicate (biological replicates) and data represent mean relative expression ± standard deviation. Transcription expression between vascular and leaf tissues (paired-samples t -test): For Z4 and Z5lines, *** P = 0.001

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    Strength of promoters Px and 35S-Px. a Quantitative PCR analysis of β -glucuronidase (GUS) transcriptional expression. Total RNA was extracted from the apical vascular tissues of 50–60-day-old plants. Relative transcription was calculated using the comparative C T ). *** P = 0.000 (Duncan’s test). b Quantitative enzymatic assay of GUS activity in transgenic tobacco lines. The apical vascular tissues of 50–60-day-old plants were also used for soluble protein extraction and measurement of GUS activity. The reaction was carried out at 37 °C for 4 h, and 0.43 mg soluble protein was obtained from each sample. The experiment was repeated at least three times (biological replicates). Bars and whiskers represent mean GUS activity ± standard deviation (SD). *** P = 0.000 (Duncan’s test)
    Figure Legend Snippet: Strength of promoters Px and 35S-Px. a Quantitative PCR analysis of β -glucuronidase (GUS) transcriptional expression. Total RNA was extracted from the apical vascular tissues of 50–60-day-old plants. Relative transcription was calculated using the comparative C T ). *** P = 0.000 (Duncan’s test). b Quantitative enzymatic assay of GUS activity in transgenic tobacco lines. The apical vascular tissues of 50–60-day-old plants were also used for soluble protein extraction and measurement of GUS activity. The reaction was carried out at 37 °C for 4 h, and 0.43 mg soluble protein was obtained from each sample. The experiment was repeated at least three times (biological replicates). Bars and whiskers represent mean GUS activity ± standard deviation (SD). *** P = 0.000 (Duncan’s test)

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Enzymatic Assay, Activity Assay, Transgenic Assay, Protein Extraction, Standard Deviation

    6) Product Images from "Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes"

    Article Title: Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.02.049

    Pcf11 Expression Is Regulated by IPA (A) Schematic of mouse Pcf11 gene. Multiple PASs are indicated. EPA, internal exonic polyadenylation; IPA, intronic polyadenylation; TPA, 3′ terminal exon polyadenylation. 3′READS data of brain, heart, and testis are shown. (B) Features of intron 1 compared with other introns in the mouse genome. Each feature has ten bins, and the bin containing intron 1 of Pcf11 is highlighted in red. Distance to TSS is based on all IPA sites in PolyA_DB. (C) RT-qPCR analysis of IPA and TPA isoform expression in siPcf11 versus siCtrl cells. Error bars are SD on the basis of three replicates. (D) Top: schematic showing two sgRNAs used to remove a region flanking the IPA site. Bottom: validation of IPA site knockout (IPA Pcf11 -KO) by genomic DNA PCR using primers flanking the KO region. WT, wild-type 4T1 cells. (E) RT-qPCR analysis of Pcf11 isoform expression in WT and IPA Pcf11 -KO cells. Error bars are SD on the basis of three replicates. (F) Western blot analysis of PCF11 in WT and IPA Pcf11 -KO cells. (G) 3′UTR APA changes in IPA Pcf11 -KO cells. (H) IPA changes in IPA Pcf11 -KO cells. IPA isoforms (all combined) were compared with TPA isoforms (all combined) in analysis. (I) IPA distribution maps of activated IPA events in IPA Pcf11 -KO cells. (J) Left: schematic of PASs around the transcription start site (TSS); right: metagene analysis of PAS usage around the TSS in WT and IPA Pcf1 1 -KO cells. (K) Gene expression changes in IPA Pcf11 -KO cells for different groups of genes on the basis of IPA site frequency in PolyA_DB. ***p
    Figure Legend Snippet: Pcf11 Expression Is Regulated by IPA (A) Schematic of mouse Pcf11 gene. Multiple PASs are indicated. EPA, internal exonic polyadenylation; IPA, intronic polyadenylation; TPA, 3′ terminal exon polyadenylation. 3′READS data of brain, heart, and testis are shown. (B) Features of intron 1 compared with other introns in the mouse genome. Each feature has ten bins, and the bin containing intron 1 of Pcf11 is highlighted in red. Distance to TSS is based on all IPA sites in PolyA_DB. (C) RT-qPCR analysis of IPA and TPA isoform expression in siPcf11 versus siCtrl cells. Error bars are SD on the basis of three replicates. (D) Top: schematic showing two sgRNAs used to remove a region flanking the IPA site. Bottom: validation of IPA site knockout (IPA Pcf11 -KO) by genomic DNA PCR using primers flanking the KO region. WT, wild-type 4T1 cells. (E) RT-qPCR analysis of Pcf11 isoform expression in WT and IPA Pcf11 -KO cells. Error bars are SD on the basis of three replicates. (F) Western blot analysis of PCF11 in WT and IPA Pcf11 -KO cells. (G) 3′UTR APA changes in IPA Pcf11 -KO cells. (H) IPA changes in IPA Pcf11 -KO cells. IPA isoforms (all combined) were compared with TPA isoforms (all combined) in analysis. (I) IPA distribution maps of activated IPA events in IPA Pcf11 -KO cells. (J) Left: schematic of PASs around the transcription start site (TSS); right: metagene analysis of PAS usage around the TSS in WT and IPA Pcf1 1 -KO cells. (K) Gene expression changes in IPA Pcf11 -KO cells for different groups of genes on the basis of IPA site frequency in PolyA_DB. ***p

    Techniques Used: Expressing, Indirect Immunoperoxidase Assay, Quantitative RT-PCR, Knock-Out, Polymerase Chain Reaction, Western Blot

    7) Product Images from "The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function"

    Article Title: The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function

    Journal: PeerJ

    doi: 10.7717/peerj.4093

    qPCR analysis of α-crystallin expression in zebrafish embryos. Box and whisker plot shows delta Ct for the three zebrafish α-crystallins, indicating mRNA levels relative to three endogenous controls from 12 h post fertilization (0.5 dpf) to 5 dpf. Lower numerical Ct values on these inverted y -axes indicate increased expression. All three graphs show low initial baseline expression that increases in αA (A) and αBa-crystallin (B), but stays consistently low in αBb-crystallin (C). Alpha A-crystallin expression increased earlier than αBa-crystallin expression. Asterisks indicate statistically significant differences in expression compared to the 0.5 dpf timepoint ( p
    Figure Legend Snippet: qPCR analysis of α-crystallin expression in zebrafish embryos. Box and whisker plot shows delta Ct for the three zebrafish α-crystallins, indicating mRNA levels relative to three endogenous controls from 12 h post fertilization (0.5 dpf) to 5 dpf. Lower numerical Ct values on these inverted y -axes indicate increased expression. All three graphs show low initial baseline expression that increases in αA (A) and αBa-crystallin (B), but stays consistently low in αBb-crystallin (C). Alpha A-crystallin expression increased earlier than αBa-crystallin expression. Asterisks indicate statistically significant differences in expression compared to the 0.5 dpf timepoint ( p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    8) Product Images from "H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development"

