cdna  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    cDNA Size Fractionation Columns
    Description:
    cDNA Size Fractionation Columns offer an efficient and convenient method for size fractioning double stranded cDNA 1 • Pre packed disposable columns efficiently remove small 500 bp cDNAs including residual adapters linkers or other fragments that could interfere with ligation of cDNA to the vector• Columns size fractionate cDNA 500 bpPerformance and Quality Testing Wash time drop volume during elution of fragments and the ability to clearly and cleanly separate a mixture of dyes including xylene cyanol and bromophenol blue are evaluated
    Catalog Number:
    18092015
    Price:
    None
    Applications:
    Cloning|cDNA Libraries & Library Construction
    Category:
    Chromatography Columns Resins Spin Filters
    Buy from Supplier


    Structured Review

    Thermo Fisher cdna
    Improved hierarchical clustering of combined NCI-60 cell-lines profiled by <t>Affymetrix</t> gene-chip and <t>cDNA</t> microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.
    cDNA Size Fractionation Columns offer an efficient and convenient method for size fractioning double stranded cDNA 1 • Pre packed disposable columns efficiently remove small 500 bp cDNAs including residual adapters linkers or other fragments that could interfere with ligation of cDNA to the vector• Columns size fractionate cDNA 500 bpPerformance and Quality Testing Wash time drop volume during elution of fragments and the ability to clearly and cleanly separate a mixture of dyes including xylene cyanol and bromophenol blue are evaluated
    https://www.bioz.com/result/cdna/product/Thermo Fisher
    Average 99 stars, based on 10468 article reviews
    Price from $9.99 to $1999.99
    cdna - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements"

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    Journal: BMC Bioinformatics

    doi: 10.1186/1471-2105-6-107

    Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.
    Figure Legend Snippet: Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.

    Techniques Used: Chromatin Immunoprecipitation, Microarray, Sequencing, Expressing, Transformation Assay, Produced

    Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.
    Figure Legend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.

    Techniques Used: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced, Amplification, Immunohistochemistry

    Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.
    Figure Legend Snippet: Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.

    Techniques Used: Sequencing, Expressing, Microarray

    Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.
    Figure Legend Snippet: Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.

    Techniques Used: Microarray

    Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.
    Figure Legend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.

    Techniques Used: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced

    Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.
    Figure Legend Snippet: Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.

    Techniques Used: Sequencing, Microarray

    2) Product Images from "Assembly of a Notch Transcriptional Activation Complex Requires Multimerization ▿"

    Article Title: Assembly of a Notch Transcriptional Activation Complex Requires Multimerization ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00360-10

    A functional ΦWΦP motif in N ICD is required for efficient multimer-monomer conversion and further active-complex assembly. (A) Flag-tagged N ICD and N ICD RAM point mutants were cotransfected into 293T cells with N ICDΔRΔ2444 Myc tag in either the presence or absence of CSL. Forty-eight hours posttransfection, whole-cell lysates were immunoprecipitated with anti-Flag antibody and subsequently analyzed by SDS-PAGE and Western blotting. Anti-Notch antibody was used to detect N ICD and N ICD RAM point mutants, anti-Myc antibody to detect N ICDΔRΔ2444 -Myc, and anti-HA antibody to detect CSL. (B) Schematic representation of N ICDΔRΔ2444 -Myc and N ICD -Flag tagged proteins. An alignment of N ICD RAM point mutations used in the experiment is also shown. The boldface denotes amino acid conservation. (C) H1299 cells were transfected with 0.2 μg of each construct. For titrations, H1299 cells were transfected with 0.2 μg of N ICDΔRΔ2444 or N ICD1767-70A cDNA and increasing amounts of N ICDΔ2105 , N ICDΔRΔ2444 , or N ICD1767-70A cDNA (0.2 to 0.8 μg). Forty-eight hours posttransfection, the cells were lysed and luciferase assays were performed in triplicate. The results are shown in relative luciferase units. The error bars represent the standard deviations from each set.
    Figure Legend Snippet: A functional ΦWΦP motif in N ICD is required for efficient multimer-monomer conversion and further active-complex assembly. (A) Flag-tagged N ICD and N ICD RAM point mutants were cotransfected into 293T cells with N ICDΔRΔ2444 Myc tag in either the presence or absence of CSL. Forty-eight hours posttransfection, whole-cell lysates were immunoprecipitated with anti-Flag antibody and subsequently analyzed by SDS-PAGE and Western blotting. Anti-Notch antibody was used to detect N ICD and N ICD RAM point mutants, anti-Myc antibody to detect N ICDΔRΔ2444 -Myc, and anti-HA antibody to detect CSL. (B) Schematic representation of N ICDΔRΔ2444 -Myc and N ICD -Flag tagged proteins. An alignment of N ICD RAM point mutations used in the experiment is also shown. The boldface denotes amino acid conservation. (C) H1299 cells were transfected with 0.2 μg of each construct. For titrations, H1299 cells were transfected with 0.2 μg of N ICDΔRΔ2444 or N ICD1767-70A cDNA and increasing amounts of N ICDΔ2105 , N ICDΔRΔ2444 , or N ICD1767-70A cDNA (0.2 to 0.8 μg). Forty-eight hours posttransfection, the cells were lysed and luciferase assays were performed in triplicate. The results are shown in relative luciferase units. The error bars represent the standard deviations from each set.

    Techniques Used: Functional Assay, Immunoprecipitation, SDS Page, Western Blot, Transfection, Construct, Luciferase

    3) Product Images from "The invertebrate microtubule-associated protein PTL-1 functions in mechanosensation and development in Caenorhabditis elegans"

    Article Title: The invertebrate microtubule-associated protein PTL-1 functions in mechanosensation and development in Caenorhabditis elegans

    Journal:

    doi: 10.1007/s00427-008-0250-z

    ptl-1 mRNA is not expressed in ptl-1∷(ok621) null mutant worms. mRNA was extracted from mixed stage worms (wild type and ptl-1(ok621) homozygotes) using Invitrogen’s Microfast-track kit (Invitrogen, Inc.) and converted to cDNA using Invitrogen’s
    Figure Legend Snippet: ptl-1 mRNA is not expressed in ptl-1∷(ok621) null mutant worms. mRNA was extracted from mixed stage worms (wild type and ptl-1(ok621) homozygotes) using Invitrogen’s Microfast-track kit (Invitrogen, Inc.) and converted to cDNA using Invitrogen’s

    Techniques Used: Mutagenesis

    4) Product Images from "Investigations into a putative role for the novel BRASSIKIN pseudokinases in compatible pollen-stigma interactions in Arabidopsis thaliana"

    Article Title: Investigations into a putative role for the novel BRASSIKIN pseudokinases in compatible pollen-stigma interactions in Arabidopsis thaliana

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-019-2160-9

    A. thaliana BKN1 gene models and ecotype polymorphisms. a. A. thaliana BKN1 gene models are shown with carpel RNA-Seq mapping coverage from Araport [ 45 ]. Yellow astericks (*) mark two in/del SNPs in Col-0 BKN1 when compared to Hh-0 BKN1 and A. lyrata BKN1 . For the BNK1 Col-0 gene annotations and cDNA, the first asterisk marks a 1 bp deletion (T) resulting in an adjacent premature stop codon and the second asterisk marks a 1 bp insertion (A) that would result in a downstream premature stop codon for the At5g11400.1 annotation. Based on the reduced carpel RNA-Seq coverage at the 5′ end of the BKN1 exon 2, there may be alternate splice sites in use (yellow arrow) and some of these potential alternate splice junctions would restore the BKN1 Col-0 reading frame to produce a longer protein as predicted for the At5g11400.1 and At5g11400.2 annotations. See also Additional file 1 : Figure S3 and S4. The orange arrow delineates the third intron that is not properly spliced in the top RT-PCR band in Fig. 1b . b. A. thaliana BKN1 polymorphisms in different ecotypes. In addition to Hh-0, Västervik and Dju-1 are predicted to encode a full length BKN1 protein (based on genomic sequencing). Bela-1 and Bik-1 displayed other SNPs that disrupt the BKN1 open reading frame (see also Additional file 1 : Figure S6)
    Figure Legend Snippet: A. thaliana BKN1 gene models and ecotype polymorphisms. a. A. thaliana BKN1 gene models are shown with carpel RNA-Seq mapping coverage from Araport [ 45 ]. Yellow astericks (*) mark two in/del SNPs in Col-0 BKN1 when compared to Hh-0 BKN1 and A. lyrata BKN1 . For the BNK1 Col-0 gene annotations and cDNA, the first asterisk marks a 1 bp deletion (T) resulting in an adjacent premature stop codon and the second asterisk marks a 1 bp insertion (A) that would result in a downstream premature stop codon for the At5g11400.1 annotation. Based on the reduced carpel RNA-Seq coverage at the 5′ end of the BKN1 exon 2, there may be alternate splice sites in use (yellow arrow) and some of these potential alternate splice junctions would restore the BKN1 Col-0 reading frame to produce a longer protein as predicted for the At5g11400.1 and At5g11400.2 annotations. See also Additional file 1 : Figure S3 and S4. The orange arrow delineates the third intron that is not properly spliced in the top RT-PCR band in Fig. 1b . b. A. thaliana BKN1 polymorphisms in different ecotypes. In addition to Hh-0, Västervik and Dju-1 are predicted to encode a full length BKN1 protein (based on genomic sequencing). Bela-1 and Bik-1 displayed other SNPs that disrupt the BKN1 open reading frame (see also Additional file 1 : Figure S6)

    Techniques Used: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Genomic Sequencing

    5) Product Images from "Potential involvement of P2Y2 receptor in diuresis of postobstructive uropathy in rats"

    Article Title: Potential involvement of P2Y2 receptor in diuresis of postobstructive uropathy in rats

    Journal:

    doi: 10.1152/ajprenal.00382.2009

    Changes in relative expression of P2Y receptor subtypes in rat inner medulla during postobstructive uropathy. BUO/R ( n = 4) and sham ( n = 3) rats were euthanized on day 8 . RNA was extracted and reverse transcribed, and cDNA samples were amplified with
    Figure Legend Snippet: Changes in relative expression of P2Y receptor subtypes in rat inner medulla during postobstructive uropathy. BUO/R ( n = 4) and sham ( n = 3) rats were euthanized on day 8 . RNA was extracted and reverse transcribed, and cDNA samples were amplified with

    Techniques Used: Expressing, Amplification

    Changes in relative expression of cyclooxygenases 1 and 2 (COX-1 and COX-2) in rat inner medulla during postobstructive uropathy. BUO/R and sham rats ( n = 4/group) were euthanized on day 12 . RNA was extracted and reverse transcribed, and cDNA samples
    Figure Legend Snippet: Changes in relative expression of cyclooxygenases 1 and 2 (COX-1 and COX-2) in rat inner medulla during postobstructive uropathy. BUO/R and sham rats ( n = 4/group) were euthanized on day 12 . RNA was extracted and reverse transcribed, and cDNA samples

    Techniques Used: Expressing

    6) Product Images from "Target Genes of Neuron-Restrictive Silencer Factor Are Abnormally Up-Regulated in Human Myotilinopathy"

    Article Title: Target Genes of Neuron-Restrictive Silencer Factor Are Abnormally Up-Regulated in Human Myotilinopathy

    Journal:

    doi: 10.2353/ajpath.2007.070520

    NRSF/REST overexpression reduces α-internexin, SNAP25, synaptophysin, and UCHL1 mRNA levels in the DMS53 cell line. A: One μg of REEX1 vector, which encodes human full-length NRSF cDNA, was transfected in DMS53 cells using Lipofectamine
    Figure Legend Snippet: NRSF/REST overexpression reduces α-internexin, SNAP25, synaptophysin, and UCHL1 mRNA levels in the DMS53 cell line. A: One μg of REEX1 vector, which encodes human full-length NRSF cDNA, was transfected in DMS53 cells using Lipofectamine

    Techniques Used: Over Expression, Plasmid Preparation, Transfection

    7) Product Images from "Tolerogenic bone marrow-derived dendritic cells induce neuroprotective regulatory T cells in a model of Parkinson’s disease"

    Article Title: Tolerogenic bone marrow-derived dendritic cells induce neuroprotective regulatory T cells in a model of Parkinson’s disease

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-018-0255-7

    Gene expression in midbrain after treatment with MPTP or BMDCs and MPTP. BMDCs were differentiated for 8 days in 20 ng/ml GM-CSF prior to 3 days of pretreatment in media alone. These BMDCs were transferred i.v. one and two weeks prior to intoxication with four doses of 16 mg/kg MPTP. Two days after intoxication, PBS, MPTP and BMDC + MPTP mice were sacrificed and the brain was removed, hemisected and the midbrain was incubated in RNAlater for 24 h prior to freezing at − 80 °C. RNA was isolated from the midbrain, converted to cDNA PCR arrays of proinflammatory genes were run. a Gene expression was determined relative to the PBS control midbrains; n = 3 for PBS and n = 4 for MPTP and MPTP+BMDCs. Fold change was determined using SA Bioscience software. b Ingenuity Pathway Analysis was used to determine the expression of genes associated with the inflammatory response in BMDCs + MPTP midbrain compared to MPTP control mice
    Figure Legend Snippet: Gene expression in midbrain after treatment with MPTP or BMDCs and MPTP. BMDCs were differentiated for 8 days in 20 ng/ml GM-CSF prior to 3 days of pretreatment in media alone. These BMDCs were transferred i.v. one and two weeks prior to intoxication with four doses of 16 mg/kg MPTP. Two days after intoxication, PBS, MPTP and BMDC + MPTP mice were sacrificed and the brain was removed, hemisected and the midbrain was incubated in RNAlater for 24 h prior to freezing at − 80 °C. RNA was isolated from the midbrain, converted to cDNA PCR arrays of proinflammatory genes were run. a Gene expression was determined relative to the PBS control midbrains; n = 3 for PBS and n = 4 for MPTP and MPTP+BMDCs. Fold change was determined using SA Bioscience software. b Ingenuity Pathway Analysis was used to determine the expression of genes associated with the inflammatory response in BMDCs + MPTP midbrain compared to MPTP control mice

    Techniques Used: Expressing, Mouse Assay, Incubation, Isolation, Polymerase Chain Reaction, Software

    Gene expression of GM-CSF, N-α-Syn, or GM-CSF/N-α-Syn-cultured BMDCs . GM-CSF-generated BMDCs were cultured for 2 days in media or 20 ng/ml GM-CSF ( GM-CSF ). BMDC cultures from each group were stimulated with 30 μg/ml N-α-Syn for 6 h ( N-α-Syn or GM-CSF + N-α-Syn , respectively). RNA was isolated, converted to cDNA and assessed by PCR arrays for proinflammatory genes. Gene expression from BMDCs from treatment groups was determined relative to BMDCs treated with media alone. Each treatment group was composed of 6 replicates and fold change was determined by SA Bioscience array software
    Figure Legend Snippet: Gene expression of GM-CSF, N-α-Syn, or GM-CSF/N-α-Syn-cultured BMDCs . GM-CSF-generated BMDCs were cultured for 2 days in media or 20 ng/ml GM-CSF ( GM-CSF ). BMDC cultures from each group were stimulated with 30 μg/ml N-α-Syn for 6 h ( N-α-Syn or GM-CSF + N-α-Syn , respectively). RNA was isolated, converted to cDNA and assessed by PCR arrays for proinflammatory genes. Gene expression from BMDCs from treatment groups was determined relative to BMDCs treated with media alone. Each treatment group was composed of 6 replicates and fold change was determined by SA Bioscience array software

