complementary dna cdna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher complementary dna cdna
    Summary of RNAi depletion experiments. (A) , (B) Semi-quantitive RT-PCR validation of the effects of RNAi knockdown on the Dnmt1 (A) and Dnmt2 (B) , indicating obvious decrease of expression level of Dnmt1 (A) and Dnmt2 (B) . Lane 1 indicate amplification using <t>cDNA</t> from Dnmt1 RNAi eggs (A) and Dnmt2 RNAi eggs (B) , respectively; Lane 2 indicate amplification using cDNA from Non-specific RNAi control (by embryonic microinjection of gfp dsRNA) eggs. gDNA, PCR using genomic <t>DNA</t> as template to control DNA contamination; M, DNA marker DL2000 (TakaRa, Japan). Actin3 is used as the internal control for Semi-quantitive RT-PCR. (C) Hatching rate of the treated eggs with Dnmt1 RNAi and Dnmt2 RNAi, indicating that RNAi knockdown of Dnmt1 significantly reduces hatched eggs compared to control, but not for Dnmt2 . * significant differences as determined by chi-squared test (p
    Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 594 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 594 article reviews
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    Images

    1) Product Images from "Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication"

    Article Title: Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-646

    Summary of RNAi depletion experiments. (A) , (B) Semi-quantitive RT-PCR validation of the effects of RNAi knockdown on the Dnmt1 (A) and Dnmt2 (B) , indicating obvious decrease of expression level of Dnmt1 (A) and Dnmt2 (B) . Lane 1 indicate amplification using cDNA from Dnmt1 RNAi eggs (A) and Dnmt2 RNAi eggs (B) , respectively; Lane 2 indicate amplification using cDNA from Non-specific RNAi control (by embryonic microinjection of gfp dsRNA) eggs. gDNA, PCR using genomic DNA as template to control DNA contamination; M, DNA marker DL2000 (TakaRa, Japan). Actin3 is used as the internal control for Semi-quantitive RT-PCR. (C) Hatching rate of the treated eggs with Dnmt1 RNAi and Dnmt2 RNAi, indicating that RNAi knockdown of Dnmt1 significantly reduces hatched eggs compared to control, but not for Dnmt2 . * significant differences as determined by chi-squared test (p
    Figure Legend Snippet: Summary of RNAi depletion experiments. (A) , (B) Semi-quantitive RT-PCR validation of the effects of RNAi knockdown on the Dnmt1 (A) and Dnmt2 (B) , indicating obvious decrease of expression level of Dnmt1 (A) and Dnmt2 (B) . Lane 1 indicate amplification using cDNA from Dnmt1 RNAi eggs (A) and Dnmt2 RNAi eggs (B) , respectively; Lane 2 indicate amplification using cDNA from Non-specific RNAi control (by embryonic microinjection of gfp dsRNA) eggs. gDNA, PCR using genomic DNA as template to control DNA contamination; M, DNA marker DL2000 (TakaRa, Japan). Actin3 is used as the internal control for Semi-quantitive RT-PCR. (C) Hatching rate of the treated eggs with Dnmt1 RNAi and Dnmt2 RNAi, indicating that RNAi knockdown of Dnmt1 significantly reduces hatched eggs compared to control, but not for Dnmt2 . * significant differences as determined by chi-squared test (p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Polymerase Chain Reaction, Marker

    2) Product Images from "Molecular Insights Reveal Psy1, SGR, and SlMYB12 Genes are Associated with Diverse Fruit Color Pigments in Tomato (Solanum lycopersicum L.)"

    Article Title: Molecular Insights Reveal Psy1, SGR, and SlMYB12 Genes are Associated with Diverse Fruit Color Pigments in Tomato (Solanum lycopersicum L.)