    Article Title: H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky982

    H3.3K4M prevents MLL3/4-mediated enhancer activation in adipogenesis. 4OHT-treated LSL-K4M; Cre-ER brown preadipocytes were collected at D4 of adipogenesis for ChIP-seq of H3K4me1 and H3K27ac. Drosophila spike-in control was used to normalize ChIP-seq data. (A–C), ChIP-qPCR of MLL4, H3K4me1, H3K27ac, BRD4, MED1 and Pol II, and qRT-PCR of eRNAs (D, E). (A, B) Heat maps ( A ) and average profiles ( B ) around MLL4 + active enhancers during adipogenesis. ( C ), as well as current data set GSE110972) on Pparg and Cebpa gene loci during adipogenesis with schematic of genomic locations of representative MLL4 + active enhancers (e1–e5). ( D ) ChIP-qPCR analyses of indicated factors are shown on enhancers e1–e5 at D0 and D4 of adipogenesis. An enhancer of constitutively expressed gene Jak1 was chosen as a negative control ( n ). ( E ) qRT-PCR of eRNA transcription on enhancers e1-e5 at D0 and D4 of adipogenesis. –4OHT at D4 was compared to +4OHT ( ∗∗ P
    Figure Legend Snippet: H3.3K4M prevents MLL3/4-mediated enhancer activation in adipogenesis. 4OHT-treated LSL-K4M; Cre-ER brown preadipocytes were collected at D4 of adipogenesis for ChIP-seq of H3K4me1 and H3K27ac. Drosophila spike-in control was used to normalize ChIP-seq data. (A–C), ChIP-qPCR of MLL4, H3K4me1, H3K27ac, BRD4, MED1 and Pol II, and qRT-PCR of eRNAs (D, E). (A, B) Heat maps ( A ) and average profiles ( B ) around MLL4 + active enhancers during adipogenesis. ( C ), as well as current data set GSE110972) on Pparg and Cebpa gene loci during adipogenesis with schematic of genomic locations of representative MLL4 + active enhancers (e1–e5). ( D ) ChIP-qPCR analyses of indicated factors are shown on enhancers e1–e5 at D0 and D4 of adipogenesis. An enhancer of constitutively expressed gene Jak1 was chosen as a negative control ( n ). ( E ) qRT-PCR of eRNA transcription on enhancers e1-e5 at D0 and D4 of adipogenesis. –4OHT at D4 was compared to +4OHT ( ∗∗ P

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control

    Histone H3.3K4M and H3.3K36M mutations impair adipogenesis. ( A ) A schematic of the process of preadipocyte derivation from interscapular BAT of newborn pup, followed by adipogenesis assay in culture. (B–D) Immortalized brown preadipocytes were infected with retroviral vector (Vec) expressing FLAG-tagged wild type (WT) or K-to-M mutant histone H3.3, followed by adipogenesis assay. ( B ) Histone extracts from preadipocytes were subjected to western blot analyses using antibodies indicated on the left. Long exposure of histone H3 Western blot reveals the relative levels of ectopic H3.3 and endogenous H3. ( C ) Six days after induction of differentiation, cells were stained with Oil Red O. Upper panels, stained dishes; lower panels, representative fields under microscope. ( D ) qRT-PCR of Pparg, Cebpa , and Fabp4 expression at day 0 (D0) and day 6 (D6) of adipogenesis. Quantitative PCR data in all figures are presented as means ± SEM. Statistical comparison between groups was performed using Student's t -test in all figures. WT at D7 was compared to K4M and K36M ( ∗∗ P
    Figure Legend Snippet: Histone H3.3K4M and H3.3K36M mutations impair adipogenesis. ( A ) A schematic of the process of preadipocyte derivation from interscapular BAT of newborn pup, followed by adipogenesis assay in culture. (B–D) Immortalized brown preadipocytes were infected with retroviral vector (Vec) expressing FLAG-tagged wild type (WT) or K-to-M mutant histone H3.3, followed by adipogenesis assay. ( B ) Histone extracts from preadipocytes were subjected to western blot analyses using antibodies indicated on the left. Long exposure of histone H3 Western blot reveals the relative levels of ectopic H3.3 and endogenous H3. ( C ) Six days after induction of differentiation, cells were stained with Oil Red O. Upper panels, stained dishes; lower panels, representative fields under microscope. ( D ) qRT-PCR of Pparg, Cebpa , and Fabp4 expression at day 0 (D0) and day 6 (D6) of adipogenesis. Quantitative PCR data in all figures are presented as means ± SEM. Statistical comparison between groups was performed using Student's t -test in all figures. WT at D7 was compared to K4M and K36M ( ∗∗ P

    Techniques Used: Infection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Staining, Microscopy, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    9) Product Images from "Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation"

    Article Title: Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

    Journal: Hepatology Communications

    doi: 10.1002/hep4.1315

    Ex vivo reprogramming of cultured hepatocytes up‐regulates DNA repair pathways. (A) Averaged relative quantification values showing expression levels of five genes involved in homologous recombination ( Brca1 , Brca2 , Rad51 , Rad52 , and Pcna ) relative to the length of culture as determined by comparative qPCR on RNA isolated from primary hepatocytes. Samples are standardized to B2m expression. (B) Deconvolution of these five genes, showing consistent up‐regulation over time. (C) Representative histogram of Ki‐67 intensity in primary hepatocytes after increasing time in culture, with peak heights normalized to mode of sample. (D) Quantification of mean fluorescence intensity in biological replicates (n = 2). (E) Hierarchical clustering of six RNA‐Seq samples at three different time points (0 hours, 24 hours, and 48 hours). The 24‐hour and 48‐hour time points are more similar to one another than either is to 0 hours. (F) Heat map of gene expression in all samples for differentially expressed genes between times 0 hours and 48 hours (n = 5,254). (G) Enrichment analysis of highly affected gene sets clustered by biologic functions between cells in culture for 48 hours relative to 0 hours. (H) Detailed enrichment summary illustrating differences in the regulation of two selected Gene Ontology function annotations of interest—DNA repair (horizontal black bars on the left) and cellular amino acid catabolic process (horizontal green bars on the left)—among the three time points collected (presented by column). The heat map includes the top and bottom 500 differentially expressed genes ranked by fold change between times 0 hours and 48 hours. Individual DNA repair genes are up‐regulated, whereas individual cellular and amino acid catabolic process genes are down‐regulated in the 24‐hour and 48‐hour groups. Abbreviations: FDR, false discovery rate; and ns, not significant at α = 0.05.
    Figure Legend Snippet: Ex vivo reprogramming of cultured hepatocytes up‐regulates DNA repair pathways. (A) Averaged relative quantification values showing expression levels of five genes involved in homologous recombination ( Brca1 , Brca2 , Rad51 , Rad52 , and Pcna ) relative to the length of culture as determined by comparative qPCR on RNA isolated from primary hepatocytes. Samples are standardized to B2m expression. (B) Deconvolution of these five genes, showing consistent up‐regulation over time. (C) Representative histogram of Ki‐67 intensity in primary hepatocytes after increasing time in culture, with peak heights normalized to mode of sample. (D) Quantification of mean fluorescence intensity in biological replicates (n = 2). (E) Hierarchical clustering of six RNA‐Seq samples at three different time points (0 hours, 24 hours, and 48 hours). The 24‐hour and 48‐hour time points are more similar to one another than either is to 0 hours. (F) Heat map of gene expression in all samples for differentially expressed genes between times 0 hours and 48 hours (n = 5,254). (G) Enrichment analysis of highly affected gene sets clustered by biologic functions between cells in culture for 48 hours relative to 0 hours. (H) Detailed enrichment summary illustrating differences in the regulation of two selected Gene Ontology function annotations of interest—DNA repair (horizontal black bars on the left) and cellular amino acid catabolic process (horizontal green bars on the left)—among the three time points collected (presented by column). The heat map includes the top and bottom 500 differentially expressed genes ranked by fold change between times 0 hours and 48 hours. Individual DNA repair genes are up‐regulated, whereas individual cellular and amino acid catabolic process genes are down‐regulated in the 24‐hour and 48‐hour groups. Abbreviations: FDR, false discovery rate; and ns, not significant at α = 0.05.