    Techniques Used: Expressing, Cell Culture, Generated, Isolation, Polymerase Chain Reaction, Software

    8) Product Images from "Increased leukotoxin production: Characterization of 100 base pairs within the 530 base pair leukotoxin promoter region of Aggregatibacter actinomycetemcomitans"

    Article Title: Increased leukotoxin production: Characterization of 100 base pairs within the 530 base pair leukotoxin promoter region of Aggregatibacter actinomycetemcomitans

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01692-6

    Transcriptional fusion of orf X with ltx operon due to promoter region deletion. A representative RT-PCR gel picture showing the transcriptional fusion in RhAa- ltx P530, but not in RhAa3 and RhAa- ltx P298-397 is seen. 1 – Genomic DNA template (+ve control); 2 – RNA template with no RT (−ve control); 3 – cDNA template ( A ). qRT-PCR analysis was carried out to assess the expression level of ltx A due to promoter deletion. There was a 10.7-fold increase in ltx A expression in RhAa- ltx P530, 5.9 fold increase in RhAa- ltx P298-530 and 7.4 fold increase in RhAa- ltx P298-397 compared to RhAa3. Values are means from a triplicate experiment. Error bars indicate ± SD. The significant fold changes (* P
    Figure Legend Snippet: Transcriptional fusion of orf X with ltx operon due to promoter region deletion. A representative RT-PCR gel picture showing the transcriptional fusion in RhAa- ltx P530, but not in RhAa3 and RhAa- ltx P298-397 is seen. 1 – Genomic DNA template (+ve control); 2 – RNA template with no RT (−ve control); 3 – cDNA template ( A ). qRT-PCR analysis was carried out to assess the expression level of ltx A due to promoter deletion. There was a 10.7-fold increase in ltx A expression in RhAa- ltx P530, 5.9 fold increase in RhAa- ltx P298-530 and 7.4 fold increase in RhAa- ltx P298-397 compared to RhAa3. Values are means from a triplicate experiment. Error bars indicate ± SD. The significant fold changes (* P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    9) Product Images from "Secretion and Organization of a Cornea-like Tissue In Vitro by Stem Cells from Human Corneal Stroma"

    Article Title: Secretion and Organization of a Cornea-like Tissue In Vitro by Stem Cells from Human Corneal Stroma

    Journal:

    doi: 10.1167/iovs.07-0587

    Differential gene expression profiles of hCSSC and keratocytes. mRNA abundance relative to 18s ribosomal RNA was determined by qRT-PCR for a panel of 18 genes using cDNA from primary human keratocytes cultured in protein-free DME-F12 medium and from hCSSC
    Figure Legend Snippet: Differential gene expression profiles of hCSSC and keratocytes. mRNA abundance relative to 18s ribosomal RNA was determined by qRT-PCR for a panel of 18 genes using cDNA from primary human keratocytes cultured in protein-free DME-F12 medium and from hCSSC

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture

    10) Product Images from "Effect of Nuclear Factor ?B Inhibition on Serotype 9 Adeno-Associated Viral (AAV9) Minidystrophin Gene Transfer to the mdx Mouse"

    Article Title: Effect of Nuclear Factor ?B Inhibition on Serotype 9 Adeno-Associated Viral (AAV9) Minidystrophin Gene Transfer to the mdx Mouse

    Journal: Molecular Medicine

    doi: 10.2119/molmed.2011.00404

    Quantification of vector DNA and mRNA transcripts in quadriceps. DNA and RNA were isolated from quadriceps muscle tissues from AAV + NBD peptide–and AAV + saline–treated mdx mice and analyzed by real-time qPCR. Vector DNA levels were quantified by primers/probes specific for AAV9 minidystrophin DNA, using isolated AAV9 minidystrophin vector DNA to generate a standard curve. The numbers of nuclei per sample were quantified by real-time qPCR using primers/ probes specific for the ApoB gene. (A) Vector copy numbers were normalized to nuclei copy number to yield vector genomes/nucleus. Isolated muscle mRNA was reverse-transcribed, and cDNA samples were analyzed by real-time qPCR with primers/probe specific for minidystrophin cDNA to assess levels of vector mRNA and also with primers/probe specific for GAPDH. (B) Vector mRNA expression was normalized to GAPDH. Data are shown as mean ± SEM; *significant difference from AAV + saline–treated mdx mice ( P
    Figure Legend Snippet: Quantification of vector DNA and mRNA transcripts in quadriceps. DNA and RNA were isolated from quadriceps muscle tissues from AAV + NBD peptide–and AAV + saline–treated mdx mice and analyzed by real-time qPCR. Vector DNA levels were quantified by primers/probes specific for AAV9 minidystrophin DNA, using isolated AAV9 minidystrophin vector DNA to generate a standard curve. The numbers of nuclei per sample were quantified by real-time qPCR using primers/ probes specific for the ApoB gene. (A) Vector copy numbers were normalized to nuclei copy number to yield vector genomes/nucleus. Isolated muscle mRNA was reverse-transcribed, and cDNA samples were analyzed by real-time qPCR with primers/probe specific for minidystrophin cDNA to assess levels of vector mRNA and also with primers/probe specific for GAPDH. (B) Vector mRNA expression was normalized to GAPDH. Data are shown as mean ± SEM; *significant difference from AAV + saline–treated mdx mice ( P

    Techniques Used: Plasmid Preparation, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    Quantification of vector DNA and mRNA transcripts in quadriceps. DNA and RNA were isolated from quadriceps muscle tissues from AAV + NBD peptide–and AAV + saline–treated mdx mice and analyzed by real-time qPCR. Vector DNA levels were quantified by primers/probes specific for AAV9 minidystrophin DNA, using isolated AAV9 minidystrophin vector DNA to generate a standard curve. The numbers of nuclei per sample were quantified by real-time qPCR using primers/ probes specific for the ApoB gene. (A) Vector copy numbers were normalized to nuclei copy number to yield vector genomes/nucleus. Isolated muscle mRNA was reverse-transcribed, and cDNA samples were analyzed by real-time qPCR with primers/probe specific for minidystrophin cDNA to assess levels of vector mRNA and also with primers/probe specific for GAPDH. (B) Vector mRNA expression was normalized to GAPDH. Data are shown as mean ± SEM; *significant difference from AAV + saline–treated mdx mice ( P
    Figure Legend Snippet: Quantification of vector DNA and mRNA transcripts in quadriceps. DNA and RNA were isolated from quadriceps muscle tissues from AAV + NBD peptide–and AAV + saline–treated mdx mice and analyzed by real-time qPCR. Vector DNA levels were quantified by primers/probes specific for AAV9 minidystrophin DNA, using isolated AAV9 minidystrophin vector DNA to generate a standard curve. The numbers of nuclei per sample were quantified by real-time qPCR using primers/ probes specific for the ApoB gene. (A) Vector copy numbers were normalized to nuclei copy number to yield vector genomes/nucleus. Isolated muscle mRNA was reverse-transcribed, and cDNA samples were analyzed by real-time qPCR with primers/probe specific for minidystrophin cDNA to assess levels of vector mRNA and also with primers/probe specific for GAPDH. (B) Vector mRNA expression was normalized to GAPDH. Data are shown as mean ± SEM; *significant difference from AAV + saline–treated mdx mice ( P

    Techniques Used: Plasmid Preparation, Isolation, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing

    11) Product Images from "Cross-platform comparison and visualisation of gene expression data using co-inertia analysis"

    Article Title: Cross-platform comparison and visualisation of gene expression data using co-inertia analysis

    Journal: BMC Bioinformatics

    doi: 10.1186/1471-2105-4-59

    Cross-platform comparison of Affymetrix and spotted cDNA expression profiles using CIA. The first two axes of a CIA of gene expression profiles of the complete gene set from the Ross spotted cDNA array dataset (closed circles) and 1517 genes from the Staunton Affymetrix dataset (arrows) are shown. Circles and arrow represent the projected co-ordinates of each dataset, and these are joined by a line, where the length of the line is proportional to the divergence between the different gene expression profiles. The cell lines are coloured as in Figure 1 . The cell lines are derived from breast (BR), melanoma (ME), colon (CO), ovarian (OV), renal (RE), lung (LC), central nervous system (CNS, glioblastoma), prostate (PR) cancers and leukaemia (LE). Colon and leukaemia cells were separated from those with mesenchymal or stromal features (glioblastoma and renal tumour cell lines) on the first axis (F1, horizontal), and melanoma cell lines were distinguished from the other cell lines on the second axis (F2, vertical). A histogram of the main factors which explain the total variability of this CIA is superimposed on the top right corner. The first three axes represented 42%, 21% and 8% of the inertia.
    Figure Legend Snippet: Cross-platform comparison of Affymetrix and spotted cDNA expression profiles using CIA. The first two axes of a CIA of gene expression profiles of the complete gene set from the Ross spotted cDNA array dataset (closed circles) and 1517 genes from the Staunton Affymetrix dataset (arrows) are shown. Circles and arrow represent the projected co-ordinates of each dataset, and these are joined by a line, where the length of the line is proportional to the divergence between the different gene expression profiles. The cell lines are coloured as in Figure 1 . The cell lines are derived from breast (BR), melanoma (ME), colon (CO), ovarian (OV), renal (RE), lung (LC), central nervous system (CNS, glioblastoma), prostate (PR) cancers and leukaemia (LE). Colon and leukaemia cells were separated from those with mesenchymal or stromal features (glioblastoma and renal tumour cell lines) on the first axis (F1, horizontal), and melanoma cell lines were distinguished from the other cell lines on the second axis (F2, vertical). A histogram of the main factors which explain the total variability of this CIA is superimposed on the top right corner. The first three axes represented 42%, 21% and 8% of the inertia.

    Techniques Used: Expressing, Derivative Assay

    Hierarchical clustering of Affymetrix and spotted cDNA expression profiles of 60 cell lines. Dendrograms showings average linkage hierarchical clustering of NCI60 human cancer cell lines using Spearman Rank correlations. Cluster analyses of the 60 cell lines based on A) gene expression profiles of 1415 genes from the Ross spotted cDNA array dataset and B) 1517 genes from the Staunton Affymetrix dataset are shown. The cell lines are coloured as in Figure 1 . The colon tumour cell line HT29 and cluster of colon tumour cell lines are highlighted by a green arrow and bar respectively.
    Figure Legend Snippet: Hierarchical clustering of Affymetrix and spotted cDNA expression profiles of 60 cell lines. Dendrograms showings average linkage hierarchical clustering of NCI60 human cancer cell lines using Spearman Rank correlations. Cluster analyses of the 60 cell lines based on A) gene expression profiles of 1415 genes from the Ross spotted cDNA array dataset and B) 1517 genes from the Staunton Affymetrix dataset are shown. The cell lines are coloured as in Figure 1 . The colon tumour cell line HT29 and cluster of colon tumour cell lines are highlighted by a green arrow and bar respectively.

    Techniques Used: Expressing

    Detecting genes defining major trends identified using CIA. The central panel (B) is the CIA from Figure 2 . The co-ordinates of the genes in each ordination are shown in the side panels A) Ross cDNA and C) Staunton Affymetrix. The top ten genes at the end of axes F1 and F2 are labelled, where red gene labels indicate genes that were present in both datasets. Genes labelled in bold describe genes that were replicated on the microarray. Genes labelled in blue represent genes that were not contained in the top ten genes, but were in the top thirty genes at the end of each axes and are of biological interest.
    Figure Legend Snippet: Detecting genes defining major trends identified using CIA. The central panel (B) is the CIA from Figure 2 . The co-ordinates of the genes in each ordination are shown in the side panels A) Ross cDNA and C) Staunton Affymetrix. The top ten genes at the end of axes F1 and F2 are labelled, where red gene labels indicate genes that were present in both datasets. Genes labelled in bold describe genes that were replicated on the microarray. Genes labelled in blue represent genes that were not contained in the top ten genes, but were in the top thirty genes at the end of each axes and are of biological interest.

    Techniques Used: Microarray

    12) Product Images from "HOXA1 drives melanoma tumor growth and metastasis and elicits an invasion gene expression signature that prognosticates clinical outcome"

    Article Title: HOXA1 drives melanoma tumor growth and metastasis and elicits an invasion gene expression signature that prognosticates clinical outcome

    Journal: Oncogene

    doi: 10.1038/onc.2013.30

    HOXA1 promotes invasion through enhanced TGFβ signaling (A) Focused TGFβ/BMP PCR profiling array on cDNA isolated from WM115 cells stably expressing vector control or HOXA1 . Shown at right are genes up-regulated (red) and down-regulated (green) greater than 4-fold in HOXA1 -expressing cells compared to vector control. (B) WM115 cells expressing vector control (Vec) or HOXA1 were transfected with the TGFβ-inducible p3TP-Lux luciferase reporter construct, followed by treatment with or without TGFβ to assess responsiveness. Error bars indicate +/− s.d.; p-values calculated by t-test. (C) Whole cell lysates from WM115 cells stably expressing vector control or HOXA1 were propagated in 1% serum with or without TGFβ for immunoblot analysis using the indicated antibodies. P-SMAD3 = phosphorylated SMAD3, Ser423+Ser425. * = denotes tubulin band. (D) WM115 cells expressing vector control or HOXA1 were treated with or without SMAD3 shRNA (shSMAD3) or non-targeting shRNA (shNT) and loaded onto transwell invasion chambers. P-values calculated by t-test.
    Figure Legend Snippet: HOXA1 promotes invasion through enhanced TGFβ signaling (A) Focused TGFβ/BMP PCR profiling array on cDNA isolated from WM115 cells stably expressing vector control or HOXA1 . Shown at right are genes up-regulated (red) and down-regulated (green) greater than 4-fold in HOXA1 -expressing cells compared to vector control. (B) WM115 cells expressing vector control (Vec) or HOXA1 were transfected with the TGFβ-inducible p3TP-Lux luciferase reporter construct, followed by treatment with or without TGFβ to assess responsiveness. Error bars indicate +/− s.d.; p-values calculated by t-test. (C) Whole cell lysates from WM115 cells stably expressing vector control or HOXA1 were propagated in 1% serum with or without TGFβ for immunoblot analysis using the indicated antibodies. P-SMAD3 = phosphorylated SMAD3, Ser423+Ser425. * = denotes tubulin band. (D) WM115 cells expressing vector control or HOXA1 were treated with or without SMAD3 shRNA (shSMAD3) or non-targeting shRNA (shNT) and loaded onto transwell invasion chambers. P-values calculated by t-test.