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules22122180

    Alignment of DNA and protein sequences, and the position of the mutation of the SGR gene. ( a ) Sequence alignment of SGR amplified from genomic DNA of the red-fruited line (KNR3) and the green-fruited line (BUC30) compared with the reference genotype Heinz1706, showing a 1-bp substitution at position 1679 bp (highlighted in red). Alternative splicing was found in the following 31 base pairs highlighted in blue, which was confirmed in the CDS of BUC30; ( b ) Sequence alignment of SGR cDNA of the red-fruited line (KNR3) and the green-fruited line (BUC30) compared with the reference genotype Heinz1706, showing a 1-bp alteration at position 461 bp (highlighted in red) followed by a 31-bp insertion, which resulted in alternative splicing in the mRNA due to the conversion of the intron to an exon (the orange box). This insertion resulted in an early stop codon (green box inside the orange box); ( c )Amino acid alignment showing one amino acid substitution (green star) due to the non-synonymous SNP and termination of translation (red star) of the protein in the green-fruited line BUC30; ( d ) Graphical representation of the SGR gene based on the genomic sequence, where the T to C substitution is positioned at the end of the third exon.
    Figure Legend Snippet: Alignment of DNA and protein sequences, and the position of the mutation of the SGR gene. ( a ) Sequence alignment of SGR amplified from genomic DNA of the red-fruited line (KNR3) and the green-fruited line (BUC30) compared with the reference genotype Heinz1706, showing a 1-bp substitution at position 1679 bp (highlighted in red). Alternative splicing was found in the following 31 base pairs highlighted in blue, which was confirmed in the CDS of BUC30; ( b ) Sequence alignment of SGR cDNA of the red-fruited line (KNR3) and the green-fruited line (BUC30) compared with the reference genotype Heinz1706, showing a 1-bp alteration at position 461 bp (highlighted in red) followed by a 31-bp insertion, which resulted in alternative splicing in the mRNA due to the conversion of the intron to an exon (the orange box). This insertion resulted in an early stop codon (green box inside the orange box); ( c )Amino acid alignment showing one amino acid substitution (green star) due to the non-synonymous SNP and termination of translation (red star) of the protein in the green-fruited line BUC30; ( d ) Graphical representation of the SGR gene based on the genomic sequence, where the T to C substitution is positioned at the end of the third exon.

    Techniques Used: Mutagenesis, Sequencing, Amplification

    3) Product Images from "The erythropoietin receptor is a downstream effector of Klotho-induced cytoprotection"

    Article Title: The erythropoietin receptor is a downstream effector of Klotho-induced cytoprotection

    Journal: Kidney international

    doi: 10.1038/ki.2013.149

    Expression of EpoR protein and mRNA in rat kidney ( A ) EpoR mRNA expression in the rat kidney or microdissected glomeruli and renal tubules and from normal adult rats at age of 3 months old by RT-PCT. Total RNA was extracted, and complimentary DNA (cDNA) generated with Oligo dT. Specific target genes were examined by PCR with rat specific primers (shown in method section). AQP2: aquaporin-2; CCD: cortical collecting duct; DCT: distal convaluted tubules; Glo: glomeruli; IMCD: inner medullary collecting duct; K: Rat kidney tissue containing cortex and medullar; NaPi-2a: Na-Pi dependent cotransporter-2a; NKCC2: Na-K-2Cl cotransporter; PT: proximal tubules; TAL: thick ascending limb; K-RT: kidney sample with reverse transcriptase omitted; ( B ) EpoR protein expression in BaF3 cell transfected with HA-tagged mouse EpoR or empty vector. Total lysates from native BaF3 and BaF3-HA-EpoR cells were subjected to immunoprecipitation by HA-Resin followed by immunobloting for EpoR with several anti-EpoR antibodies: A82, HA, M-20, Fab 6. Asterisks depict the specific HA-EpoR band. ( C ) Total proteins were extracted from human and rodent kidneys and murine FLC and Ter119-cells, and subjected to immunobloting EpoR with several antibodies against EpoR. A82 is monoclonal rabbit antibody kindly provided by Dr. Steve Elliot; M-20 is polyclonal antibody purchased from Santa Cruz Biotech. Fab 6 is a synthetic human Fab against human EpoR isolated from a phage-displayed library. Asterisks indicate multiple forms of glycosylated EpoR or EpoR fragments. Hu: human; Mu: mouse; FLC: fetal liver cells at E13.5
    Figure Legend Snippet: Expression of EpoR protein and mRNA in rat kidney ( A ) EpoR mRNA expression in the rat kidney or microdissected glomeruli and renal tubules and from normal adult rats at age of 3 months old by RT-PCT. Total RNA was extracted, and complimentary DNA (cDNA) generated with Oligo dT. Specific target genes were examined by PCR with rat specific primers (shown in method section). AQP2: aquaporin-2; CCD: cortical collecting duct; DCT: distal convaluted tubules; Glo: glomeruli; IMCD: inner medullary collecting duct; K: Rat kidney tissue containing cortex and medullar; NaPi-2a: Na-Pi dependent cotransporter-2a; NKCC2: Na-K-2Cl cotransporter; PT: proximal tubules; TAL: thick ascending limb; K-RT: kidney sample with reverse transcriptase omitted; ( B ) EpoR protein expression in BaF3 cell transfected with HA-tagged mouse EpoR or empty vector. Total lysates from native BaF3 and BaF3-HA-EpoR cells were subjected to immunoprecipitation by HA-Resin followed by immunobloting for EpoR with several anti-EpoR antibodies: A82, HA, M-20, Fab 6. Asterisks depict the specific HA-EpoR band. ( C ) Total proteins were extracted from human and rodent kidneys and murine FLC and Ter119-cells, and subjected to immunobloting EpoR with several antibodies against EpoR. A82 is monoclonal rabbit antibody kindly provided by Dr. Steve Elliot; M-20 is polyclonal antibody purchased from Santa Cruz Biotech. Fab 6 is a synthetic human Fab against human EpoR isolated from a phage-displayed library. Asterisks indicate multiple forms of glycosylated EpoR or EpoR fragments. Hu: human; Mu: mouse; FLC: fetal liver cells at E13.5