    Techniques Used: Ex Vivo, Cell Culture, Expressing, Homologous Recombination, Real-time Polymerase Chain Reaction, Isolation, Fluorescence, RNA Sequencing Assay

    10) Product Images from "TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism"

    Article Title: TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

    Journal: Open Biology

    doi: 10.1098/rsob.170134

    Timed induction of bmp4 affects expression of BMP antagonists. ( a ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of tg(hsp70:bmp4, cmlc2:GFP) embryos at 30 hpf after heat shock at 24 hpf as represented by the log2(fold change) of individual samples. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 0.170 and 0.049, respectively. ( b , c ) WMISH for fsta (30 hpf) in tg(hsp70:bmp4, cmlc2:GFP) after heat shock at 24 hpf ( b ) and control embryos ( c ). Strong upregulation after the heat shock is seen in the optic fissure and other expression domains, but not the ciliary marginal zone (arrow). WMISHs in ( b ) and ( c ) were stained in parallel for the same amount of time.
    Figure Legend Snippet: Timed induction of bmp4 affects expression of BMP antagonists. ( a ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of tg(hsp70:bmp4, cmlc2:GFP) embryos at 30 hpf after heat shock at 24 hpf as represented by the log2(fold change) of individual samples. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 0.170 and 0.049, respectively. ( b , c ) WMISH for fsta (30 hpf) in tg(hsp70:bmp4, cmlc2:GFP) after heat shock at 24 hpf ( b ) and control embryos ( c ). Strong upregulation after the heat shock is seen in the optic fissure and other expression domains, but not the ciliary marginal zone (arrow). WMISHs in ( b ) and ( c ) were stained in parallel for the same amount of time.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Staining

    BMP antagonists grem2b and fsta are expressed in the optic fissure. WMISHs were performed at 30 hpf and are shown in lateral view. ( a ) bmp4 is expressed in the dorsal optic cup (arrow). ( b ) Expression of vax2 in the ventral retina. ( c , d ) Expression of grem2b in the optic cup is restricted to the optic fissure (arrow). ( d ) Confocal view of grem2b expression (red) with DAPI counterstaining (blue). ( e , f ) fsta is expressed in the optic fissure (arrows), as well as the ciliary marginal zone (CMZ, arrowhead). ( f ) Confocal view of fsta expression (red) with DAPI counterstaining (blue). ( g ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of SIS3-treated embryos (30 hpf) as represented by the log2(fold change) of individual samples. Embryos were treated from 24 hpf onward, controls were treated with DMSO. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 4.3 × 10 −3 and 0.877, respectively. ( h ) Model of the proposed role of TGFβ and BMP antagonism during optic fissure fusion. TGFβ signalling domains in the optic fissure margins are shielded from BMP by induced BMP antagonists. ( i ) Scheme of a heat shock inducible BMP construct used to create the transgenic line tg(hsp70:bmp4, cmlc2:GFP) . GFP expressed under the cardiac cmlc2 promoter serves as transgenesis marker. Experimental procedure using heat shocks at different time points between 21 and 26 hpf to induce bmp4 expression, aiming at disrupting optic fissure fusion. Analysis of phenotypes was scheduled for 3 dpf.
    Figure Legend Snippet: BMP antagonists grem2b and fsta are expressed in the optic fissure. WMISHs were performed at 30 hpf and are shown in lateral view. ( a ) bmp4 is expressed in the dorsal optic cup (arrow). ( b ) Expression of vax2 in the ventral retina. ( c , d ) Expression of grem2b in the optic cup is restricted to the optic fissure (arrow). ( d ) Confocal view of grem2b expression (red) with DAPI counterstaining (blue). ( e , f ) fsta is expressed in the optic fissure (arrows), as well as the ciliary marginal zone (CMZ, arrowhead). ( f ) Confocal view of fsta expression (red) with DAPI counterstaining (blue). ( g ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of SIS3-treated embryos (30 hpf) as represented by the log2(fold change) of individual samples. Embryos were treated from 24 hpf onward, controls were treated with DMSO. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 4.3 × 10 −3 and 0.877, respectively. ( h ) Model of the proposed role of TGFβ and BMP antagonism during optic fissure fusion. TGFβ signalling domains in the optic fissure margins are shielded from BMP by induced BMP antagonists. ( i ) Scheme of a heat shock inducible BMP construct used to create the transgenic line tg(hsp70:bmp4, cmlc2:GFP) . GFP expressed under the cardiac cmlc2 promoter serves as transgenesis marker. Experimental procedure using heat shocks at different time points between 21 and 26 hpf to induce bmp4 expression, aiming at disrupting optic fissure fusion. Analysis of phenotypes was scheduled for 3 dpf.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Construct, Transgenic Assay, Marker

    Loss of TGFβ2 ligand results in coloboma. ( a , b ) Frontal sections (E18.5, H E) of ( b ) TGFβ2 KO and ( a ) wild-type mouse embryos from mixed genetic background. Note the persisting optic fissure (boxed in b ), scale bars 200 µm. ( c , d ) Frontal sections (E16.5, H E) of ( d ) TGFβ2 KO and ( c ) wild-type mouse embryos from a sole genetic background, mild coloboma phenotype boxed in ( d ), Scale bars 200 µm. ( e ) Expression analysis of gremlin and follistatin, decrease in TGFβ2 KO (TGFβ2/GDNF KO) as represented by the log2(fold change). Error bars represent the 95% confidence interval. Corrected p -values of control gene expression compared to KO for Grem1 and Fst, 5.5 × 10 −3 and 1.2 × 10 −3 , respectively. ( f ) Expression analysis of gremlin and follistatin by quantitative PCR, decrease in TGFβ2 KO (TGFβ2 −/−, GDNF +/− ) compared to controls (TGFβ2 +/+ , GDNF +/− ) as represented by the log2(fold change) of individual samples; n = 3, horizontal bars represent the arithmetic mean. p -values for Grem1 and Fst, 7.8 × 10 −3 and 0.029, respectively. ( g ) Selected terms enriched in the set of downregulated genes in TGFβ2/GDNF KO microarray based on gProfiler analysis. BP, biological process; CC, cellular component; MF, molecular function. ( h ) Expression analysis of several ECM-related genes. Error bars represent the 95% confidence interval. ( i ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in TGFβ2 KO (sole genetic background) compared to wild-type as represented by the log2(fold change) of individual samples; n = 5, one KO sample was excluded as an outlier. Horizontal bars represent the arithmetic mean. p -Values for Grem1 and Fst, 0.312 and 0.027, respectively.
    Figure Legend Snippet: Loss of TGFβ2 ligand results in coloboma. ( a , b ) Frontal sections (E18.5, H E) of ( b ) TGFβ2 KO and ( a ) wild-type mouse embryos from mixed genetic background. Note the persisting optic fissure (boxed in b ), scale bars 200 µm. ( c , d ) Frontal sections (E16.5, H E) of ( d ) TGFβ2 KO and ( c ) wild-type mouse embryos from a sole genetic background, mild coloboma phenotype boxed in ( d ), Scale bars 200 µm. ( e ) Expression analysis of gremlin and follistatin, decrease in TGFβ2 KO (TGFβ2/GDNF KO) as represented by the log2(fold change). Error bars represent the 95% confidence interval. Corrected p -values of control gene expression compared to KO for Grem1 and Fst, 5.5 × 10 −3 and 1.2 × 10 −3 , respectively. ( f ) Expression analysis of gremlin and follistatin by quantitative PCR, decrease in TGFβ2 KO (TGFβ2 −/−, GDNF +/− ) compared to controls (TGFβ2 +/+ , GDNF +/− ) as represented by the log2(fold change) of individual samples; n = 3, horizontal bars represent the arithmetic mean. p -values for Grem1 and Fst, 7.8 × 10 −3 and 0.029, respectively. ( g ) Selected terms enriched in the set of downregulated genes in TGFβ2/GDNF KO microarray based on gProfiler analysis. BP, biological process; CC, cellular component; MF, molecular function. ( h ) Expression analysis of several ECM-related genes. Error bars represent the 95% confidence interval. ( i ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in TGFβ2 KO (sole genetic background) compared to wild-type as represented by the log2(fold change) of individual samples; n = 5, one KO sample was excluded as an outlier. Horizontal bars represent the arithmetic mean. p -Values for Grem1 and Fst, 0.312 and 0.027, respectively.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Microarray