    Techniques Used: Polymerase Chain Reaction, Isolation, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Luciferase, Construct, shRNA

    HOXA1 expression down-regulates genes important for melanocyte differentiation and pigmentation (A) Molecule network generated using Ingenuity Pathways Analysis. The network is displayed graphically as nodes (genes) and edges (the biological relationships between nodes). Solid lines represent direct interactions and dashed lines represent indirect interactions. Green colors denote genes that were under-expressed > 10-fold in WM115 expressing HOXA1 versus control. Red box surrounds MITF node and known MITF target genes differentially expressed > 10-fold in WM115- HOXA1 cells. (B) Whole cell lysates from WM115 and SkMel30 cells stably expressing vector control (Vec) or HOXA1 were processed for immunoblot analysis using the indicated antibodies. Load = tubulin (WM115 panel) and GAPDH (SkMel30 panel). (C) Heat map representing Affymetrix probe expression for genes boxed in (A). Expression values at right indicate fold change in gene expression ( HOXA1 versus vector control). (D) Expression validation of select genes by RT-qPCR analysis of cDNA prepared from WM115 and SkMel30 (high MITF) and WM278 and CHL-1 (low MITF) cells expressing vector control or HOXA1 . All values are normalized based on Actin B expression and plotted as fold change compared to vector control (set as 1.0 for each gene). Values indicate fold change for genes found up-regulated (red) and down-regulated (green) in WM115 transcriptome analysis.
    Figure Legend Snippet: HOXA1 expression down-regulates genes important for melanocyte differentiation and pigmentation (A) Molecule network generated using Ingenuity Pathways Analysis. The network is displayed graphically as nodes (genes) and edges (the biological relationships between nodes). Solid lines represent direct interactions and dashed lines represent indirect interactions. Green colors denote genes that were under-expressed > 10-fold in WM115 expressing HOXA1 versus control. Red box surrounds MITF node and known MITF target genes differentially expressed > 10-fold in WM115- HOXA1 cells. (B) Whole cell lysates from WM115 and SkMel30 cells stably expressing vector control (Vec) or HOXA1 were processed for immunoblot analysis using the indicated antibodies. Load = tubulin (WM115 panel) and GAPDH (SkMel30 panel). (C) Heat map representing Affymetrix probe expression for genes boxed in (A). Expression values at right indicate fold change in gene expression ( HOXA1 versus vector control). (D) Expression validation of select genes by RT-qPCR analysis of cDNA prepared from WM115 and SkMel30 (high MITF) and WM278 and CHL-1 (low MITF) cells expressing vector control or HOXA1 . All values are normalized based on Actin B expression and plotted as fold change compared to vector control (set as 1.0 for each gene). Values indicate fold change for genes found up-regulated (red) and down-regulated (green) in WM115 transcriptome analysis.

    Techniques Used: Expressing, Generated, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR

    13) Product Images from "Growth hormone is produced within the hippocampus where it responds to age, sex, and stress"

    Article Title: Growth hormone is produced within the hippocampus where it responds to age, sex, and stress

    Journal:

    doi: 10.1073/pnas.0507776103

    Estrogen stimulates GH production. ( A ) GH mRNA levels were increased in cortical neurons upon exposure to 10 nM 17-β-estradiol relative to untreated controls. ( B ) RT-PCR products from amplification of cDNA generated from the total RNA of untreated
    Figure Legend Snippet: Estrogen stimulates GH production. ( A ) GH mRNA levels were increased in cortical neurons upon exposure to 10 nM 17-β-estradiol relative to untreated controls. ( B ) RT-PCR products from amplification of cDNA generated from the total RNA of untreated

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Generated

    14) Product Images from "Cytoprotective effect of selective small-molecule caspase inhibitors against staurosporine-induced apoptosis"

    Article Title: Cytoprotective effect of selective small-molecule caspase inhibitors against staurosporine-induced apoptosis

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S60283

    Hierarchical clustering of 200 upregulated and downregulated genes from NIH3T3 samples treated with DMSO, RBC1023, STAU, or RBC1023 plus STAU. Notes: NIH3T3 cells were treated with DMSO, 20 μM of RBC1023 (c23), 1 μM of STAU, or 20 μM of RBC1023 (c23) plus 1 μM of STAU for 20 hours. Total RNA was isolated and converted to complementaryDNA by reverse transcriptase reaction. The cDNA products were fragmented and hybridized to Affymetrix GeneChip Mouse Gene 1.0ST (Affymetrix Inc., Santa Clara, CA, USA). 15 Clustering of 200 upregulated and downregulated genes that exhibit significant differences in all groups was determined by one-way analysis of variance. The clustering was based on fold changes within each paired data set. The color scale of the log2 ratios is shown at the bottom. Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.
    Figure Legend Snippet: Hierarchical clustering of 200 upregulated and downregulated genes from NIH3T3 samples treated with DMSO, RBC1023, STAU, or RBC1023 plus STAU. Notes: NIH3T3 cells were treated with DMSO, 20 μM of RBC1023 (c23), 1 μM of STAU, or 20 μM of RBC1023 (c23) plus 1 μM of STAU for 20 hours. Total RNA was isolated and converted to complementaryDNA by reverse transcriptase reaction. The cDNA products were fragmented and hybridized to Affymetrix GeneChip Mouse Gene 1.0ST (Affymetrix Inc., Santa Clara, CA, USA). 15 Clustering of 200 upregulated and downregulated genes that exhibit significant differences in all groups was determined by one-way analysis of variance. The clustering was based on fold changes within each paired data set. The color scale of the log2 ratios is shown at the bottom. Abbreviations: DMSO, dimethyl sulfoxide; STAU, staurosporine.

    Techniques Used: Isolation

    15) Product Images from "Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling"

    Article Title: Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-10-246

    Quality of input RNA and targets synthesised from different amounts of input RNA . A , Bioanalyzer electrophoretic profile of the diluted Universal Human Reference RNA used as input for all amplifications. This profile corresponds to a classical and non-degraded human RNA with two fine characteristic peaks corresponding to 18S and 28S RNAs. 8.4 corresponds to the RNA Integrity Number (RIN) and reflects the high quality of this RNA B , electrophoretic profiles of cRNA obtained after one- and two-round Affymetrix amplification using 2 μg, and 100 ng of input RNA respectively or water as negative control. C , D , E , F , electrophoretic profiles of cRNA or cDNA obtained using Ambion ( C ), Arcturus ( D ), Epicentre ( E ) and Nugen ( F ) amplification systems from 250 pg and 500 pg of input RNA, or water as negative control.
    Figure Legend Snippet: Quality of input RNA and targets synthesised from different amounts of input RNA . A , Bioanalyzer electrophoretic profile of the diluted Universal Human Reference RNA used as input for all amplifications. This profile corresponds to a classical and non-degraded human RNA with two fine characteristic peaks corresponding to 18S and 28S RNAs. 8.4 corresponds to the RNA Integrity Number (RIN) and reflects the high quality of this RNA B , electrophoretic profiles of cRNA obtained after one- and two-round Affymetrix amplification using 2 μg, and 100 ng of input RNA respectively or water as negative control. C , D , E , F , electrophoretic profiles of cRNA or cDNA obtained using Ambion ( C ), Arcturus ( D ), Epicentre ( E ) and Nugen ( F ) amplification systems from 250 pg and 500 pg of input RNA, or water as negative control.

    Techniques Used: Amplification, Negative Control

    16) Product Images from "Loss of SOCS3 in myeloid cells prolongs survival in a syngeneic model of glioma"

    Article Title: Loss of SOCS3 in myeloid cells prolongs survival in a syngeneic model of glioma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7992

    SOCS3 −/− macrophages display enhanced M1 gene expression when exposed to GL261 conditioned medium ( A – E ) SOCS3 fl/fl and SOCS3 −/− BMDM were harvested from the femurs of 7–8 week old mice and cultured in RPMI 1640 containing 10% FBS and 10 ng/ml murine M-CSF for 5–7 days to expand. Cells were plated and at 24 h treated with GL261 conditioned medium (50% volume) for the indicated times. RNA was isolated, cDNA generated and qRT-PCR performed for the indicated genes. * p
    Figure Legend Snippet: SOCS3 −/− macrophages display enhanced M1 gene expression when exposed to GL261 conditioned medium ( A – E ) SOCS3 fl/fl and SOCS3 −/− BMDM were harvested from the femurs of 7–8 week old mice and cultured in RPMI 1640 containing 10% FBS and 10 ng/ml murine M-CSF for 5–7 days to expand. Cells were plated and at 24 h treated with GL261 conditioned medium (50% volume) for the indicated times. RNA was isolated, cDNA generated and qRT-PCR performed for the indicated genes. * p

    Techniques Used: Expressing, Mouse Assay, Cell Culture, Isolation, Generated, Quantitative RT-PCR

    SOCS3-deficient macrophages exhibit prolonged activation of STAT3 when exposed to GL261 conditioned medium ( A and B ) SOCS3 fl/fl and SOCS3 −/− BMDM were harvested from the femurs of 7–8 week old mice and cultured in RPMI 1640 containing 10% FBS and 10 ng/ml murine M-CSF for 5–7 days to expand. Cells were plated and at 24 h treated with GCM (50% volume) for the indicated times. Cells were lysed and immunoblotted with the indicated Abs (A) or RNA was isolated, cDNA generated and qRT-PCR performed for the indicated genes (B) Densitometric analysis is displayed as percent increase of SOCS3 −/− compared to control SOCS3 fl/fl macrophages. For example, at 1 h the SOCS3 −/− macrophages display 30% higher p-STAT3 than the corresponding SOCS3 fl/fl 1 h time point. * p
    Figure Legend Snippet: SOCS3-deficient macrophages exhibit prolonged activation of STAT3 when exposed to GL261 conditioned medium ( A and B ) SOCS3 fl/fl and SOCS3 −/− BMDM were harvested from the femurs of 7–8 week old mice and cultured in RPMI 1640 containing 10% FBS and 10 ng/ml murine M-CSF for 5–7 days to expand. Cells were plated and at 24 h treated with GCM (50% volume) for the indicated times. Cells were lysed and immunoblotted with the indicated Abs (A) or RNA was isolated, cDNA generated and qRT-PCR performed for the indicated genes (B) Densitometric analysis is displayed as percent increase of SOCS3 −/− compared to control SOCS3 fl/fl macrophages. For example, at 1 h the SOCS3 −/− macrophages display 30% higher p-STAT3 than the corresponding SOCS3 fl/fl 1 h time point. * p

    Techniques Used: Activation Assay, Mouse Assay, Cell Culture, Isolation, Generated, Quantitative RT-PCR

    17) Product Images from "Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance"

    Article Title: Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065535

    Generation of CsDFR and CsANR transgenic tobacco. A, Graphic representation of pCAMBIA 1302 vector with cDNA of CsDFR and CsANR . CsDFR and CsANR cDNA was inserted in-between the NcoI and BglII restriction site of pCAMBIA 1302. B, Genomic DNA PCR confirmed the insertion of CsDFR and CsANR cDNA in plant genome of transgenic lines. C, Semi-quantitative PCR documented the transcript expression levels of CsDFR and CsANR in transgenic tobacco lines. Housekeeping gene 26S rRNA was used as internal control for expression study and experiments were repeated at least three times with similar results.
    Figure Legend Snippet: Generation of CsDFR and CsANR transgenic tobacco. A, Graphic representation of pCAMBIA 1302 vector with cDNA of CsDFR and CsANR . CsDFR and CsANR cDNA was inserted in-between the NcoI and BglII restriction site of pCAMBIA 1302. B, Genomic DNA PCR confirmed the insertion of CsDFR and CsANR cDNA in plant genome of transgenic lines. C, Semi-quantitative PCR documented the transcript expression levels of CsDFR and CsANR in transgenic tobacco lines. Housekeeping gene 26S rRNA was used as internal control for expression study and experiments were repeated at least three times with similar results.

    Techniques Used: Transgenic Assay, Plasmid Preparation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing

    18) Product Images from "Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus"

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus

    Journal: Molecular Plant Pathology

    doi: 10.1111/mpp.12877

    HrpX is required for pathogenicity and regulates expression of T3S and T3E genes in Acidovorax citrulli M6. (A) Disease lesions produced in a melon leaf inoculated with wild‐type M6, but not with mutant strains defective in hrpX or hrcV (encoding a core component of the T3SS) genes. The picture was taken at 3 days after inoculation (dai). (B) Cell death observed in a pepper leaf following inoculation with wild‐type M6, but not with hrpX and hrcV mutants. The picture was taken at 4 dai. In (A) and (B), leaves were syringe‐infiltrated with a bacterial suspension of 10 8 cfu/mL. (C) Qualitative assessment of differential gene expression between wild‐type M6 and the M6 hrpX mutant after 72 h of growth in XVM2 minimal medium at 28 °C. gDNA, genomic DNA. cDNA, reverse‐trancriptase (RT)‐PCR of RNA extracts. Genes: hrcV ( APS58_2306 ), hrcT ( APS58_2309 ), hrcJ ( APS58_2321 ) and hrcC ( APS58_2331 ), encoding core T3SS components; APS58_3289 , encoding a T3E similar to Pseudomonas syringae hopW1‐1 ; GAPDH , glyceraldehyde‐3‐phosphate dehydrogenase ( APS58_1610 ; control).
    Figure Legend Snippet: HrpX is required for pathogenicity and regulates expression of T3S and T3E genes in Acidovorax citrulli M6. (A) Disease lesions produced in a melon leaf inoculated with wild‐type M6, but not with mutant strains defective in hrpX or hrcV (encoding a core component of the T3SS) genes. The picture was taken at 3 days after inoculation (dai). (B) Cell death observed in a pepper leaf following inoculation with wild‐type M6, but not with hrpX and hrcV mutants. The picture was taken at 4 dai. In (A) and (B), leaves were syringe‐infiltrated with a bacterial suspension of 10 8 cfu/mL. (C) Qualitative assessment of differential gene expression between wild‐type M6 and the M6 hrpX mutant after 72 h of growth in XVM2 minimal medium at 28 °C. gDNA, genomic DNA. cDNA, reverse‐trancriptase (RT)‐PCR of RNA extracts. Genes: hrcV ( APS58_2306 ), hrcT ( APS58_2309 ), hrcJ ( APS58_2321 ) and hrcC ( APS58_2331 ), encoding core T3SS components; APS58_3289 , encoding a T3E similar to Pseudomonas syringae hopW1‐1 ; GAPDH , glyceraldehyde‐3‐phosphate dehydrogenase ( APS58_1610 ; control).

    Techniques Used: Expressing, Produced, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

    19) Product Images from "E2F6 is essential for cell viability in breast cancer cells during replication stress"

    Article Title: E2F6 is essential for cell viability in breast cancer cells during replication stress

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-1905-6

    E2F6 cDNA levels were increased in breast cancer cell lines and Jurkat cells relative to MCF-10A. cDNAs prepared from cancer cell lines (MCF-7, T-47D, MDA-MB-231, MDA-MB-468, and Jurkat cells) and normal breast cells (MCF-10A) were subjected to qRT-PCR for E2F6. In this experiment we used four different primer pairs. The primer pairs (E2F6# com2, E2F6# var a, and E2F6# var b) are the same primers used in Figure 1. Primer pairs E2F6# com1 and E2F6# com2 detect a conserved region across E2F6 transcript variants, while primer pair E2F6# var a is designed to detect only E2F6 transcript variant a and primer E2F6# var b detects only E2F6 transcript variant b. In each case expression was first normalized to 18S rRNA. The level of each cDNA is shown relative to that of the same gene in MCF-10A (fold change). FLI1 cDNA level was checked using primers Fli1#1 and Fli1#2 as a negative control. Mean expression and error bars representing the corresponding standard deviation of 5 independent repeats are shown. The stars represent the P-values for the significant difference between the normal and cancer cell lines using unpaired Student t-tests.
    Figure Legend Snippet: E2F6 cDNA levels were increased in breast cancer cell lines and Jurkat cells relative to MCF-10A. cDNAs prepared from cancer cell lines (MCF-7, T-47D, MDA-MB-231, MDA-MB-468, and Jurkat cells) and normal breast cells (MCF-10A) were subjected to qRT-PCR for E2F6. In this experiment we used four different primer pairs. The primer pairs (E2F6# com2, E2F6# var a, and E2F6# var b) are the same primers used in Figure 1. Primer pairs E2F6# com1 and E2F6# com2 detect a conserved region across E2F6 transcript variants, while primer pair E2F6# var a is designed to detect only E2F6 transcript variant a and primer E2F6# var b detects only E2F6 transcript variant b. In each case expression was first normalized to 18S rRNA. The level of each cDNA is shown relative to that of the same gene in MCF-10A (fold change). FLI1 cDNA level was checked using primers Fli1#1 and Fli1#2 as a negative control. Mean expression and error bars representing the corresponding standard deviation of 5 independent repeats are shown. The stars represent the P-values for the significant difference between the normal and cancer cell lines using unpaired Student t-tests.