    Techniques Used: Expressing, Generated, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Isolation

    4) Product Images from "Differential Gene Expression in CD8+ Cells from HIV-1 Infected Subjects Showing Suppression of HIV Replication"

    Article Title: Differential Gene Expression in CD8+ Cells from HIV-1 Infected Subjects Showing Suppression of HIV Replication

    Journal: Virology

    doi: 10.1016/j.virol.2006.12.007

    Preparation of cRNA for microarray hybridization. Total RNA was isolated and purified from CD8+ cells from HIV+ subjects showing high CNAR and seronegative control subjects with none or low CNAR using a combined Trizol / RNeasy method. Complementary DNA (cDNA) was synthesized with the SuperScript™ Double Stranded cDNA Synthesis Kit. The cDNA was then subjected to an in vitro for details.
    Figure Legend Snippet: Preparation of cRNA for microarray hybridization. Total RNA was isolated and purified from CD8+ cells from HIV+ subjects showing high CNAR and seronegative control subjects with none or low CNAR using a combined Trizol / RNeasy method. Complementary DNA (cDNA) was synthesized with the SuperScript™ Double Stranded cDNA Synthesis Kit. The cDNA was then subjected to an in vitro for details.

    Techniques Used: Microarray, Hybridization, Isolation, Purification, Synthesized, In Vitro

    5) Product Images from "Discovery of stromal regulatory networks that suppress Ras-sensitized epithelial cell proliferation"

    Article Title: Discovery of stromal regulatory networks that suppress Ras-sensitized epithelial cell proliferation