    11) Product Images from "H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development"

    Article Title: H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky982

    H3.3K4M prevents MLL3/4-mediated enhancer activation in adipogenesis. 4OHT-treated LSL-K4M; Cre-ER brown preadipocytes were collected at D4 of adipogenesis for ChIP-seq of H3K4me1 and H3K27ac. Drosophila spike-in control was used to normalize ChIP-seq data. (A–C), ChIP-qPCR of MLL4, H3K4me1, H3K27ac, BRD4, MED1 and Pol II, and qRT-PCR of eRNAs (D, E). (A, B) Heat maps ( A ) and average profiles ( B ) around MLL4 + active enhancers during adipogenesis. ( C ) Genome browser view of PPARγ, C/EBPα, MLL4, CBP, BRD4, MED1, H3K4me1 and H3K27ac (data obtained from published data sets GSE50466, GSE74189, and GSE99101 ( 4 , 6 , 7 ), as well as current data set GSE110972) on Pparg and Cebpa gene loci during adipogenesis with schematic of genomic locations of representative MLL4 + active enhancers (e1–e5). ( D ) ChIP-qPCR analyses of indicated factors are shown on enhancers e1–e5 at D0 and D4 of adipogenesis. An enhancer of constitutively expressed gene Jak1 was chosen as a negative control ( n ). ( E ) qRT-PCR of eRNA transcription on enhancers e1-e5 at D0 and D4 of adipogenesis. –4OHT at D4 was compared to +4OHT ( ∗∗ P
    Figure Legend Snippet: H3.3K4M prevents MLL3/4-mediated enhancer activation in adipogenesis. 4OHT-treated LSL-K4M; Cre-ER brown preadipocytes were collected at D4 of adipogenesis for ChIP-seq of H3K4me1 and H3K27ac. Drosophila spike-in control was used to normalize ChIP-seq data. (A–C), ChIP-qPCR of MLL4, H3K4me1, H3K27ac, BRD4, MED1 and Pol II, and qRT-PCR of eRNAs (D, E). (A, B) Heat maps ( A ) and average profiles ( B ) around MLL4 + active enhancers during adipogenesis. ( C ) Genome browser view of PPARγ, C/EBPα, MLL4, CBP, BRD4, MED1, H3K4me1 and H3K27ac (data obtained from published data sets GSE50466, GSE74189, and GSE99101 ( 4 , 6 , 7 ), as well as current data set GSE110972) on Pparg and Cebpa gene loci during adipogenesis with schematic of genomic locations of representative MLL4 + active enhancers (e1–e5). ( D ) ChIP-qPCR analyses of indicated factors are shown on enhancers e1–e5 at D0 and D4 of adipogenesis. An enhancer of constitutively expressed gene Jak1 was chosen as a negative control ( n ). ( E ) qRT-PCR of eRNA transcription on enhancers e1-e5 at D0 and D4 of adipogenesis. –4OHT at D4 was compared to +4OHT ( ∗∗ P

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control

    Histone H3.3K4M and H3.3K36M mutations impair adipogenesis. ( A ) A schematic of the process of preadipocyte derivation from interscapular BAT of newborn pup, followed by adipogenesis assay in culture. (B–D) Immortalized brown preadipocytes were infected with retroviral vector (Vec) expressing FLAG-tagged wild type (WT) or K-to-M mutant histone H3.3, followed by adipogenesis assay. ( B ) Histone extracts from preadipocytes were subjected to western blot analyses using antibodies indicated on the left. Long exposure of histone H3 Western blot reveals the relative levels of ectopic H3.3 and endogenous H3. ( C ) Six days after induction of differentiation, cells were stained with Oil Red O. Upper panels, stained dishes; lower panels, representative fields under microscope. ( D ) qRT-PCR of Pparg, Cebpa , and Fabp4 expression at day 0 (D0) and day 6 (D6) of adipogenesis. Quantitative PCR data in all figures are presented as means ± SEM. Statistical comparison between groups was performed using Student's t -test in all figures. WT at D7 was compared to K4M and K36M ( ∗∗ P
    Figure Legend Snippet: Histone H3.3K4M and H3.3K36M mutations impair adipogenesis. ( A ) A schematic of the process of preadipocyte derivation from interscapular BAT of newborn pup, followed by adipogenesis assay in culture. (B–D) Immortalized brown preadipocytes were infected with retroviral vector (Vec) expressing FLAG-tagged wild type (WT) or K-to-M mutant histone H3.3, followed by adipogenesis assay. ( B ) Histone extracts from preadipocytes were subjected to western blot analyses using antibodies indicated on the left. Long exposure of histone H3 Western blot reveals the relative levels of ectopic H3.3 and endogenous H3. ( C ) Six days after induction of differentiation, cells were stained with Oil Red O. Upper panels, stained dishes; lower panels, representative fields under microscope. ( D ) qRT-PCR of Pparg, Cebpa , and Fabp4 expression at day 0 (D0) and day 6 (D6) of adipogenesis. Quantitative PCR data in all figures are presented as means ± SEM. Statistical comparison between groups was performed using Student's t -test in all figures. WT at D7 was compared to K4M and K36M ( ∗∗ P

    Techniques Used: Infection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot, Staining, Microscopy, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    12) Product Images from "CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding"

    Article Title: CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0288-7

    Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments
    Figure Legend Snippet: Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments

    Techniques Used: Polymerase Chain Reaction, Diagnostic Assay, Activation Assay, Real-time Polymerase Chain Reaction, Positive Control, In Vitro, Negative Control, DNA Extraction

    13) Product Images from "Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation"

    Article Title: Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03030

    Transcriptional analysis of modABC genes. (A) Expression of β-galactosidase from a P modABC :: lacZ fusion in P. putida J5. Cells were grown aerobically in NI medium supplemented with different concentrations of molybdate. (B) RT-qPCR analysis of the modA gene transcript produced in P. putida J5 that was grown as described above. Results presented in these histograms are the means of three independent experiments, and error bars indicate the standard deviations. Different letters represent significant differences between the treatments.
    Figure Legend Snippet: Transcriptional analysis of modABC genes. (A) Expression of β-galactosidase from a P modABC :: lacZ fusion in P. putida J5. Cells were grown aerobically in NI medium supplemented with different concentrations of molybdate. (B) RT-qPCR analysis of the modA gene transcript produced in P. putida J5 that was grown as described above. Results presented in these histograms are the means of three independent experiments, and error bars indicate the standard deviations. Different letters represent significant differences between the treatments.