    Techniques Used: Multiple Displacement Amplification, Quantitative RT-PCR, Variant Assay, Expressing, Negative Control, Standard Deviation

    20) Product Images from "E2F6 is essential for cell viability in breast cancer cells during replication stress"

    Article Title: E2F6 is essential for cell viability in breast cancer cells during replication stress

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-1905-6

    E2F6 cDNA levels were increased in breast cancer cell lines and Jurkat cells relative to MCF-10A. cDNAs prepared from cancer cell lines (MCF-7, T-47D, MDA-MB-231, MDA-MB-468, and Jurkat cells) and normal breast cells (MCF-10A) were subjected to qRT-PCR for E2F6. In this experiment we used four different primer pairs. The primer pairs (E2F6# com2, E2F6# var a, and E2F6# var b) are the same primers used in Figure 1. Primer pairs E2F6# com1 and E2F6# com2 detect a conserved region across E2F6 transcript variants, while primer pair E2F6# var a is designed to detect only E2F6 transcript variant a and primer E2F6# var b detects only E2F6 transcript variant b. In each case expression was first normalized to 18S rRNA. The level of each cDNA is shown relative to that of the same gene in MCF-10A (fold change). FLI1 cDNA level was checked using primers Fli1#1 and Fli1#2 as a negative control. Mean expression and error bars representing the corresponding standard deviation of 5 independent repeats are shown. The stars represent the P-values for the significant difference between the normal and cancer cell lines using unpaired Student t-tests.
    Figure Legend Snippet: E2F6 cDNA levels were increased in breast cancer cell lines and Jurkat cells relative to MCF-10A. cDNAs prepared from cancer cell lines (MCF-7, T-47D, MDA-MB-231, MDA-MB-468, and Jurkat cells) and normal breast cells (MCF-10A) were subjected to qRT-PCR for E2F6. In this experiment we used four different primer pairs. The primer pairs (E2F6# com2, E2F6# var a, and E2F6# var b) are the same primers used in Figure 1. Primer pairs E2F6# com1 and E2F6# com2 detect a conserved region across E2F6 transcript variants, while primer pair E2F6# var a is designed to detect only E2F6 transcript variant a and primer E2F6# var b detects only E2F6 transcript variant b. In each case expression was first normalized to 18S rRNA. The level of each cDNA is shown relative to that of the same gene in MCF-10A (fold change). FLI1 cDNA level was checked using primers Fli1#1 and Fli1#2 as a negative control. Mean expression and error bars representing the corresponding standard deviation of 5 independent repeats are shown. The stars represent the P-values for the significant difference between the normal and cancer cell lines using unpaired Student t-tests.

    Techniques Used: Multiple Displacement Amplification, Quantitative RT-PCR, Variant Assay, Expressing, Negative Control, Standard Deviation

    21) Product Images from "Rapid and ultrasensitive digital PCR (dPCR) profiling of EGFRvIII in tumor cells and tissues"

    Article Title: Rapid and ultrasensitive digital PCR (dPCR) profiling of EGFRvIII in tumor cells and tissues

    Journal: Neuro-Oncology Advances

    doi: 10.1093/noajnl/vdz030

    Lower limit of detection of EGFRvIII digital PCR assay. (A) The digital PCR assay containing probe and primer pairs against EGFR wild type was able to detect wild-type copies in U87 WT as well as U87 vIII cDNA at concentrations as low as 1.25E-3 ng/µL. (B) Changing the probe and primer pairs allowed for the detection of EGFRvIII in U87 vIII cDNA at concentrations as low as 1.25E-3 ng/μL. EGFRvIII was not detected in U87 WT cDNA. Representative scatter plots of vIII expression (red) of U87 EGFR WT(C) and U87 EGFRvIII (D) cDNA at a concentration of 2.5E-3 ng/µL, respectively. Non-amplified wells are denoted in yellow.
    Figure Legend Snippet: Lower limit of detection of EGFRvIII digital PCR assay. (A) The digital PCR assay containing probe and primer pairs against EGFR wild type was able to detect wild-type copies in U87 WT as well as U87 vIII cDNA at concentrations as low as 1.25E-3 ng/µL. (B) Changing the probe and primer pairs allowed for the detection of EGFRvIII in U87 vIII cDNA at concentrations as low as 1.25E-3 ng/μL. EGFRvIII was not detected in U87 WT cDNA. Representative scatter plots of vIII expression (red) of U87 EGFR WT(C) and U87 EGFRvIII (D) cDNA at a concentration of 2.5E-3 ng/µL, respectively. Non-amplified wells are denoted in yellow.

    Techniques Used: Digital PCR, Expressing, Concentration Assay, Amplification

    Schematic representation of a potential diagnostic digital PCR assay. A tumor specimen is collected at the time of resection. This specimen is either snap-frozen in liquid nitrogen, stored at −20 o C for immediate processing, or stored in PreservCyt® media. RNA is extracted from the sample mentioned as well as from FFPE slides and then undergoes first-strand cDNA synthesis followed by magnetic bead separation. cDNA is analyzed via a digital PCR platform using unique primer-probe combinations to detect EGFR WT and EGFRvIII. The highly multiplex nature of digital PCR allows for the quantification of EGFR WT and EGFRvIII copy numbers. Total assay time is less than 24 hours.
    Figure Legend Snippet: Schematic representation of a potential diagnostic digital PCR assay. A tumor specimen is collected at the time of resection. This specimen is either snap-frozen in liquid nitrogen, stored at −20 o C for immediate processing, or stored in PreservCyt® media. RNA is extracted from the sample mentioned as well as from FFPE slides and then undergoes first-strand cDNA synthesis followed by magnetic bead separation. cDNA is analyzed via a digital PCR platform using unique primer-probe combinations to detect EGFR WT and EGFRvIII. The highly multiplex nature of digital PCR allows for the quantification of EGFR WT and EGFRvIII copy numbers. Total assay time is less than 24 hours.

    Techniques Used: Diagnostic Assay, Digital PCR, Formalin-fixed Paraffin-Embedded, Multiplex Assay

    Detection of EGFRvIII in patient-derived glioma neurospheres. (A) cDNA extracted from various patient-derived glioma neurosphere cell lines were amplified using EGFR ORF primers ( upper panel ), β-Actin primers ( lower panel). (B) Western blot analysis confirms the expression of EGFR WT and EGFRvIII in various patient-derived glioma neurospheres. (C) Graphical representation of copies per microliter generated when dPCR assay was run on cDNA extracted from various patient-derived glioma neurospheres-EGFR WT (blue) and EGFRvIII (red). NS039 and HK296 both have abundant copies of vIII, whereas HK301 and HK296 have an abundance of EGFR WT. (D) Copies per microliter of EGFR WT and EGFRvIII generated when our dPCR assay was run on cDNA extracted from NS039 and T4213 cells spiked into mouse blood. These results emphasize that additional cell types do not affect the outcome of our assay. (E) Representative scatter plots of vIII expression (red) from NS039 cells (top) and T4213 (bottom) spiked into mouse blood following our digital PCR assay. Non-amplified wells are denoted in yellow.
    Figure Legend Snippet: Detection of EGFRvIII in patient-derived glioma neurospheres. (A) cDNA extracted from various patient-derived glioma neurosphere cell lines were amplified using EGFR ORF primers ( upper panel ), β-Actin primers ( lower panel). (B) Western blot analysis confirms the expression of EGFR WT and EGFRvIII in various patient-derived glioma neurospheres. (C) Graphical representation of copies per microliter generated when dPCR assay was run on cDNA extracted from various patient-derived glioma neurospheres-EGFR WT (blue) and EGFRvIII (red). NS039 and HK296 both have abundant copies of vIII, whereas HK301 and HK296 have an abundance of EGFR WT. (D) Copies per microliter of EGFR WT and EGFRvIII generated when our dPCR assay was run on cDNA extracted from NS039 and T4213 cells spiked into mouse blood. These results emphasize that additional cell types do not affect the outcome of our assay. (E) Representative scatter plots of vIII expression (red) from NS039 cells (top) and T4213 (bottom) spiked into mouse blood following our digital PCR assay. Non-amplified wells are denoted in yellow.

    Techniques Used: Derivative Assay, Amplification, Western Blot, Expressing, Generated, Digital PCR

    22) Product Images from "Impact of Fli-1 transcription factor on autoantibody and lupus nephritis in NZM2410 mice"

    Article Title: Impact of Fli-1 transcription factor on autoantibody and lupus nephritis in NZM2410 mice

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/j.1365-2249.2010.04245.x

    Decreased Egr-1 expression in kidneys from Fli-1 +/− NZM2410 mice compared to wild-type controls. Total RNA was prepared from kidneys at the age of 18 weeks ( n = 4 in each group). Total RNA was converted to cDNA with the SuperScript First-Strand
    Figure Legend Snippet: Decreased Egr-1 expression in kidneys from Fli-1 +/− NZM2410 mice compared to wild-type controls. Total RNA was prepared from kidneys at the age of 18 weeks ( n = 4 in each group). Total RNA was converted to cDNA with the SuperScript First-Strand

    Techniques Used: Expressing, Mouse Assay

    23) Product Images from "Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements"

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    Journal: BMC Bioinformatics

    doi: 10.1186/1471-2105-6-107

    Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.
    Figure Legend Snippet: Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.

    Techniques Used: Chromatin Immunoprecipitation, Microarray, Sequencing, Expressing, Transformation Assay, Produced

    Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.
    Figure Legend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.

    Techniques Used: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced, Amplification, Immunohistochemistry

    Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.
    Figure Legend Snippet: Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.

    Techniques Used: Sequencing, Expressing, Microarray

    Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.
    Figure Legend Snippet: Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.

    Techniques Used: Microarray

    Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.
    Figure Legend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.

    Techniques Used: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced

    Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.
    Figure Legend Snippet: Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.

    Techniques Used: Sequencing, Microarray

    24) Product Images from "Autoregulation of GLD-2 cytoplasmic poly(A) polymerase"

    Article Title: Autoregulation of GLD-2 cytoplasmic poly(A) polymerase

    Journal: RNA

    doi: 10.1261/rna.333507

    GLD-2 protein is bound to XlGLD-2A(L) mRNA. Oocyte extracts were incubated with α-GLD-2 or pre-immune guinea pig serum and bound to Protein A–Sepharose. RNA was extracted and used as a template for reverse transcription. mRNAs were detected by semiquantitative PCR on cDNA with gene-specific primers. XlGLD-2A(L) mRNA is detected in RNAs immunoprecipitated by α-GLD-2, but not by pre-immune serum. The lengths of products are consistent with the known 3′-termini of the mRNAs.
    Figure Legend Snippet: GLD-2 protein is bound to XlGLD-2A(L) mRNA. Oocyte extracts were incubated with α-GLD-2 or pre-immune guinea pig serum and bound to Protein A–Sepharose. RNA was extracted and used as a template for reverse transcription. mRNAs were detected by semiquantitative PCR on cDNA with gene-specific primers. XlGLD-2A(L) mRNA is detected in RNAs immunoprecipitated by α-GLD-2, but not by pre-immune serum. The lengths of products are consistent with the known 3′-termini of the mRNAs.

    Techniques Used: Incubation, Polymerase Chain Reaction, Immunoprecipitation

    Polyadenylation of endogenous XlGLD-2A(L) mRNA during oocyte maturation. (A) Measurements of poly(A) length. Total RNA from Xenopus oocytes, maturing oocytes, and eggs was ligated to the 5′-end of a modified DNA oligo. Reverse transcriptase was used to synthesize DNA complementary to the mRNA, using a primer anti-sense to the ligated DNA oligonucleotides; this newly synthesized DNA contained the poly(A) tail region. A gene-specific primer, along with a primer anti-sense to the ligated DNA oligo, was used to amplify the 3′-end of the gene of interest by PCR. The lengths of PCR products from gene-specific primers were determined using 2.5% agarose gels. ( B ) XlGLD-2A(L) is polyadenylated during oocyte maturation. mRNA was prepared at the indicated times after addition of progesterone or from unfertilized eggs. To determine poly(A) lengths of each endogenous mRNA, reactions were performed using primers specific for each of the four indicated mRNAs. The lengths of products obtained in resting oocytes were consistent with the known ends of the mRNA. The PCR products of XlGLD-2A(L) and cyclin B1 show slower electrophoretic mobility during maturation, indicating that those mRNAs are polyadenylated during maturation. The PCR products obtained with XlGLD-2A(S) and ribosomal protein L1 primers do not increase in length, indicating that these mRNAs are not polyadenylated. ( C ) Analysis of mRNAs encoding factors involved in translational regulation during oocyte maturation. The poly(A) tail of mRNAs for CPEB , CPSF-160 , CPSF-100 , CPSF-73 , PABP-1 , Xcat-2 , and maskin were analyzed during oocyte maturation, using the same protocol as in A and B . The lengths of the specific 3′-end fragments, as determined by EST analysis, are indicated with arrowheads.
    Figure Legend Snippet: Polyadenylation of endogenous XlGLD-2A(L) mRNA during oocyte maturation. (A) Measurements of poly(A) length. Total RNA from Xenopus oocytes, maturing oocytes, and eggs was ligated to the 5′-end of a modified DNA oligo. Reverse transcriptase was used to synthesize DNA complementary to the mRNA, using a primer anti-sense to the ligated DNA oligonucleotides; this newly synthesized DNA contained the poly(A) tail region. A gene-specific primer, along with a primer anti-sense to the ligated DNA oligo, was used to amplify the 3′-end of the gene of interest by PCR. The lengths of PCR products from gene-specific primers were determined using 2.5% agarose gels. ( B ) XlGLD-2A(L) is polyadenylated during oocyte maturation. mRNA was prepared at the indicated times after addition of progesterone or from unfertilized eggs. To determine poly(A) lengths of each endogenous mRNA, reactions were performed using primers specific for each of the four indicated mRNAs. The lengths of products obtained in resting oocytes were consistent with the known ends of the mRNA. The PCR products of XlGLD-2A(L) and cyclin B1 show slower electrophoretic mobility during maturation, indicating that those mRNAs are polyadenylated during maturation. The PCR products obtained with XlGLD-2A(S) and ribosomal protein L1 primers do not increase in length, indicating that these mRNAs are not polyadenylated. ( C ) Analysis of mRNAs encoding factors involved in translational regulation during oocyte maturation. The poly(A) tail of mRNAs for CPEB , CPSF-160 , CPSF-100 , CPSF-73 , PABP-1 , Xcat-2 , and maskin were analyzed during oocyte maturation, using the same protocol as in A and B . The lengths of the specific 3′-end fragments, as determined by EST analysis, are indicated with arrowheads.