    Techniques Used: Expressing, Quantitative RT-PCR, Produced

    14) Product Images from "Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2"

    Article Title: Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2

    Journal: eLife

    doi: 10.7554/eLife.47691

    Dominant negative Lsr2 variant (Lsr2*) inhibits DNA binding by wild type Lsr2 and promotes antibiotic production in S. venezuelae . ( A ) Electrophoretic mobility shift assay illustrating DNA binding by wild type Lsr2, no DNA binding by the R82A mutant allele (mutant Lsr2*), and effective inhibition of Lsr2 binding by the mutant variant, based on the increasing abundance of ‘free probe’ as mutant concentrations rise. Probe: 1.5 kb region corresponding to the Lsr2 binding site in the chloramphenicol biosynthetic cluster. Reactions were separated on a 1% agarose gel stained with ethidium bromide. ( B ) Bioactivity of S. venezuelae extracts against Micrococcus luteus . Wild type S. venezuelae carrying either a control (empty) plasmid (left), or one overexpressing the mutant 9R82A) Lsr2 variant (right; Lsr2*) were cultured for 18 hr prior to extraction in methanol and reconstitution in DMSO. Extracts were applied to Whatman filter discs, and cultures were grown overnight. ( C ) Quantitative PCR analysis of Lsr2-3xFLAG-binding to the validated target site sven_0926 , following ChIP, for a strain carrying an empty plasmid (control) or overexpressing Lsr2* (O/E Lsr2*). Overexpressing Lsr2* led to a 40% drop in binding by Lsr2. ChIP experiments were conducted in duplicate, and qPCR was done for each replicate in technical triplicate.
    Figure Legend Snippet: Dominant negative Lsr2 variant (Lsr2*) inhibits DNA binding by wild type Lsr2 and promotes antibiotic production in S. venezuelae . ( A ) Electrophoretic mobility shift assay illustrating DNA binding by wild type Lsr2, no DNA binding by the R82A mutant allele (mutant Lsr2*), and effective inhibition of Lsr2 binding by the mutant variant, based on the increasing abundance of ‘free probe’ as mutant concentrations rise. Probe: 1.5 kb region corresponding to the Lsr2 binding site in the chloramphenicol biosynthetic cluster. Reactions were separated on a 1% agarose gel stained with ethidium bromide. ( B ) Bioactivity of S. venezuelae extracts against Micrococcus luteus . Wild type S. venezuelae carrying either a control (empty) plasmid (left), or one overexpressing the mutant 9R82A) Lsr2 variant (right; Lsr2*) were cultured for 18 hr prior to extraction in methanol and reconstitution in DMSO. Extracts were applied to Whatman filter discs, and cultures were grown overnight. ( C ) Quantitative PCR analysis of Lsr2-3xFLAG-binding to the validated target site sven_0926 , following ChIP, for a strain carrying an empty plasmid (control) or overexpressing Lsr2* (O/E Lsr2*). Overexpressing Lsr2* led to a 40% drop in binding by Lsr2. ChIP experiments were conducted in duplicate, and qPCR was done for each replicate in technical triplicate.

    Techniques Used: Dominant Negative Mutation, Variant Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis, Inhibition, Agarose Gel Electrophoresis, Staining, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    15) Product Images from "The cardiovascular protective effects of rooibos (Aspalathus linearis) extract on diesel exhaust particles induced inflammation and oxidative stress involve NF-κB- and Nrf2-dependent pathways modulation"

    Article Title: The cardiovascular protective effects of rooibos (Aspalathus linearis) extract on diesel exhaust particles induced inflammation and oxidative stress involve NF-κB- and Nrf2-dependent pathways modulation

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2019.e01426

    Effect of rooibos extract on the inflammatory effect of DEP: The gene expressions of inflammatory cytokine-IL-8, IL-10, TNFα and IL-1β were determined in (A) aorta and (B) heart; by RT-qPCR using primers against the cDNA as described in the materials and methods. Values are mean ± SEM of six experiment animals done in triplicate (n = 3). *p
    Figure Legend Snippet: Effect of rooibos extract on the inflammatory effect of DEP: The gene expressions of inflammatory cytokine-IL-8, IL-10, TNFα and IL-1β were determined in (A) aorta and (B) heart; by RT-qPCR using primers against the cDNA as described in the materials and methods. Values are mean ± SEM of six experiment animals done in triplicate (n = 3). *p

    Techniques Used: Quantitative RT-PCR

    16) Product Images from "Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation"

    Article Title: Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03030

    Transcriptional analysis of modABC genes. (A) Expression of β-galactosidase from a P modABC :: lacZ fusion in P. putida J5. Cells were grown aerobically in NI medium supplemented with different concentrations of molybdate. (B) RT-qPCR analysis of the modA gene transcript produced in P. putida J5 that was grown as described above. Results presented in these histograms are the means of three independent experiments, and error bars indicate the standard deviations. Different letters represent significant differences between the treatments.
    Figure Legend Snippet: Transcriptional analysis of modABC genes. (A) Expression of β-galactosidase from a P modABC :: lacZ fusion in P. putida J5. Cells were grown aerobically in NI medium supplemented with different concentrations of molybdate. (B) RT-qPCR analysis of the modA gene transcript produced in P. putida J5 that was grown as described above. Results presented in these histograms are the means of three independent experiments, and error bars indicate the standard deviations. Different letters represent significant differences between the treatments.

    Techniques Used: Expressing, Quantitative RT-PCR, Produced

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    Article Title: H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development
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    Article Title: TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism
    Article Snippet: .. Quantitative realtime PCR was performed with a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories) and the Luna Universal qPCR Master Mix (New England Biolabs) in technical triplicates with 20 µl reaction volume and 2 µl of a 1:10 dilution of the cDNA template. .. Gapdh was used as reference gene for mouse qPCR and eef1a1l was used for zebrafish qPCR.

    Article Title: The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function
    Article Snippet: .. All cDNA samples (three biological replicates for each timepoint) were amplified using Luna Universal qPCR Master Mix (NEB) on an Applied Biosystems StepOne Real-Time PCR System (Thermo Fisher). ..

    Article Title: Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes
    Article Snippet: .. Real-time quantitative PCR (qPCR) Total RNA was extracted using TRIzol (Thermo Fisher) and treated with Turbo DNase (Thermo Fisher). cDNA was synthesized using oligo(dT) and M-MLV reverse transcriptase with 1.5–2 μg of RNA. cDNA was then mixed with real-time PCR primers and Luna qPCR master mix (NEB). .. PCR was run on an Applied Biosciences StepOne Plus Real-Time PCR system.