    Techniques Used: Modification, Synthesized, Polymerase Chain Reaction

    25) Product Images from "Analyses of Group III Secreted Phospholipase A2 Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis * Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis * S⃞"

    Article Title: Analyses of Group III Secreted Phospholipase A2 Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis * Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M804628200

    Generation of PLA2G3 Tg mice. A , schematic diagram of the construction of PLA2G3 Tg mice. The human PLA2G3 transgene was inserted at the EcoRI site downstream of the neo r / pA cassette. This fragment, in which the PLA2G3 transgene is silent, was introduced into mice, and the transgene-positive offspring were then mated with CAG-Cre Tg mice. At this stage, the LNL (for loxP-neo r /pA-loxP ) cassette was excised by Cre recombinase, and the PLA2G3 transgene was activated under control of the CAG promoter in the transgene-positive pups. The positions of primers for PCR genotyping and the sizes of the amplified fragments are shown. B , PCR genotyping of PLA2G3 Tg mice. After mating of LNL-III Tg mice with CAG- Cre Tg mice, PCR genotyping was performed on their F1 progeny. Fragments of 2.2 and 0.7 kb were amplified for LNL-III Tg and CAG-PLA2G3 Tg (III-Tg) mice, respectively, whereas these products were not detected in WT littermates. C , detection of PLA2G3 mRNA in tissues of WT and III-Tg neonates by Northern blotting. The membrane was hybridized with human PLA2G3 cDNA and exposed to x-ray film for 1 day. D , PLA 2 enzymatic activity in the homogenates of tissues from WT and III-Tg mice shown in C. E , PLA 2 enzymatic activity in sera of WT and III-Tg mice. PCR genotyping of the corresponding mice is shown in the top. F , detection of PLA2G3 protein in the serum. Sera from WT and PLA2G3 Tg mice were immunoprecipitated and immunoblotted with anti-PLA2G3 antibodies, as described under “Experimental Procedures.” The major band is indicated by an arrow on the right .
    Figure Legend Snippet: Generation of PLA2G3 Tg mice. A , schematic diagram of the construction of PLA2G3 Tg mice. The human PLA2G3 transgene was inserted at the EcoRI site downstream of the neo r / pA cassette. This fragment, in which the PLA2G3 transgene is silent, was introduced into mice, and the transgene-positive offspring were then mated with CAG-Cre Tg mice. At this stage, the LNL (for loxP-neo r /pA-loxP ) cassette was excised by Cre recombinase, and the PLA2G3 transgene was activated under control of the CAG promoter in the transgene-positive pups. The positions of primers for PCR genotyping and the sizes of the amplified fragments are shown. B , PCR genotyping of PLA2G3 Tg mice. After mating of LNL-III Tg mice with CAG- Cre Tg mice, PCR genotyping was performed on their F1 progeny. Fragments of 2.2 and 0.7 kb were amplified for LNL-III Tg and CAG-PLA2G3 Tg (III-Tg) mice, respectively, whereas these products were not detected in WT littermates. C , detection of PLA2G3 mRNA in tissues of WT and III-Tg neonates by Northern blotting. The membrane was hybridized with human PLA2G3 cDNA and exposed to x-ray film for 1 day. D , PLA 2 enzymatic activity in the homogenates of tissues from WT and III-Tg mice shown in C. E , PLA 2 enzymatic activity in sera of WT and III-Tg mice. PCR genotyping of the corresponding mice is shown in the top. F , detection of PLA2G3 protein in the serum. Sera from WT and PLA2G3 Tg mice were immunoprecipitated and immunoblotted with anti-PLA2G3 antibodies, as described under “Experimental Procedures.” The major band is indicated by an arrow on the right .

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Amplification, Northern Blot, Proximity Ligation Assay, Activity Assay, Immunoprecipitation

    26) Product Images from "cDNA Microarrays as a Tool for Identification of Biomineralization Proteins in the Coccolithophorid Emiliania huxleyi (Haptophyta)"

    Article Title: cDNA Microarrays as a Tool for Identification of Biomineralization Proteins in the Coccolithophorid Emiliania huxleyi (Haptophyta)

    Journal:

    doi: 10.1128/AEM.00343-06

    Real-time RT-PCR relative gene expression for 63 transcripts significantly up- or down-regulated in calcifying B39 relative to noncalcifying E. huxleyi 1516 cultures grown in phosphate-replete media (red bars). The corresponding cDNA microarray FC data
    Figure Legend Snippet: Real-time RT-PCR relative gene expression for 63 transcripts significantly up- or down-regulated in calcifying B39 relative to noncalcifying E. huxleyi 1516 cultures grown in phosphate-replete media (red bars). The corresponding cDNA microarray FC data

    Techniques Used: Quantitative RT-PCR, Expressing, Microarray

    Real-time RT-PCR validation of cDNA microarray. (A) Comparison of relative expression (FC) for 20 randomly chosen transcripts in phosphate-limiting relative to phosphate-replete E. huxleyi cultures determined by cDNA microarray and real-time RT-PCR. Three
    Figure Legend Snippet: Real-time RT-PCR validation of cDNA microarray. (A) Comparison of relative expression (FC) for 20 randomly chosen transcripts in phosphate-limiting relative to phosphate-replete E. huxleyi cultures determined by cDNA microarray and real-time RT-PCR. Three

    Techniques Used: Quantitative RT-PCR, Microarray, Expressing

    Images and statistical analysis of competitively hybridized cDNA microarrays. (a) Image of the entire cDNA microarray after dye swap experiment. Phosphate-limited f/2 1516 cultures are green, and phosphate-replete f/2 1516 cultures are red). (b) Upper
    Figure Legend Snippet: Images and statistical analysis of competitively hybridized cDNA microarrays. (a) Image of the entire cDNA microarray after dye swap experiment. Phosphate-limited f/2 1516 cultures are green, and phosphate-replete f/2 1516 cultures are red). (b) Upper

    Techniques Used: Microarray

    27) Product Images from "Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus"

    Article Title: Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus

    Journal: Molecular Plant Pathology

    doi: 10.1111/mpp.12877

    HrpX is required for pathogenicity and regulates expression of T3S and T3E genes in Acidovorax citrulli M6. (A) Disease lesions produced in a melon leaf inoculated with wild‐type M6, but not with mutant strains defective in hrpX or hrcV (encoding a core component of the T3SS) genes. The picture was taken at 3 days after inoculation (dai). (B) Cell death observed in a pepper leaf following inoculation with wild‐type M6, but not with hrpX and hrcV mutants. The picture was taken at 4 dai. In (A) and (B), leaves were syringe‐infiltrated with a bacterial suspension of 10 8 cfu/mL. (C) Qualitative assessment of differential gene expression between wild‐type M6 and the M6 hrpX mutant after 72 h of growth in XVM2 minimal medium at 28 °C. gDNA, genomic DNA. cDNA, reverse‐trancriptase (RT)‐PCR of RNA extracts. Genes: hrcV ( APS58_2306 ), hrcT ( APS58_2309 ), hrcJ ( APS58_2321 ) and hrcC ( APS58_2331 ), encoding core T3SS components; APS58_3289 , encoding a T3E similar to Pseudomonas syringae hopW1‐1 ; GAPDH , glyceraldehyde‐3‐phosphate dehydrogenase ( APS58_1610 ; control).
    Figure Legend Snippet: HrpX is required for pathogenicity and regulates expression of T3S and T3E genes in Acidovorax citrulli M6. (A) Disease lesions produced in a melon leaf inoculated with wild‐type M6, but not with mutant strains defective in hrpX or hrcV (encoding a core component of the T3SS) genes. The picture was taken at 3 days after inoculation (dai). (B) Cell death observed in a pepper leaf following inoculation with wild‐type M6, but not with hrpX and hrcV mutants. The picture was taken at 4 dai. In (A) and (B), leaves were syringe‐infiltrated with a bacterial suspension of 10 8 cfu/mL. (C) Qualitative assessment of differential gene expression between wild‐type M6 and the M6 hrpX mutant after 72 h of growth in XVM2 minimal medium at 28 °C. gDNA, genomic DNA. cDNA, reverse‐trancriptase (RT)‐PCR of RNA extracts. Genes: hrcV ( APS58_2306 ), hrcT ( APS58_2309 ), hrcJ ( APS58_2321 ) and hrcC ( APS58_2331 ), encoding core T3SS components; APS58_3289 , encoding a T3E similar to Pseudomonas syringae hopW1‐1 ; GAPDH , glyceraldehyde‐3‐phosphate dehydrogenase ( APS58_1610 ; control).

    Techniques Used: Expressing, Produced, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

    28) Product Images from "Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles"

    Article Title: Dissociation of heterochromatin protein 1 from lamin B receptor induced by human polyomavirus agnoprotein: role in nuclear egress of viral particles

    Journal:

    doi: 10.1038/sj.embor.7400406

    Interaction of JCV Agno with HP1 in vivo . ( A ) Schematic representation of human HP1α and HP1γ showing the regions (bars) encoded by cDNA fragments isolated by a yeast two-hybrid assay with the N-terminal region of Agno as a bait. CD and
    Figure Legend Snippet: Interaction of JCV Agno with HP1 in vivo . ( A ) Schematic representation of human HP1α and HP1γ showing the regions (bars) encoded by cDNA fragments isolated by a yeast two-hybrid assay with the N-terminal region of Agno as a bait. CD and

    Techniques Used: In Vivo, Isolation, Y2H Assay

    29) Product Images from "Expression of S100P and Its Novel Binding Partner S100PBPR in Early Pancreatic Cancer"

    Article Title: Expression of S100P and Its Novel Binding Partner S100PBPR in Early Pancreatic Cancer

    Journal:

    doi:

    Expression of S100P and S100PBPR in PanINs, PDACs, CPs, and pancreatic cancer cell lines, as determined by quantitative real-time PCR. Quantitative real-time PCR was conducted on 10-ng cDNA samples using the SYBR Green method and the ABI7700 sequence
    Figure Legend Snippet: Expression of S100P and S100PBPR in PanINs, PDACs, CPs, and pancreatic cancer cell lines, as determined by quantitative real-time PCR. Quantitative real-time PCR was conducted on 10-ng cDNA samples using the SYBR Green method and the ABI7700 sequence

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

    30) Product Images from "Manipulating insulin signaling to enhance mosquito reproduction"

    Article Title: Manipulating insulin signaling to enhance mosquito reproduction

    Journal: BMC Physiology

    doi: 10.1186/1472-6793-9-15

    Knockdown of AaegPTEN6 in the abdomen/fat body and ovary : Female mosquitoes (48 h post eclosion) were injected with 2 μg of dsRNA (0.5 μl of a 4 μg/μl solution). Injected mosquitoes were allowed to recover for 48 h before being mated with male mosquitoes and provided with a bloodmeal. At 48 h post-bloodmeal (96 h post-injection) we generated cDNA from the total RNA of abdomens/fat body and ovaries to assess the transcript knockdown after specific dsRNA injections. qRT-PCR was performed using primers specific to AaegPTEN6 and actin as a control. Abdomens/fat body injected with universal AaegPTEN dsRNA had a 90% reduction in AaegPTEN6 transcript, while those injected with AaegPTEN6 specific dsRNA had a 98% reduction in AaegPTEN6 transcript. Similarily, ovaries injected with universal AaegPTEN dsRNA had a 51% reduction in AaegPTEN6 transcript, while those injected with AaegPTEN6 specific dsRNA had a 99% reduction in AaegPTEN6 transcript relative to control mosquitoes injected with DsRed dsRNA. Different letters indicate a significant difference between samples.
    Figure Legend Snippet: Knockdown of AaegPTEN6 in the abdomen/fat body and ovary : Female mosquitoes (48 h post eclosion) were injected with 2 μg of dsRNA (0.5 μl of a 4 μg/μl solution). Injected mosquitoes were allowed to recover for 48 h before being mated with male mosquitoes and provided with a bloodmeal. At 48 h post-bloodmeal (96 h post-injection) we generated cDNA from the total RNA of abdomens/fat body and ovaries to assess the transcript knockdown after specific dsRNA injections. qRT-PCR was performed using primers specific to AaegPTEN6 and actin as a control. Abdomens/fat body injected with universal AaegPTEN dsRNA had a 90% reduction in AaegPTEN6 transcript, while those injected with AaegPTEN6 specific dsRNA had a 98% reduction in AaegPTEN6 transcript. Similarily, ovaries injected with universal AaegPTEN dsRNA had a 51% reduction in AaegPTEN6 transcript, while those injected with AaegPTEN6 specific dsRNA had a 99% reduction in AaegPTEN6 transcript relative to control mosquitoes injected with DsRed dsRNA. Different letters indicate a significant difference between samples.

    Techniques Used: Injection, Generated, Quantitative RT-PCR

    31) Product Images from "Nuclear Degradation of Wilms Tumor 1-associating Protein and Survivin Splice Variant Switching Underlie IGF-1-mediated Survival *"

    Article Title: Nuclear Degradation of Wilms Tumor 1-associating Protein and Survivin Splice Variant Switching Underlie IGF-1-mediated Survival *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.034629

    WTAP regulates pre-mRNA splicing of survivin, a process that underlies IGF-1-regulated SMC survival. A , ethidium bromide-stained cDNA amplified by RT-PCR using primers specific for survivin, including its splice variants. Total RNA was harvested from
    Figure Legend Snippet: WTAP regulates pre-mRNA splicing of survivin, a process that underlies IGF-1-regulated SMC survival. A , ethidium bromide-stained cDNA amplified by RT-PCR using primers specific for survivin, including its splice variants. Total RNA was harvested from

    Techniques Used: Staining, Amplification, Reverse Transcription Polymerase Chain Reaction

    32) Product Images from "The Golgi matrix protein giantin is required for normal cilia function in zebrafish"

    Article Title: The Golgi matrix protein giantin is required for normal cilia function in zebrafish