    Amplification:

    Article Title: Nutrient Sensing, Nutrition, and Metabolism: Hepatic ZIP8 deficiency is associated with disrupted selenium homeostasis, liver pathology, and tumor formation
    Article Snippet: .. The quantitative (q)PCR for individual gene expression was carried out with Luna Universal qPCR Master Mix (New England Biolabs) and gene-specific primers; cycling conditions included 95°C for 1 min, followed by amplification for 40 cycles at 95°C for 15 s, and then 60°C for 30 s in the CFX96 Real-Time PCR system (Bio-Rad). .. Relative expression of mRNA levels was normalized to that of the housekeeping control gene Gapdh , using the ΔΔCq method.

    Article Title: Analysis of Global Collection of Group A Streptococcus Genomes Reveals that the Majority Encode a Trio of M and M-Like Proteins
    Article Snippet: .. Target genes from 5 μl of cDNA were amplified in 20-μl reactions with Luna Universal SYBR qPCR master mix (NEB; M3003) in a Bio-Rad CX96 real-time PCR detection system with conditions as follows: 95°C for 60 s, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Dissociation curves were calculated for each reaction to confirm product specificity. ..

    Article Title: The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function
    Article Snippet: .. All cDNA samples (three biological replicates for each timepoint) were amplified using Luna Universal qPCR Master Mix (NEB) on an Applied Biosystems StepOne Real-Time PCR System (Thermo Fisher). ..

    In Vitro:

    Article Title: CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding
    Article Snippet: .. DNA from symptomatic field plant of Agbagba plant and asymptomatic in vitro plantlet of Cavendish Williams were used as positive and negative controls, respectively. qPCR was performed in a 20 µL reaction volume containing 60 ng of genomic DNA, 10 µL of Luna Universal qPCR master mix (New England Biolab), and 0.3 µL of 10 µM of each primer (P4F and P4R, Supplementary Table ). qPCR was performed using the ABI 7500 real-time machine (Applied Biosystem, USA). .. The means and standard deviation were calculated for CT values of six technical replicates from two independent experiments, using Minitab 14 statistical software, 2012.

    Synthesized:

    Article Title: Enhanced vascular activity of a new chimeric promoter containing the full CaMV 35S promoter and the plant XYLOGEN PROTEIN 1 promoter
    Article Snippet: .. Quantitative real-time PCR (qPCR) was performed using the Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) and 0.5 µL of synthesized cDNA as template following the manufacturer’s protocol. ..

    Article Title: Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes
    Article Snippet: .. Real-time quantitative PCR (qPCR) Total RNA was extracted using TRIzol (Thermo Fisher) and treated with Turbo DNase (Thermo Fisher). cDNA was synthesized using oligo(dT) and M-MLV reverse transcriptase with 1.5–2 μg of RNA. cDNA was then mixed with real-time PCR primers and Luna qPCR master mix (NEB). .. PCR was run on an Applied Biosciences StepOne Plus Real-Time PCR system.

    SYBR Green Assay:

    Article Title: H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development
    Article Snippet: .. SYBR green primers for other genes were described previously ( , ), and PCR was done using Luna® Universal qPCR Master Mix according to the manufacturers’ instructions (NEB). .. RNA-seq and ChIP-seq were performed as described in detail previously with the use of Illumina HiSeq 2500 ( , , , ).

    Expressing:

    Article Title: Nutrient Sensing, Nutrition, and Metabolism: Hepatic ZIP8 deficiency is associated with disrupted selenium homeostasis, liver pathology, and tumor formation
    Article Snippet: .. The quantitative (q)PCR for individual gene expression was carried out with Luna Universal qPCR Master Mix (New England Biolabs) and gene-specific primers; cycling conditions included 95°C for 1 min, followed by amplification for 40 cycles at 95°C for 15 s, and then 60°C for 30 s in the CFX96 Real-Time PCR system (Bio-Rad). .. Relative expression of mRNA levels was normalized to that of the housekeeping control gene Gapdh , using the ΔΔCq method.

    Polymerase Chain Reaction:

    Article Title: H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development
    Article Snippet: .. SYBR green primers for other genes were described previously ( , ), and PCR was done using Luna® Universal qPCR Master Mix according to the manufacturers’ instructions (NEB). .. RNA-seq and ChIP-seq were performed as described in detail previously with the use of Illumina HiSeq 2500 ( , , , ).

    Article Title: TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism
    Article Snippet: .. Quantitative realtime PCR was performed with a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories) and the Luna Universal qPCR Master Mix (New England Biolabs) in technical triplicates with 20 µl reaction volume and 2 µl of a 1:10 dilution of the cDNA template. .. Gapdh was used as reference gene for mouse qPCR and eef1a1l was used for zebrafish qPCR.

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    New England Biolabs luna universal qpcr master mix
    Liver pathology showing significant nodular tumors in Slc39a8(+/neo) mice. A : wild-type (WT) mouse ( far left column ) was 20 mo of age; Slc39a8(+/neo) mice ( three right columns ) were 13, 18 and 21 mo of age, respectively. Macroscopic livers ( top row ) and microscopic hematoxylin-eosin (H E) staining ( bottom two rows ) is shown. Nodular tumors are visualized in parts or in the entire liver of Slc39a8(+/neo) mice. Out of 22 hypomorph mice, 10 were identified with similar nodular tumors. Scale bars: 500 μm ( middle row ); 100 μm ( bottom row ). B : hepatic Slc39a8 mRNA expression in WT ( n = 6) and Slc39a8(+/neo) mice ages 13–21 mo ( n = 7). C : typical immunohistochemistry (IHC) staining for α-fetoprotein (AFP) and proliferating cell nuclear antigen (PCNA) in WT and Slc39a8(+/neo) mice (20 mo of age) ( top ). Bottom : quantitative <t>PCR</t> analysis of Afp in WT and in nontumor portion and tumor of Slc39a8(+/neo) ( n = 3 for each of the three histograms). All data represent means ± SE, with 1.0 designated for WT mean expression. * P ≤ 0.05 by Student’s t -test. T, tumor.
    Luna Universal Qpcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Liver pathology showing significant nodular tumors in Slc39a8(+/neo) mice. A : wild-type (WT) mouse ( far left column ) was 20 mo of age; Slc39a8(+/neo) mice ( three right columns ) were 13, 18 and 21 mo of age, respectively. Macroscopic livers ( top row ) and microscopic hematoxylin-eosin (H E) staining ( bottom two rows ) is shown. Nodular tumors are visualized in parts or in the entire liver of Slc39a8(+/neo) mice. Out of 22 hypomorph mice, 10 were identified with similar nodular tumors. Scale bars: 500 μm ( middle row ); 100 μm ( bottom row ). B : hepatic Slc39a8 mRNA expression in WT ( n = 6) and Slc39a8(+/neo) mice ages 13–21 mo ( n = 7). C : typical immunohistochemistry (IHC) staining for α-fetoprotein (AFP) and proliferating cell nuclear antigen (PCNA) in WT and Slc39a8(+/neo) mice (20 mo of age) ( top ). Bottom : quantitative PCR analysis of Afp in WT and in nontumor portion and tumor of Slc39a8(+/neo) ( n = 3 for each of the three histograms). All data represent means ± SE, with 1.0 designated for WT mean expression. * P ≤ 0.05 by Student’s t -test. T, tumor.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Nutrient Sensing, Nutrition, and Metabolism: Hepatic ZIP8 deficiency is associated with disrupted selenium homeostasis, liver pathology, and tumor formation