    Journal: Biology Open

    doi: 10.1242/bio.025502

    Experimental knockdown and knockout of giantin in vivo . (A) Schematic representation of the zebrafish golgb1 transcript (ENSDART00000131402.2) showing the binding sites of the designed morpholinos (ATG and E14), the relative exonic location of the human annotated p115 binding site, transmembrane domain (TMD), the various mouse mutants (black asterisks) and the ocd rat allele (white asterisk) as described in Lan et al. (2016) , and Katayama et al. (2011) , respectively. Coloured bars show the interspecies conserved sequence regions as assigned in Ensembl (release 87). Red asterisks indicate the location of zebrafish giantin mutant alleles. (B) Alternative spliced PCR products (blue arrows) from 32 hpf RT-PCR cDNA of two amplicons in E14 MO binding region. Black arrows indicate the expected size of PCR product. (C) Sa11389 line carrying a point mutation from C to T (yellow highlight, red letters) resulting in a premature stop codon at Q2948 ( golgb1 Q2948X ) from the EZRC. (D) TALEN site directed mutagenesis resulted in an 8 bp deletion (spacer sequence in yellow, red letters), resulting in golgb1 X3078 mutant line. (E) Alignment of Golgb1 WT protein sequence (Ensembl release 87) with predicted Golgb1 X3078 protein sequence showing translated spacer sequence (red) and site of deletion T3028 and A3029 with a subsequent frameshift from E3027 (red asterisk) changing 51 amino acids to a predicted stop codon at position 3078. Part of cDNA exon 14 sequence. (F) Stereomicroscope images of 48 hpf control, ATG, and E14 morphants exhibiting defects in the eye, heart, cranium, and various axis orientations (blue, green, black, red arrow, respectively). (G) Percentile quantification of scored phenotypes. (H) Stereomicroscope images of heterozygote in-cross 5 dpf larvae from both mutant lines and (I) 7 wpf female adults. (J) Dot plot for body length at 7 wpf, and (K) 44-46 wpf for golgb1 Q2948X and 41-43 wpf for golgb1 X3078 alleles. Scale bars: (F) 100 µm, (H) 200 µm, (I) 500 µm. (J, K) One-way ANOVA with Tukey's multiple comparison test. All experiments of three replicates. Bars show means with standard deviation. * P
    Figure Legend Snippet: Experimental knockdown and knockout of giantin in vivo . (A) Schematic representation of the zebrafish golgb1 transcript (ENSDART00000131402.2) showing the binding sites of the designed morpholinos (ATG and E14), the relative exonic location of the human annotated p115 binding site, transmembrane domain (TMD), the various mouse mutants (black asterisks) and the ocd rat allele (white asterisk) as described in Lan et al. (2016) , and Katayama et al. (2011) , respectively. Coloured bars show the interspecies conserved sequence regions as assigned in Ensembl (release 87). Red asterisks indicate the location of zebrafish giantin mutant alleles. (B) Alternative spliced PCR products (blue arrows) from 32 hpf RT-PCR cDNA of two amplicons in E14 MO binding region. Black arrows indicate the expected size of PCR product. (C) Sa11389 line carrying a point mutation from C to T (yellow highlight, red letters) resulting in a premature stop codon at Q2948 ( golgb1 Q2948X ) from the EZRC. (D) TALEN site directed mutagenesis resulted in an 8 bp deletion (spacer sequence in yellow, red letters), resulting in golgb1 X3078 mutant line. (E) Alignment of Golgb1 WT protein sequence (Ensembl release 87) with predicted Golgb1 X3078 protein sequence showing translated spacer sequence (red) and site of deletion T3028 and A3029 with a subsequent frameshift from E3027 (red asterisk) changing 51 amino acids to a predicted stop codon at position 3078. Part of cDNA exon 14 sequence. (F) Stereomicroscope images of 48 hpf control, ATG, and E14 morphants exhibiting defects in the eye, heart, cranium, and various axis orientations (blue, green, black, red arrow, respectively). (G) Percentile quantification of scored phenotypes. (H) Stereomicroscope images of heterozygote in-cross 5 dpf larvae from both mutant lines and (I) 7 wpf female adults. (J) Dot plot for body length at 7 wpf, and (K) 44-46 wpf for golgb1 Q2948X and 41-43 wpf for golgb1 X3078 alleles. Scale bars: (F) 100 µm, (H) 200 µm, (I) 500 µm. (J, K) One-way ANOVA with Tukey's multiple comparison test. All experiments of three replicates. Bars show means with standard deviation. * P

    Techniques Used: Knock-Out, In Vivo, Binding Assay, Sequencing, Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    33) Product Images from "The G9a Histone Methyltransferase Inhibitor BIX-01294 Modulates Gene Expression during Plasmodium falciparum Gametocyte Development and Transmission"

    Article Title: The G9a Histone Methyltransferase Inhibitor BIX-01294 Modulates Gene Expression during Plasmodium falciparum Gametocyte Development and Transmission

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20205087

    De-regulation of gene expression following BIX-01294 treatment of gametocytes. Immature (imGC) and mature (mGC) gametocytes as well as gametocytes at 1 h post-activation (aGC) were treated with BIX-01294 at IC 90 concentrations or 0.5% vol. DMSO (untreated) for 1 and 6 h, total RNA was isolated and cDNA synthesized to be employed in microarray assays. Genes with a relative expression levels greater than 1.5-fold for at least one of the two time-points combined with a consistent up- or downregulation for both time-points were used for further analysis. ( a ) bar charts showing total up- and downregulated genes in imGC, mGC and aGC; ( b ) bar chart showing the mean fold changes in imGC, mGC and aGC following BIX-01294 treatment; ( c ) pie chart showing detailed number of upregulated genes in gametocytes following BIX-01294 treatment based on the predicted function; ( d ) genes 2-fold downregulated in aGC with high expression in gametocytes (GC) and/or ookinetes (OK) grouped by function (according to PlasmoDB; www.plasmodb.org ).
    Figure Legend Snippet: De-regulation of gene expression following BIX-01294 treatment of gametocytes. Immature (imGC) and mature (mGC) gametocytes as well as gametocytes at 1 h post-activation (aGC) were treated with BIX-01294 at IC 90 concentrations or 0.5% vol. DMSO (untreated) for 1 and 6 h, total RNA was isolated and cDNA synthesized to be employed in microarray assays. Genes with a relative expression levels greater than 1.5-fold for at least one of the two time-points combined with a consistent up- or downregulation for both time-points were used for further analysis. ( a ) bar charts showing total up- and downregulated genes in imGC, mGC and aGC; ( b ) bar chart showing the mean fold changes in imGC, mGC and aGC following BIX-01294 treatment; ( c ) pie chart showing detailed number of upregulated genes in gametocytes following BIX-01294 treatment based on the predicted function; ( d ) genes 2-fold downregulated in aGC with high expression in gametocytes (GC) and/or ookinetes (OK) grouped by function (according to PlasmoDB; www.plasmodb.org ).

    Techniques Used: Expressing, Activation Assay, Isolation, Synthesized, Microarray

    34) Product Images from "Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria"

    Article Title: Ms1, a novel sRNA interacting with the RNA polymerase core in mycobacteria

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku793

    Mycobacterial Ms1 sRNA is expressed in amounts comparable to 6S RNAs. ( A ) Total RNA was isolated from Bacillus subtilis ( B.s. ), Escherichia coli ( E.c. ) and Mycobacterium smegmatis ( M.s. ) in exponential (EX) or stationary (ST) phase. RNAs were resolved on denaturing polyacrylamide gels and stained with GelRed. In M. smegmatis , an ∼300 nt sRNA was present in stationary phase cells in amounts comparable to B. subtilis or E. coli 6S RNAs. ( B ) Before loading onto the gel, total RNA from M. smegmatis stationary phase was incubated either with a complementary DNA oligonucleotide (anti-Ms1 oligo) or nonspecific control oligonucleotides (ns-oligo 1 and ns-oligo 2) and treated with RNase H. ( C ) The first nucleotide of Ms1 is adenine transcribed from position 6 242 368 in the genome. The putative −10 and −35 promoter sequences (framed) are perfectly conserved in M. smegmatis , Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium avium . The 5′ end sequences of previously identified Ms1 homologs in these species are highlighted in bold. The consensus promoter sequence was adopted from ( 36 ). ( D ) The flanking genes of Ms1 in M. smegmatis are shown. ( E ) Scheme of Ms1's position in the genome of M. smegmatis with respect to the origin of replication (ori).
    Figure Legend Snippet: Mycobacterial Ms1 sRNA is expressed in amounts comparable to 6S RNAs. ( A ) Total RNA was isolated from Bacillus subtilis ( B.s. ), Escherichia coli ( E.c. ) and Mycobacterium smegmatis ( M.s. ) in exponential (EX) or stationary (ST) phase. RNAs were resolved on denaturing polyacrylamide gels and stained with GelRed. In M. smegmatis , an ∼300 nt sRNA was present in stationary phase cells in amounts comparable to B. subtilis or E. coli 6S RNAs. ( B ) Before loading onto the gel, total RNA from M. smegmatis stationary phase was incubated either with a complementary DNA oligonucleotide (anti-Ms1 oligo) or nonspecific control oligonucleotides (ns-oligo 1 and ns-oligo 2) and treated with RNase H. ( C ) The first nucleotide of Ms1 is adenine transcribed from position 6 242 368 in the genome. The putative −10 and −35 promoter sequences (framed) are perfectly conserved in M. smegmatis , Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium avium . The 5′ end sequences of previously identified Ms1 homologs in these species are highlighted in bold. The consensus promoter sequence was adopted from ( 36 ). ( D ) The flanking genes of Ms1 in M. smegmatis are shown. ( E ) Scheme of Ms1's position in the genome of M. smegmatis with respect to the origin of replication (ori).

    Techniques Used: Isolation, Staining, Incubation, Sequencing

    35) Product Images from "On the selection of appropriate distances for gene expression data clustering"

    Article Title: On the selection of appropriate distances for gene expression data clustering

    Journal: BMC Bioinformatics

    doi: 10.1186/1471-2105-15-S2-S2

    Cancer Datasets Results: Class recovery obtained for cancer datasets regarding the three evaluation scenarios under consideration, subfigures (a), (b), and (c) . Bars display mean results for each pair of clustering method and distance function in different types of datasets: cDNA (left) and Affymetrix (right).
    Figure Legend Snippet: Cancer Datasets Results: Class recovery obtained for cancer datasets regarding the three evaluation scenarios under consideration, subfigures (a), (b), and (c) . Bars display mean results for each pair of clustering method and distance function in different types of datasets: cDNA (left) and Affymetrix (right).

    Techniques Used:

    36) Product Images from "Folic acid inhibits COLO-205 colon cancer cell proliferation through activating the FRα/c-SRC/ERK1/2/NFκB/TP53 pathway: in vitro and in vivo studies"

    Article Title: Folic acid inhibits COLO-205 colon cancer cell proliferation through activating the FRα/c-SRC/ERK1/2/NFκB/TP53 pathway: in vitro and in vivo studies

    Journal: Scientific Reports

    doi: 10.1038/srep11187

    FA induces TP53-dependent up-regulations in CDKN1A and CDKN1B and the G0/G1 arrest in COLO-205. ( A ) Pre-transfection with DN- TP53 cDNA prevented FA-induced increases in the levels of CDKN1A and CDKN1B protein. In the control group, the cell was transfected with pcDNA 3.1(+) expression vector. The gels have been run in the same experimental conditions and the cropped blots were shown. The entire gel pictures of Fig. 2A were shown in the Supplemental Fig. 1B . Values (means ± s.e.mean.) shown in parentheses represent the relative protein abundance of CDKN1A, CDKN1B and TP53, which has been normalized with corresponding G3PDH and expressed as fold of its own control. Three samples were analyzed in each group * p
    Figure Legend Snippet: FA induces TP53-dependent up-regulations in CDKN1A and CDKN1B and the G0/G1 arrest in COLO-205. ( A ) Pre-transfection with DN- TP53 cDNA prevented FA-induced increases in the levels of CDKN1A and CDKN1B protein. In the control group, the cell was transfected with pcDNA 3.1(+) expression vector. The gels have been run in the same experimental conditions and the cropped blots were shown. The entire gel pictures of Fig. 2A were shown in the Supplemental Fig. 1B . Values (means ± s.e.mean.) shown in parentheses represent the relative protein abundance of CDKN1A, CDKN1B and TP53, which has been normalized with corresponding G3PDH and expressed as fold of its own control. Three samples were analyzed in each group * p

    Techniques Used: Transfection, Expressing, Plasmid Preparation

    37) Product Images from "Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2"

    Article Title: Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13194-2

    Effect of PALB2 variants on protein expression and/or stability. a Western blot analysis of the expression of human PALB2 variants in Trp53 KO /Palb2 KO mES cells using an antibody directed against the N-terminus of PALB2. Wild-type (WT) human PALB2 and empty vector (Ev) served as controls on each blot. Tubulin was a loading control. Marked PALB2 variants (red *) showed low levels of protein expression. b RT-qPCR analysis of human PALB2 variants from ( a ) with low expression levels (red *). Primers specific for human PALB2 cDNA and the Pim1 control locus were used. Data represent the mean percentage (±SEM) of PALB2 mRNA relative to WT, which was set to 100%, from two independent RNA isolation experiments. Variants/conditions are categorized by color as either WT (black), truncating variant (red), VUS (blue) or empty vector (Ev, grey). Ev-1, -2, -3 refer to Ev controls from three different replicates. c Partial structures of the PALB2 WD40 domain showing the effect of 4 PALB2 variants exhibiting low protein expression as shown in ( a ). Partial structures without and with variant are shown side by side for each variant, indicating loss of stabilizing interactions (but not any possible conformational changes). Source data are provided as a Source Data file.
    Figure Legend Snippet: Effect of PALB2 variants on protein expression and/or stability. a Western blot analysis of the expression of human PALB2 variants in Trp53 KO /Palb2 KO mES cells using an antibody directed against the N-terminus of PALB2. Wild-type (WT) human PALB2 and empty vector (Ev) served as controls on each blot. Tubulin was a loading control. Marked PALB2 variants (red *) showed low levels of protein expression. b RT-qPCR analysis of human PALB2 variants from ( a ) with low expression levels (red *). Primers specific for human PALB2 cDNA and the Pim1 control locus were used. Data represent the mean percentage (±SEM) of PALB2 mRNA relative to WT, which was set to 100%, from two independent RNA isolation experiments. Variants/conditions are categorized by color as either WT (black), truncating variant (red), VUS (blue) or empty vector (Ev, grey). Ev-1, -2, -3 refer to Ev controls from three different replicates. c Partial structures of the PALB2 WD40 domain showing the effect of 4 PALB2 variants exhibiting low protein expression as shown in ( a ). Partial structures without and with variant are shown side by side for each variant, indicating loss of stabilizing interactions (but not any possible conformational changes). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Quantitative RT-PCR, Isolation, Variant Assay

    38) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1091/mbc.E07-05-0496

    SYT-SSX2 induces an increase in the expression and activation of the EphB2 receptor. (A) Transcript levels of Eph/ephrin pathway components in SYT- and SYT-SSX2–expressing cells examined by RT-PCR. GAPDH cDNA was used as control. (B) Eph/ephrin
    Figure Legend Snippet: SYT-SSX2 induces an increase in the expression and activation of the EphB2 receptor. (A) Transcript levels of Eph/ephrin pathway components in SYT- and SYT-SSX2–expressing cells examined by RT-PCR. GAPDH cDNA was used as control. (B) Eph/ephrin

    Techniques Used: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction

    39) Product Images from "Overexpression of the Kaposi's Sarcoma-Associated Herpesvirus Transactivator K-Rta Can Complement a K-bZIP Deletion BACmid and Yields an Enhanced Growth Phenotype ▿"

    Article Title: Overexpression of the Kaposi's Sarcoma-Associated Herpesvirus Transactivator K-Rta Can Complement a K-bZIP Deletion BACmid and Yields an Enhanced Growth Phenotype ▿

    Journal:

    doi: 10.1128/JVI.00832-07

    qPCR analysis of several virus-encoded mRNAs from Ad50-induced BAC-containing cell lines. Vero cell lines harboring BAC36ΔK8 or BAC36 were treated with TPA and Ad50 (MOI = 4,500), and total cellular RNA was harvested and, following cDNA
    Figure Legend Snippet: qPCR analysis of several virus-encoded mRNAs from Ad50-induced BAC-containing cell lines. Vero cell lines harboring BAC36ΔK8 or BAC36 were treated with TPA and Ad50 (MOI = 4,500), and total cellular RNA was harvested and, following cDNA

    Techniques Used: Real-time Polymerase Chain Reaction, BAC Assay

    40) Product Images from "New Role for the Protein Tyrosine Phosphatase DEP-1 in Akt Activation and Endothelial Cell Survival ▿"

    Article Title: New Role for the Protein Tyrosine Phosphatase DEP-1 in Akt Activation and Endothelial Cell Survival ▿

    Journal:

    doi: 10.1128/MCB.01374-08

    DEP-1 leads to the global dephosphorylation of VEGFR2. (A) HEK 293 cells were transfected with 20 μg of VEGFR2 cDNA and increasing amounts of WT Myr-DEP-1 cDNA. Cells were serum starved, stimulated with VEGF (50 ng/ml) for 2 min, and lysed. VEGFR2
    Figure Legend Snippet: DEP-1 leads to the global dephosphorylation of VEGFR2. (A) HEK 293 cells were transfected with 20 μg of VEGFR2 cDNA and increasing amounts of WT Myr-DEP-1 cDNA. Cells were serum starved, stimulated with VEGF (50 ng/ml) for 2 min, and lysed. VEGFR2

    Techniques Used: De-Phosphorylation Assay, Transfection

    DEP-1 D/A traps VEGFR2 via tyrosine residues in the activation loop. (A) HEK 293 cells were transfected with empty vector (pShuttle) or cDNA constructs encoding the WT CSF-VEGFR2, single Y/F mutants of every major VEGFR2 autophosphorylation site, and
    Figure Legend Snippet: DEP-1 D/A traps VEGFR2 via tyrosine residues in the activation loop. (A) HEK 293 cells were transfected with empty vector (pShuttle) or cDNA constructs encoding the WT CSF-VEGFR2, single Y/F mutants of every major VEGFR2 autophosphorylation site, and

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Construct

    Related Articles

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Chibby Promotes Adipocyte Differentiation through Inhibition of ?-Catenin Signaling ▿
    Article Snippet: .. Total RNA was purified from epidydimal white adipose tissue (WAT), interscapular brown adipose tissue (BAT), and liver of 2-month-old C57BL/6J mice with RNeasy spin columns (QIAGEN), and synthesis of cDNA was performed with oligo(dT) primers using the ThermoScript reverse transcription (RT)-PCR System (Invitrogen) according to the manufacturer's instructions. ..

    Synthesized:

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication
    Article Snippet: .. The cDNA was synthesized using the CloneMiner cDNA Library Construction Kit (Life Technologies) from 3 μg of mRNA and fractionated with cDNA Size Fractionation Columns (Life Technologies). .. After BP recombination reaction (Life Technologies) using 100 ng of cDNA and 300 ng of an entry vector, pDONR221, the entry library containing approximately 2.5 x 107 clones, was amplified as a pool of transformants in One Shot TOP10 Electrocomp E . coli cells (Life Technologies).

    Ethanol Precipitation:

    Article Title: Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method
    Article Snippet: .. The cDNAs were separated from the linkers by a cDNA size fractionation column (Life Technologies) and subjected to the ethanol precipitation. .. Then, cDNAs were cloned into Sal I/ Not I site of pSPORT1 plasmid vector in a total volume of 20 μL reaction at room temperature for 3 h. The 100 μL of DH10B Escherichia coli host (Life Technologies) was transformed with the 5 μL ligation mixture by the chemical method according to the manufacturer's instruction.

    Mouse Assay:

    Article Title: Chibby Promotes Adipocyte Differentiation through Inhibition of ?-Catenin Signaling ▿
    Article Snippet: .. Total RNA was purified from epidydimal white adipose tissue (WAT), interscapular brown adipose tissue (BAT), and liver of 2-month-old C57BL/6J mice with RNeasy spin columns (QIAGEN), and synthesis of cDNA was performed with oligo(dT) primers using the ThermoScript reverse transcription (RT)-PCR System (Invitrogen) according to the manufacturer's instructions. ..

    Real-time Polymerase Chain Reaction:

    Article Title: HLA-DQ6 (DQB1*0601)-Restricted T Cells Protect against Experimental Autoimmune Encephalomyelitis in HLA-DR3.DQ6 Double-Transgenic Mice by Generating Anti-Inflammatory IFN-γ 1
    Article Snippet: .. RNA was extracted from cells using RNAeasy columns (Qiagen) and cDNA was prepared using RNase H-reverse transcriptase (Invitrogen). cDNA was analyzed by real-time quantitative PCR in triplicates by using SYBR GreenER qPCR reagent system (Invitrogen). .. The expression level of each gene was quantified using the threshold cycle (Ct ) method normalized for the housekeeping gene GAPDH.

    Polymerase Chain Reaction:

    Article Title: Secreted proteins of Uromyces fabae: similarities and stage specificity
    Article Snippet: .. Following addition of an Eco RI cleavage site to the cDNA using an Eco RI– Not I adapter (Stratagene, La Jolla, CA; ), size fractionation was performed using cDNA Size Fractionation Columns (Invitrogen, Carlsbad, CA). cDNA fragment size was determined by ligating aliquots of the fractions into plasmid pSuc2tM13ori ( ) and subsequent colony PCR. .. Numbers of transformants from this test were also used to determine optimal insert/vector ratios for the main ligation. cDNA fragments with a length no less than 200 bp were cloned into plasmid pSuc2tM13ori.

    Generated:

    Article Title: Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation
    Article Snippet: .. To validate the differential expression of genes identified by microarray analysis, two independent Lin− Kit+ hematopoietic progenitor cell samples were treated with 100 ng/mL IL-6, 10 ng/mL G-CSF or saline for 4 h, and total RNA was prepared using TRIzol and RNeasy columns. cDNA was generated using Superscript II reverse transcriptase (Invitrogen, CA, USA) according to the manufacturer's instructions. .. Quantitative real-time PCR was performed using an Applied Biosystems GeneCard and a 7900HT scanner (Applied Biosystems, CA, USA).

    Fractionation:

    Article Title: Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method
    Article Snippet: .. The cDNAs were separated from the linkers by a cDNA size fractionation column (Life Technologies) and subjected to the ethanol precipitation. .. Then, cDNAs were cloned into Sal I/ Not I site of pSPORT1 plasmid vector in a total volume of 20 μL reaction at room temperature for 3 h. The 100 μL of DH10B Escherichia coli host (Life Technologies) was transformed with the 5 μL ligation mixture by the chemical method according to the manufacturer's instruction.

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication
    Article Snippet: .. The cDNA was synthesized using the CloneMiner cDNA Library Construction Kit (Life Technologies) from 3 μg of mRNA and fractionated with cDNA Size Fractionation Columns (Life Technologies). .. After BP recombination reaction (Life Technologies) using 100 ng of cDNA and 300 ng of an entry vector, pDONR221, the entry library containing approximately 2.5 x 107 clones, was amplified as a pool of transformants in One Shot TOP10 Electrocomp E . coli cells (Life Technologies).

    Article Title: Analysis of expressed sequence tags and identification of genes encoding cell-wall-degrading enzymes from the fungivorous nematode Aphelenchus avenae
    Article Snippet: .. SalI/Sma I adaptors (Takara) were added to double stranded cDNA which was digested with Not I and size fractionated using a cDNA Size Fractionation Column to remove small cDNA ( < 500 bp) (Invitrogen). ..

    Article Title: Secreted proteins of Uromyces fabae: similarities and stage specificity
    Article Snippet: .. Following addition of an Eco RI cleavage site to the cDNA using an Eco RI– Not I adapter (Stratagene, La Jolla, CA; ), size fractionation was performed using cDNA Size Fractionation Columns (Invitrogen, Carlsbad, CA). cDNA fragment size was determined by ligating aliquots of the fractions into plasmid pSuc2tM13ori ( ) and subsequent colony PCR. .. Numbers of transformants from this test were also used to determine optimal insert/vector ratios for the main ligation. cDNA fragments with a length no less than 200 bp were cloned into plasmid pSuc2tM13ori.

    Expressing:

    Article Title: Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation
    Article Snippet: .. To validate the differential expression of genes identified by microarray analysis, two independent Lin− Kit+ hematopoietic progenitor cell samples were treated with 100 ng/mL IL-6, 10 ng/mL G-CSF or saline for 4 h, and total RNA was prepared using TRIzol and RNeasy columns. cDNA was generated using Superscript II reverse transcriptase (Invitrogen, CA, USA) according to the manufacturer's instructions. .. Quantitative real-time PCR was performed using an Applied Biosystems GeneCard and a 7900HT scanner (Applied Biosystems, CA, USA).

    cDNA Library Assay:

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication
    Article Snippet: .. The cDNA was synthesized using the CloneMiner cDNA Library Construction Kit (Life Technologies) from 3 μg of mRNA and fractionated with cDNA Size Fractionation Columns (Life Technologies). .. After BP recombination reaction (Life Technologies) using 100 ng of cDNA and 300 ng of an entry vector, pDONR221, the entry library containing approximately 2.5 x 107 clones, was amplified as a pool of transformants in One Shot TOP10 Electrocomp E . coli cells (Life Technologies).

    Purification:

    Article Title: Chibby Promotes Adipocyte Differentiation through Inhibition of ?-Catenin Signaling ▿
    Article Snippet: .. Total RNA was purified from epidydimal white adipose tissue (WAT), interscapular brown adipose tissue (BAT), and liver of 2-month-old C57BL/6J mice with RNeasy spin columns (QIAGEN), and synthesis of cDNA was performed with oligo(dT) primers using the ThermoScript reverse transcription (RT)-PCR System (Invitrogen) according to the manufacturer's instructions. ..

    Microarray:

    Article Title: Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation
    Article Snippet: .. To validate the differential expression of genes identified by microarray analysis, two independent Lin− Kit+ hematopoietic progenitor cell samples were treated with 100 ng/mL IL-6, 10 ng/mL G-CSF or saline for 4 h, and total RNA was prepared using TRIzol and RNeasy columns. cDNA was generated using Superscript II reverse transcriptase (Invitrogen, CA, USA) according to the manufacturer's instructions. .. Quantitative real-time PCR was performed using an Applied Biosystems GeneCard and a 7900HT scanner (Applied Biosystems, CA, USA).

    Plasmid Preparation:

    Article Title: Secreted proteins of Uromyces fabae: similarities and stage specificity
    Article Snippet: .. Following addition of an Eco RI cleavage site to the cDNA using an Eco RI– Not I adapter (Stratagene, La Jolla, CA; ), size fractionation was performed using cDNA Size Fractionation Columns (Invitrogen, Carlsbad, CA). cDNA fragment size was determined by ligating aliquots of the fractions into plasmid pSuc2tM13ori ( ) and subsequent colony PCR. .. Numbers of transformants from this test were also used to determine optimal insert/vector ratios for the main ligation. cDNA fragments with a length no less than 200 bp were cloned into plasmid pSuc2tM13ori.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Thermo Fisher otoferlin cdna fragments
    <t>Otoferlin</t> dual‐ AAV ‐ TS ‐transduced Otof −/− organs of Corti express full‐length otoferlin mRNA Schematic representation of otoferlin <t>cDNA</t> from otoferlin dual‐AAV‐transduced, wild‐type, and Otof −/− organs of Corti, displaying binding sites of primers used in PCRs to assess dual‐AAV reassembly. Otoferlin PCR amplicons from organ of Corti cDNA. A 1,753‐bp‐long amplicon (*), also present in non‐injected wild‐type controls (WTB6, WTCD1B6F1), indicates successful reassembly of the split otoferlin expression cassette in otoferlin dual‐AAV‐TS‐transduced CD1B6F1‐ Otof −/− organs of Corti (injected ear). In Otof −/− samples, three shorter products were amplified (a, b and c). Sanger sequencing confirmed correct dual‐AAV split‐site assembly (dashed line) as well as the presence of an artificial AccIII restriction site introduced in the dual‐AAV‐TS otoferlin cDNA, which is absent in the wild‐type (WT) and Otof −/− cDNA (a–c). Amplicons a‐c from Otof −/− organs of Corti all lack exons 14–15, while bands “b” (1,480 bp) and “c” (1,679 bp) still contain intron 20–21 (b) or intron 23–24 (c), respectively. Western blotting on cell lysates of WT and Otof −/− CD1B6F1 organs of Corti. Two bands of ˜210–230 kDa, corresponding to full‐length otoferlin, were detected in WT but absent in Otof −/− ears. (**) refers to an unspecific band detected in both samples. GAPDH was used as loading control. Data information: CDS: coding sequence, Ex: exon, TS: trans‐splicing, Hyb: hybrid, control ear: non‐treated ears, non‐injected ear: contralateral non‐injected Otof −/− ears. Source data are available online for this figure.
    Otoferlin Cdna Fragments, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/otoferlin cdna fragments/product/Thermo Fisher
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    otoferlin cdna fragments - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    86
    Thermo Fisher qrt pcr single stranded cdna
    Effects of HWB treatment on the expression of flavonoid biosynthesis-related genes and the occurrence of red lenticel discoloration on mango fruit cv. Shelly. (A) <t>qRT-PCR</t> profile of differentially expressed genes Ugft3, PAL, CFIL and CHSï , which are related to the flavonoid biosynthesis process, naringenin-chalcone synthase activity, and the phenylpropanoid biosynthesis pathway. (B) level of lenticel discoloration of HWB-treated and control fruits, and (C) lenticel discoloration symptoms on mango fruits, cv. Shelly following HWB treatment. qRT-PCR values were normalized to the values obtained in samples from untreated mango fruits at 0 h. Expression data are means of two replicates. Lenticel discoloration was evaluated following 2 weeks of storage at 12°C [ 9 ]. Average values followed by different letters differ significantly at P
    Qrt Pcr Single Stranded Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr single stranded cdna/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    qrt pcr single stranded cdna - by Bioz Stars, 2020-07
    86/100 stars
      Buy from Supplier

    94
    Thermo Fisher first strand complementary dna
    Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for <t>RNA-seq.</t> eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic <t>DNA</t> of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5
    First Strand Complementary Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/first strand complementary dna/product/Thermo Fisher
    Average 94 stars, based on 217 article reviews
    Price from $9.99 to $1999.99
    first strand complementary dna - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    90
    Thermo Fisher mouse multi tissue cdna
    Identification and validation of gigaxonin as a miR-501 target decreasing MYH3 levels in primary muscle cells. Primary myoblasts were transfected with control antagomir or antagomir-501 and harvested after 48 h. RNA was extracted and used for <t>cDNA</t> synthesis and RNA-seq after DNase-treatment ( n =3). (A) Venn diagram showing the overlap between predicted target genes for miR-501 in mouse and human, based on TargetScan v6.2. The 11 transcripts that were significantly upregulated, predicted as miR-501 targets in mouse and human, and conserved among mammals were considered for further analysis. (B) <t>qRT-PCR</t> confirmation of six out of the 11 selected genes as potential miR-501 targets based on inhibition or overexpression of miR-501 in primary myoblasts, respectively. Cells were harvested 48 h after transfection with the antagomirs or miRNA mimics. Values are shown relative to transfections with control mimic or antagomir as indicated by the dashed line. n =11-12. (C) Primary myoblasts were transfected with pcDNA3.1 vector encoding N-terminally FLAG-tagged gigaxonin or empty vector, and differentiation was induced by serum withdrawal for 2 days. Densitometry shows MYH3 protein normalized to GAPDH or desmin. n =6. (D) Effect of the proteasome inhibitor MG-132 on MYH3 levels after gigaxonin overexpression. MG-132 was added to the media at the indicated time points and concentrations before harvesting. (E) The human 3′ UTR of GAN was cloned into the pmirGLO vector with (mut) or without (wt) a mutation of three nucleotides in the miR-501-binding site. Constructs were transfected into HEK293 cells and luciferase activity was measured after 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. n =5. (F) qRT-PCR analysis of Gan expression in FACS-sorted MPs or regenerating muscle (CTX TA) 4 days after CTX injection. RNA derived from the same experiment shown in Fig. 5 A. Data are presented as mean±s.e.m. All qRT-PCR data are normalized to 18S rRNA. * P
    Mouse Multi Tissue Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse multi tissue cdna/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse multi tissue cdna - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