    doi: 10.1152/ajpgi.00165.2018

    Figure Lengend Snippet: Liver pathology showing significant nodular tumors in Slc39a8(+/neo) mice. A : wild-type (WT) mouse ( far left column ) was 20 mo of age; Slc39a8(+/neo) mice ( three right columns ) were 13, 18 and 21 mo of age, respectively. Macroscopic livers ( top row ) and microscopic hematoxylin-eosin (H E) staining ( bottom two rows ) is shown. Nodular tumors are visualized in parts or in the entire liver of Slc39a8(+/neo) mice. Out of 22 hypomorph mice, 10 were identified with similar nodular tumors. Scale bars: 500 μm ( middle row ); 100 μm ( bottom row ). B : hepatic Slc39a8 mRNA expression in WT ( n = 6) and Slc39a8(+/neo) mice ages 13–21 mo ( n = 7). C : typical immunohistochemistry (IHC) staining for α-fetoprotein (AFP) and proliferating cell nuclear antigen (PCNA) in WT and Slc39a8(+/neo) mice (20 mo of age) ( top ). Bottom : quantitative PCR analysis of Afp in WT and in nontumor portion and tumor of Slc39a8(+/neo) ( n = 3 for each of the three histograms). All data represent means ± SE, with 1.0 designated for WT mean expression. * P ≤ 0.05 by Student’s t -test. T, tumor.

    Article Snippet: The quantitative (q)PCR for individual gene expression was carried out with Luna Universal qPCR Master Mix (New England Biolabs) and gene-specific primers; cycling conditions included 95°C for 1 min, followed by amplification for 40 cycles at 95°C for 15 s, and then 60°C for 30 s in the CFX96 Real-Time PCR system (Bio-Rad).

    Techniques: Mouse Assay, Staining, Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction

    Expression analysis of Mga regulon genes. cDNA from 19 isolates grown to mid-log phase in rich medium were analyzed for the expression of Mga regulon genes. The isolates were selected to be representative of all possible Mga regulon configurations and emm cluster diversity where possible. Primers were designed to amplify all members of the gene family where possible ( mrp , enn , pgs , sph , and scpA ) and to amplify a subset where sequence diversity necessitates. The dot plot symbols represent the mean value of the four qPCR analyses for each isolate, and the error bars represent the standard errors for all isolates for each gene.

    Journal: mSphere

    Article Title: Analysis of Global Collection of Group A Streptococcus Genomes Reveals that the Majority Encode a Trio of M and M-Like Proteins

    doi: 10.1128/mSphere.00806-19

    Figure Lengend Snippet: Expression analysis of Mga regulon genes. cDNA from 19 isolates grown to mid-log phase in rich medium were analyzed for the expression of Mga regulon genes. The isolates were selected to be representative of all possible Mga regulon configurations and emm cluster diversity where possible. Primers were designed to amplify all members of the gene family where possible ( mrp , enn , pgs , sph , and scpA ) and to amplify a subset where sequence diversity necessitates. The dot plot symbols represent the mean value of the four qPCR analyses for each isolate, and the error bars represent the standard errors for all isolates for each gene.

    Article Snippet: Target genes from 5 μl of cDNA were amplified in 20-μl reactions with Luna Universal SYBR qPCR master mix (NEB; M3003) in a Bio-Rad CX96 real-time PCR detection system with conditions as follows: 95°C for 60 s, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Dissociation curves were calculated for each reaction to confirm product specificity.

    Techniques: Expressing, Sequencing, Real-time Polymerase Chain Reaction

    Timed induction of bmp4 affects expression of BMP antagonists. ( a ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of tg(hsp70:bmp4, cmlc2:GFP) embryos at 30 hpf after heat shock at 24 hpf as represented by the log2(fold change) of individual samples. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 0.170 and 0.049, respectively. ( b , c ) WMISH for fsta (30 hpf) in tg(hsp70:bmp4, cmlc2:GFP) after heat shock at 24 hpf ( b ) and control embryos ( c ). Strong upregulation after the heat shock is seen in the optic fissure and other expression domains, but not the ciliary marginal zone (arrow). WMISHs in ( b ) and ( c ) were stained in parallel for the same amount of time.

    Journal: Open Biology

    Article Title: TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

    doi: 10.1098/rsob.170134

    Figure Lengend Snippet: Timed induction of bmp4 affects expression of BMP antagonists. ( a ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of tg(hsp70:bmp4, cmlc2:GFP) embryos at 30 hpf after heat shock at 24 hpf as represented by the log2(fold change) of individual samples. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 0.170 and 0.049, respectively. ( b , c ) WMISH for fsta (30 hpf) in tg(hsp70:bmp4, cmlc2:GFP) after heat shock at 24 hpf ( b ) and control embryos ( c ). Strong upregulation after the heat shock is seen in the optic fissure and other expression domains, but not the ciliary marginal zone (arrow). WMISHs in ( b ) and ( c ) were stained in parallel for the same amount of time.

    Article Snippet: Quantitative realtime PCR was performed with a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories) and the Luna Universal qPCR Master Mix (New England Biolabs) in technical triplicates with 20 µl reaction volume and 2 µl of a 1:10 dilution of the cDNA template.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining

    BMP antagonists grem2b and fsta are expressed in the optic fissure. WMISHs were performed at 30 hpf and are shown in lateral view. ( a ) bmp4 is expressed in the dorsal optic cup (arrow). ( b ) Expression of vax2 in the ventral retina. ( c , d ) Expression of grem2b in the optic cup is restricted to the optic fissure (arrow). ( d ) Confocal view of grem2b expression (red) with DAPI counterstaining (blue). ( e , f ) fsta is expressed in the optic fissure (arrows), as well as the ciliary marginal zone (CMZ, arrowhead). ( f ) Confocal view of fsta expression (red) with DAPI counterstaining (blue). ( g ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of SIS3-treated embryos (30 hpf) as represented by the log2(fold change) of individual samples. Embryos were treated from 24 hpf onward, controls were treated with DMSO. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 4.3 × 10 −3 and 0.877, respectively. ( h ) Model of the proposed role of TGFβ and BMP antagonism during optic fissure fusion. TGFβ signalling domains in the optic fissure margins are shielded from BMP by induced BMP antagonists. ( i ) Scheme of a heat shock inducible BMP construct used to create the transgenic line tg(hsp70:bmp4, cmlc2:GFP) . GFP expressed under the cardiac cmlc2 promoter serves as transgenesis marker. Experimental procedure using heat shocks at different time points between 21 and 26 hpf to induce bmp4 expression, aiming at disrupting optic fissure fusion. Analysis of phenotypes was scheduled for 3 dpf.

    Journal: Open Biology

    Article Title: TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

    doi: 10.1098/rsob.170134

    Figure Lengend Snippet: BMP antagonists grem2b and fsta are expressed in the optic fissure. WMISHs were performed at 30 hpf and are shown in lateral view. ( a ) bmp4 is expressed in the dorsal optic cup (arrow). ( b ) Expression of vax2 in the ventral retina. ( c , d ) Expression of grem2b in the optic cup is restricted to the optic fissure (arrow). ( d ) Confocal view of grem2b expression (red) with DAPI counterstaining (blue). ( e , f ) fsta is expressed in the optic fissure (arrows), as well as the ciliary marginal zone (CMZ, arrowhead). ( f ) Confocal view of fsta expression (red) with DAPI counterstaining (blue). ( g ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in heads of SIS3-treated embryos (30 hpf) as represented by the log2(fold change) of individual samples. Embryos were treated from 24 hpf onward, controls were treated with DMSO. Material from three individuals was pooled for one sample; n = 3, horizontal bars represent the arithmetic mean. p -Values for grem2b and fsta , 4.3 × 10 −3 and 0.877, respectively. ( h ) Model of the proposed role of TGFβ and BMP antagonism during optic fissure fusion. TGFβ signalling domains in the optic fissure margins are shielded from BMP by induced BMP antagonists. ( i ) Scheme of a heat shock inducible BMP construct used to create the transgenic line tg(hsp70:bmp4, cmlc2:GFP) . GFP expressed under the cardiac cmlc2 promoter serves as transgenesis marker. Experimental procedure using heat shocks at different time points between 21 and 26 hpf to induce bmp4 expression, aiming at disrupting optic fissure fusion. Analysis of phenotypes was scheduled for 3 dpf.