    Image Search Results


    Otoferlin dual‐ AAV ‐ TS ‐transduced Otof −/− organs of Corti express full‐length otoferlin mRNA Schematic representation of otoferlin cDNA from otoferlin dual‐AAV‐transduced, wild‐type, and Otof −/− organs of Corti, displaying binding sites of primers used in PCRs to assess dual‐AAV reassembly. Otoferlin PCR amplicons from organ of Corti cDNA. A 1,753‐bp‐long amplicon (*), also present in non‐injected wild‐type controls (WTB6, WTCD1B6F1), indicates successful reassembly of the split otoferlin expression cassette in otoferlin dual‐AAV‐TS‐transduced CD1B6F1‐ Otof −/− organs of Corti (injected ear). In Otof −/− samples, three shorter products were amplified (a, b and c). Sanger sequencing confirmed correct dual‐AAV split‐site assembly (dashed line) as well as the presence of an artificial AccIII restriction site introduced in the dual‐AAV‐TS otoferlin cDNA, which is absent in the wild‐type (WT) and Otof −/− cDNA (a–c). Amplicons a‐c from Otof −/− organs of Corti all lack exons 14–15, while bands “b” (1,480 bp) and “c” (1,679 bp) still contain intron 20–21 (b) or intron 23–24 (c), respectively. Western blotting on cell lysates of WT and Otof −/− CD1B6F1 organs of Corti. Two bands of ˜210–230 kDa, corresponding to full‐length otoferlin, were detected in WT but absent in Otof −/− ears. (**) refers to an unspecific band detected in both samples. GAPDH was used as loading control. Data information: CDS: coding sequence, Ex: exon, TS: trans‐splicing, Hyb: hybrid, control ear: non‐treated ears, non‐injected ear: contralateral non‐injected Otof −/− ears. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: A dual‐AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock‐out mice

    doi: 10.15252/emmm.201809396

    Figure Lengend Snippet: Otoferlin dual‐ AAV ‐ TS ‐transduced Otof −/− organs of Corti express full‐length otoferlin mRNA Schematic representation of otoferlin cDNA from otoferlin dual‐AAV‐transduced, wild‐type, and Otof −/− organs of Corti, displaying binding sites of primers used in PCRs to assess dual‐AAV reassembly. Otoferlin PCR amplicons from organ of Corti cDNA. A 1,753‐bp‐long amplicon (*), also present in non‐injected wild‐type controls (WTB6, WTCD1B6F1), indicates successful reassembly of the split otoferlin expression cassette in otoferlin dual‐AAV‐TS‐transduced CD1B6F1‐ Otof −/− organs of Corti (injected ear). In Otof −/− samples, three shorter products were amplified (a, b and c). Sanger sequencing confirmed correct dual‐AAV split‐site assembly (dashed line) as well as the presence of an artificial AccIII restriction site introduced in the dual‐AAV‐TS otoferlin cDNA, which is absent in the wild‐type (WT) and Otof −/− cDNA (a–c). Amplicons a‐c from Otof −/− organs of Corti all lack exons 14–15, while bands “b” (1,480 bp) and “c” (1,679 bp) still contain intron 20–21 (b) or intron 23–24 (c), respectively. Western blotting on cell lysates of WT and Otof −/− CD1B6F1 organs of Corti. Two bands of ˜210–230 kDa, corresponding to full‐length otoferlin, were detected in WT but absent in Otof −/− ears. (**) refers to an unspecific band detected in both samples. GAPDH was used as loading control. Data information: CDS: coding sequence, Ex: exon, TS: trans‐splicing, Hyb: hybrid, control ear: non‐treated ears, non‐injected ear: contralateral non‐injected Otof −/− ears. Source data are available online for this figure.

    Article Snippet: Otoferlin cDNA fragments spanning the split‐site of the full‐length otoferlin expression cassette were amplified from the cochlear cDNA using DreamTaq Polymerase (#EP0702, Thermo Fisher Scientific).

    Techniques: Binding Assay, Polymerase Chain Reaction, Amplification, Injection, Expressing, Sequencing, Western Blot

    Effects of HWB treatment on the expression of flavonoid biosynthesis-related genes and the occurrence of red lenticel discoloration on mango fruit cv. Shelly. (A) qRT-PCR profile of differentially expressed genes Ugft3, PAL, CFIL and CHSï , which are related to the flavonoid biosynthesis process, naringenin-chalcone synthase activity, and the phenylpropanoid biosynthesis pathway. (B) level of lenticel discoloration of HWB-treated and control fruits, and (C) lenticel discoloration symptoms on mango fruits, cv. Shelly following HWB treatment. qRT-PCR values were normalized to the values obtained in samples from untreated mango fruits at 0 h. Expression data are means of two replicates. Lenticel discoloration was evaluated following 2 weeks of storage at 12°C [ 9 ]. Average values followed by different letters differ significantly at P

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Effects of HWB treatment on the expression of flavonoid biosynthesis-related genes and the occurrence of red lenticel discoloration on mango fruit cv. Shelly. (A) qRT-PCR profile of differentially expressed genes Ugft3, PAL, CFIL and CHSï , which are related to the flavonoid biosynthesis process, naringenin-chalcone synthase activity, and the phenylpropanoid biosynthesis pathway. (B) level of lenticel discoloration of HWB-treated and control fruits, and (C) lenticel discoloration symptoms on mango fruits, cv. Shelly following HWB treatment. qRT-PCR values were normalized to the values obtained in samples from untreated mango fruits at 0 h. Expression data are means of two replicates. Lenticel discoloration was evaluated following 2 weeks of storage at 12°C [ 9 ]. Average values followed by different letters differ significantly at P

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Activity Assay

    Shelly. Effect of HWB on differential expression of chlorophyll and anthocyanin accumulation-related genes and color development in mango cv. Shelly. (A, B) qRT-PCR gene-expression profiles of genes related to (A) chlorophyll accumulation ( Thl1ch , LHCIIb, Oxepch and PIRC ) and (B) anthocyanin synthesis ( 85A2 and Anthocyanin5 ). The expression profile comprises data taken from samples of mango tissues sampled from cv. Shelly at four different time points after HWB treatment. (C) Changes in color index after 16 days of storage at 12°C followed by 8 days at 20°C. Vertical bars indicate SD of five replicates. qRT-PCR values were normalized to the values obtained with untreated mango fruit samples at 0 h. Expression data are the means of two replicates.

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Shelly. Effect of HWB on differential expression of chlorophyll and anthocyanin accumulation-related genes and color development in mango cv. Shelly. (A, B) qRT-PCR gene-expression profiles of genes related to (A) chlorophyll accumulation ( Thl1ch , LHCIIb, Oxepch and PIRC ) and (B) anthocyanin synthesis ( 85A2 and Anthocyanin5 ). The expression profile comprises data taken from samples of mango tissues sampled from cv. Shelly at four different time points after HWB treatment. (C) Changes in color index after 16 days of storage at 12°C followed by 8 days at 20°C. Vertical bars indicate SD of five replicates. qRT-PCR values were normalized to the values obtained with untreated mango fruit samples at 0 h. Expression data are the means of two replicates.

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Quantitative RT-PCR

    Validation of RNA-seq results by means of qRT-PCR. Ten differentially expressed genes (two from each of clusters 1 to 5) were examined by RNA-seq and qRT-PCR at four different time points after HWB treatment: A , B (cluster 1); C , D (cluster 2); E , F (cluster 3); G , H (cluster 4); and I , J (cluster 5). Values were normalized to the values obtained with untreated mango fruit samples at 0 h and the proportional fold-change (FC) was calculated. Expression data are means of two replicates.

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Validation of RNA-seq results by means of qRT-PCR. Ten differentially expressed genes (two from each of clusters 1 to 5) were examined by RNA-seq and qRT-PCR at four different time points after HWB treatment: A , B (cluster 1); C , D (cluster 2); E , F (cluster 3); G , H (cluster 4); and I , J (cluster 5). Values were normalized to the values obtained with untreated mango fruit samples at 0 h and the proportional fold-change (FC) was calculated. Expression data are means of two replicates.

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

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

    Differential expressions of genes modulating the mechanism of resistance to A. alternata in naturally infected mango fruits. (A) Effect of HWB on alternaria black spot (ABS) symptom development on mango cvs. Palmer, Kent, Tommy Atkins, Keitt, Lily and Shelly. (B) ABS symptom development on naturally infected fruits cv. Keitt following HWB treatment. (C) qRT-PCR differential expression profiling of genes Syn121 , glutardoxin , IT1K2 and AOS of cv. Shelly. Fruit peel tissues were sampled at four different time points after HWB treatment. RNA was extracted and served as a template for cDNA followed by qRT-PCR analysis of the genes of interest. Proportional increases in relative expression values were normalized against the samples of untreated mango fruits at 0 h. Expression data are means of two replicates. ABS-covered area was evaluated after 4 weeks of storage at 12°C. Average values followed by different letters differ significantly at P

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Differential expressions of genes modulating the mechanism of resistance to A. alternata in naturally infected mango fruits. (A) Effect of HWB on alternaria black spot (ABS) symptom development on mango cvs. Palmer, Kent, Tommy Atkins, Keitt, Lily and Shelly. (B) ABS symptom development on naturally infected fruits cv. Keitt following HWB treatment. (C) qRT-PCR differential expression profiling of genes Syn121 , glutardoxin , IT1K2 and AOS of cv. Shelly. Fruit peel tissues were sampled at four different time points after HWB treatment. RNA was extracted and served as a template for cDNA followed by qRT-PCR analysis of the genes of interest. Proportional increases in relative expression values were normalized against the samples of untreated mango fruits at 0 h. Expression data are means of two replicates. ABS-covered area was evaluated after 4 weeks of storage at 12°C. Average values followed by different letters differ significantly at P

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Infection, Quantitative RT-PCR, Expressing

    Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for RNA-seq. eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic DNA of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5

    Journal: BMC Genomics

    Article Title: Identification of alternative splicing events by RNA sequencing in early growth tomato fruits

    doi: 10.1186/s12864-015-2128-6

    Figure Lengend Snippet: Validation of AS events in different tissues by RT-PCR. Twenty-two multi-exon genes that underwent AS were validated by RT-PCR. RT-PCR analysis was performed on multiple tissues including those used for RNA-seq. eIF4α6 was used as control. Sl, S.lycopersicum cv Heinz1706; Sp, S.pimpinellifolium LA1589. noRT, negative control using reaction mixture without reverse transcriptase added as templates. Genomic, genomic DNA of LA1589 as PCR templates. M, DNA marker 2 K Plus II (Transgen, Beijing). IR, intron retention; AA, alternative acceptor; AD, alternative donor; ES, exon skipping; Others, AS events with more than one of the four basic types. Function description and primer information for the 22 genes can be found in Additional file 8 : Table S5

    Article Snippet: After genomic DNA in these RNA samples was removed by RNase-free DNase according to the manufacturer’s protocol (New England BioLabs, USA), the total RNA (1 μg per sample) was used to synthesize first strand complementary DNA using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Negative Control, Polymerase Chain Reaction, Marker

    Identification and validation of gigaxonin as a miR-501 target decreasing MYH3 levels in primary muscle cells. Primary myoblasts were transfected with control antagomir or antagomir-501 and harvested after 48 h. RNA was extracted and used for cDNA synthesis and RNA-seq after DNase-treatment ( n =3). (A) Venn diagram showing the overlap between predicted target genes for miR-501 in mouse and human, based on TargetScan v6.2. The 11 transcripts that were significantly upregulated, predicted as miR-501 targets in mouse and human, and conserved among mammals were considered for further analysis. (B) qRT-PCR confirmation of six out of the 11 selected genes as potential miR-501 targets based on inhibition or overexpression of miR-501 in primary myoblasts, respectively. Cells were harvested 48 h after transfection with the antagomirs or miRNA mimics. Values are shown relative to transfections with control mimic or antagomir as indicated by the dashed line. n =11-12. (C) Primary myoblasts were transfected with pcDNA3.1 vector encoding N-terminally FLAG-tagged gigaxonin or empty vector, and differentiation was induced by serum withdrawal for 2 days. Densitometry shows MYH3 protein normalized to GAPDH or desmin. n =6. (D) Effect of the proteasome inhibitor MG-132 on MYH3 levels after gigaxonin overexpression. MG-132 was added to the media at the indicated time points and concentrations before harvesting. (E) The human 3′ UTR of GAN was cloned into the pmirGLO vector with (mut) or without (wt) a mutation of three nucleotides in the miR-501-binding site. Constructs were transfected into HEK293 cells and luciferase activity was measured after 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. n =5. (F) qRT-PCR analysis of Gan expression in FACS-sorted MPs or regenerating muscle (CTX TA) 4 days after CTX injection. RNA derived from the same experiment shown in Fig. 5 A. Data are presented as mean±s.e.m. All qRT-PCR data are normalized to 18S rRNA. * P

    Journal: Development (Cambridge, England)

    Article Title: MicroRNA deep sequencing in two adult stem cell populations identifies miR-501 as a novel regulator of myosin heavy chain during muscle regeneration

    doi: 10.1242/dev.136051

    Figure Lengend Snippet: Identification and validation of gigaxonin as a miR-501 target decreasing MYH3 levels in primary muscle cells. Primary myoblasts were transfected with control antagomir or antagomir-501 and harvested after 48 h. RNA was extracted and used for cDNA synthesis and RNA-seq after DNase-treatment ( n =3). (A) Venn diagram showing the overlap between predicted target genes for miR-501 in mouse and human, based on TargetScan v6.2. The 11 transcripts that were significantly upregulated, predicted as miR-501 targets in mouse and human, and conserved among mammals were considered for further analysis. (B) qRT-PCR confirmation of six out of the 11 selected genes as potential miR-501 targets based on inhibition or overexpression of miR-501 in primary myoblasts, respectively. Cells were harvested 48 h after transfection with the antagomirs or miRNA mimics. Values are shown relative to transfections with control mimic or antagomir as indicated by the dashed line. n =11-12. (C) Primary myoblasts were transfected with pcDNA3.1 vector encoding N-terminally FLAG-tagged gigaxonin or empty vector, and differentiation was induced by serum withdrawal for 2 days. Densitometry shows MYH3 protein normalized to GAPDH or desmin. n =6. (D) Effect of the proteasome inhibitor MG-132 on MYH3 levels after gigaxonin overexpression. MG-132 was added to the media at the indicated time points and concentrations before harvesting. (E) The human 3′ UTR of GAN was cloned into the pmirGLO vector with (mut) or without (wt) a mutation of three nucleotides in the miR-501-binding site. Constructs were transfected into HEK293 cells and luciferase activity was measured after 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. n =5. (F) qRT-PCR analysis of Gan expression in FACS-sorted MPs or regenerating muscle (CTX TA) 4 days after CTX injection. RNA derived from the same experiment shown in Fig. 5 A. Data are presented as mean±s.e.m. All qRT-PCR data are normalized to 18S rRNA. * P

    Article Snippet: ORFs of the respective genes were amplified by PCR from mouse multi-tissue cDNA using high fidelity PCR enzyme mix (Thermo Scientific) and specific primers.

    Techniques: Transfection, RNA Sequencing Assay, Quantitative RT-PCR, Inhibition, Over Expression, Plasmid Preparation, Clone Assay, Mutagenesis, Binding Assay, Construct, Luciferase, Activity Assay, Expressing, FACS, Injection, Derivative Assay