    Article Snippet: Quantitative realtime PCR was performed with a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories) and the Luna Universal qPCR Master Mix (New England Biolabs) in technical triplicates with 20 µl reaction volume and 2 µl of a 1:10 dilution of the cDNA template.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Construct, Transgenic Assay, Marker

    Loss of TGFβ2 ligand results in coloboma. ( a , b ) Frontal sections (E18.5, H E) of ( b ) TGFβ2 KO and ( a ) wild-type mouse embryos from mixed genetic background. Note the persisting optic fissure (boxed in b ), scale bars 200 µm. ( c , d ) Frontal sections (E16.5, H E) of ( d ) TGFβ2 KO and ( c ) wild-type mouse embryos from a sole genetic background, mild coloboma phenotype boxed in ( d ), Scale bars 200 µm. ( e ) Expression analysis of gremlin and follistatin, decrease in TGFβ2 KO (TGFβ2/GDNF KO) as represented by the log2(fold change). Error bars represent the 95% confidence interval. Corrected p -values of control gene expression compared to KO for Grem1 and Fst, 5.5 × 10 −3 and 1.2 × 10 −3 , respectively. ( f ) Expression analysis of gremlin and follistatin by quantitative PCR, decrease in TGFβ2 KO (TGFβ2 −/−, GDNF +/− ) compared to controls (TGFβ2 +/+ , GDNF +/− ) as represented by the log2(fold change) of individual samples; n = 3, horizontal bars represent the arithmetic mean. p -values for Grem1 and Fst, 7.8 × 10 −3 and 0.029, respectively. ( g ) Selected terms enriched in the set of downregulated genes in TGFβ2/GDNF KO microarray based on gProfiler analysis. BP, biological process; CC, cellular component; MF, molecular function. ( h ) Expression analysis of several ECM-related genes. Error bars represent the 95% confidence interval. ( i ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in TGFβ2 KO (sole genetic background) compared to wild-type as represented by the log2(fold change) of individual samples; n = 5, one KO sample was excluded as an outlier. Horizontal bars represent the arithmetic mean. p -Values for Grem1 and Fst, 0.312 and 0.027, respectively.

    Journal: Open Biology

    Article Title: TGFβ-facilitated optic fissure fusion and the role of bone morphogenetic protein antagonism

    doi: 10.1098/rsob.170134

    Figure Lengend Snippet: Loss of TGFβ2 ligand results in coloboma. ( a , b ) Frontal sections (E18.5, H E) of ( b ) TGFβ2 KO and ( a ) wild-type mouse embryos from mixed genetic background. Note the persisting optic fissure (boxed in b ), scale bars 200 µm. ( c , d ) Frontal sections (E16.5, H E) of ( d ) TGFβ2 KO and ( c ) wild-type mouse embryos from a sole genetic background, mild coloboma phenotype boxed in ( d ), Scale bars 200 µm. ( e ) Expression analysis of gremlin and follistatin, decrease in TGFβ2 KO (TGFβ2/GDNF KO) as represented by the log2(fold change). Error bars represent the 95% confidence interval. Corrected p -values of control gene expression compared to KO for Grem1 and Fst, 5.5 × 10 −3 and 1.2 × 10 −3 , respectively. ( f ) Expression analysis of gremlin and follistatin by quantitative PCR, decrease in TGFβ2 KO (TGFβ2 −/−, GDNF +/− ) compared to controls (TGFβ2 +/+ , GDNF +/− ) as represented by the log2(fold change) of individual samples; n = 3, horizontal bars represent the arithmetic mean. p -values for Grem1 and Fst, 7.8 × 10 −3 and 0.029, respectively. ( g ) Selected terms enriched in the set of downregulated genes in TGFβ2/GDNF KO microarray based on gProfiler analysis. BP, biological process; CC, cellular component; MF, molecular function. ( h ) Expression analysis of several ECM-related genes. Error bars represent the 95% confidence interval. ( i ) Expression analysis of gremlin and follistatin by quantitative PCR, differential expression in TGFβ2 KO (sole genetic background) compared to wild-type as represented by the log2(fold change) of individual samples; n = 5, one KO sample was excluded as an outlier. Horizontal bars represent the arithmetic mean. p -Values for Grem1 and Fst, 0.312 and 0.027, respectively.

    Article Snippet: Quantitative realtime PCR was performed with a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories) and the Luna Universal qPCR Master Mix (New England Biolabs) in technical triplicates with 20 µl reaction volume and 2 µl of a 1:10 dilution of the cDNA template.

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

    Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments

    Journal: Communications Biology

    Article Title: CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding

    doi: 10.1038/s42003-019-0288-7

    Figure Lengend Snippet: Evaluation of genome-edited and wild type non-edited control plants of Gonja Manjaya for induction of BSV symptoms under water stress conditions. Two-month-old plants were subjected to water stress for 2 weeks. Disease symptoms as chlorosis or yellow streaks were recorded at the end of the stress period and pictures were taken. a Pictures of asymptomatic genome-edited plants (17, 76, and 81), symptomatic-edited plants (66 and 97), and wild type control plants (WT). b PCR diagnostic to detect activation of episomal BSOLV in genome edited and control plants under water stress conditions. c qPCR analysis to detect episomal BSOLV in genome edited and control plants under water stress conditions. PC1‒2, symptomatic plant of Agbagba from field as positive control; WT, wild type non-edited control Gonja Manjaya plant under stress conditions; 17, 66, 76, 81, 97, edited plants under stress conditions; CW, asymptomatic in vitro plantlet of Cavendish Williams as negative control; NTC no template control. For PCR and qPCR, leaf samples from three symptomatic wild type non-edited control plants (WT) of Gonja Manjaya were pooled for DNA extraction. Similarly, the leaves from three replicates for symptomatic and asymptomatic-edited events were pooled for PCR and qPCR. CT values were presented as means and standard error of six technical replicates from two independent experiments

    Article Snippet: DNA from symptomatic field plant of Agbagba plant and asymptomatic in vitro plantlet of Cavendish Williams were used as positive and negative controls, respectively. qPCR was performed in a 20 µL reaction volume containing 60 ng of genomic DNA, 10 µL of Luna Universal qPCR master mix (New England Biolab), and 0.3 µL of 10 µM of each primer (P4F and P4R, Supplementary Table ). qPCR was performed using the ABI 7500 real-time machine (Applied Biosystem, USA).

    Techniques: Polymerase Chain Reaction, Diagnostic Assay, Activation Assay, Real-time Polymerase Chain Reaction, Positive Control, In Vitro, Negative Control, DNA Extraction