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2017.04.024

    In vivo deletion of Tlk1 and Tlk2 in mouse mammary fibroblasts increases proliferation of adjacent ductal epithelium (A, B) Schematic overview of the targeting vector used to generate the Tlk1 and Tlk2 conditional knock-out mice. Line 1: partial genomic locus of mouse Tlk1 or Tlk2 gene with exons represented as dark gray boxes and Southern probe as light gray bar. Line 2: targeting vector with exons 9 and 10 in Tlk1 and exon 11 in Tlk2 flanked by loxP elements (black triangles), the LacZ reporter cassette and neomycin (Neo) resistance cassette flanked by FRT sites (white triangles). Line 3: targeted genomic locus (Neo). Line 4: Neo cassette was removed by Actin-Flpe mediated recombination ( Flox (F) ). Black arrows P1–P3 indicate location of genotyping primers. Diagram not to scale. (C, D) Southern blot (left panels) of ES cells confirming targeting of Tlk1 or Tlk2 genomic loci respectively. PCR genotyping of genomic DNA (right panels) from WT ( Tlk1 +/+ or Tlk2 +/+ ) heterozygous ( Tlk1 +/F or Tlk2 +/F ) and homozygous ( Tlk1 F/F or Tlk2 F/F ) mice with primers P1/P2 following deletion of Neo cassette. (E) Left: bright field images of whole mount inguinal mammary glands from 10-week-old Tlk1 F/F ;Tlk2 F/F (control) and Fsp-cre;Tlk1 F/F ;Tlk2 F/F (Tlk1 Δ/Δ ;Tlk2 Δ/Δ ) mice stained with carmine red (bar = 2000 μm). Boxes indicate area of magnification in adjacent panel (bar = 100 μm). Middle: H E stained sections of opposite inguinal gland (bar = 50 μm). Right: representative images of sections of inguinal mammary glands immuno-stained for the proliferation marker Ki67 (brown) and counterstained with hematoxylin (blue) (bar = 50 μm). Boxes indicate area of magnification in adjacent panel (bar = 20 μm). (F) Percent Ki67 positive epithelial cells per 20X field (dots) of tissue sections described in (E). Red bar, median; black bars, interquartile range; pooled counts from 6 control mice or 5 Fsp-cre mice. P value: Mann-Whitney test of medians. (G) Left: Representative images of inguinal mammary gland sections immuno-stained for smooth muscle actin (SMA; brown) and counterstained with hematoxylin (blue) (bar = 50 μm). Insets represent consecutive sections immuno-stained with Ki67 (brown) and counterstained with hematoxylin (blue). Right: Representative images of inguinal mammary gland sections immuno-stained for smooth muscle actin (SMA; red) and Ki67 (brown); counterstained with hematoxylin (blue) (bar = 50 μm). (H) PCR genotyping of mammary fibroblasts derived from Tlk1 F/F and Tlk2 F/F mice with or without Fsp-cre to detect the Flox allele ( Tlk F ; P1/P2 primers), the deletion product formed after Fsp-cre -mediated recombination ( Tlk Δ ; P1/P3 primers) and Fsp-cre. (I) qRT-PCR for Tlk1 or Tlk2 on cDNA derived from fibroblasts isolated from Tlk1 F/F ;Tlk2 F/F mammary glands of control (−) and Fsp-cre positive (+) mice. Expression was normalized to Gapdh. (J) Mammary gland wholemounts from Fsp-cre;Rosa loxP/+ and Rosa loxP/+ (left, inset) mice stained for β-galactosidase. Higher (10×) magnification of a wholemount gland (top-right) and a cross section stained for β-galactosidase and counterstained with nuclear fast red (bottom-right). Str, stromal fibroblasts; lu, lumen; epi, epithelial cells. (K) qRT-PCR for Tlk1 expression (left) on cDNA derived from Tlk1 F/F mammary fibroblasts (MMFs) or mammary epithelial cells (MECs) lacking (−) or containing (+) the Fsp-cre transgene. qRT-PCR for Tlk2 expression (right) on cDNA derived from Tlk2 F/F mammary fibroblasts (MMFs) or mammary epithelial cells (MECs) lacking (−) or containing (+) the Fsp-cre transgene. Expression was normalized to Gapdh. .
    Figure Legend Snippet: In vivo deletion of Tlk1 and Tlk2 in mouse mammary fibroblasts increases proliferation of adjacent ductal epithelium (A, B) Schematic overview of the targeting vector used to generate the Tlk1 and Tlk2 conditional knock-out mice. Line 1: partial genomic locus of mouse Tlk1 or Tlk2 gene with exons represented as dark gray boxes and Southern probe as light gray bar. Line 2: targeting vector with exons 9 and 10 in Tlk1 and exon 11 in Tlk2 flanked by loxP elements (black triangles), the LacZ reporter cassette and neomycin (Neo) resistance cassette flanked by FRT sites (white triangles). Line 3: targeted genomic locus (Neo). Line 4: Neo cassette was removed by Actin-Flpe mediated recombination ( Flox (F) ). Black arrows P1–P3 indicate location of genotyping primers. Diagram not to scale. (C, D) Southern blot (left panels) of ES cells confirming targeting of Tlk1 or Tlk2 genomic loci respectively. PCR genotyping of genomic DNA (right panels) from WT ( Tlk1 +/+ or Tlk2 +/+ ) heterozygous ( Tlk1 +/F or Tlk2 +/F ) and homozygous ( Tlk1 F/F or Tlk2 F/F ) mice with primers P1/P2 following deletion of Neo cassette. (E) Left: bright field images of whole mount inguinal mammary glands from 10-week-old Tlk1 F/F ;Tlk2 F/F (control) and Fsp-cre;Tlk1 F/F ;Tlk2 F/F (Tlk1 Δ/Δ ;Tlk2 Δ/Δ ) mice stained with carmine red (bar = 2000 μm). Boxes indicate area of magnification in adjacent panel (bar = 100 μm). Middle: H E stained sections of opposite inguinal gland (bar = 50 μm). Right: representative images of sections of inguinal mammary glands immuno-stained for the proliferation marker Ki67 (brown) and counterstained with hematoxylin (blue) (bar = 50 μm). Boxes indicate area of magnification in adjacent panel (bar = 20 μm). (F) Percent Ki67 positive epithelial cells per 20X field (dots) of tissue sections described in (E). Red bar, median; black bars, interquartile range; pooled counts from 6 control mice or 5 Fsp-cre mice. P value: Mann-Whitney test of medians. (G) Left: Representative images of inguinal mammary gland sections immuno-stained for smooth muscle actin (SMA; brown) and counterstained with hematoxylin (blue) (bar = 50 μm). Insets represent consecutive sections immuno-stained with Ki67 (brown) and counterstained with hematoxylin (blue). Right: Representative images of inguinal mammary gland sections immuno-stained for smooth muscle actin (SMA; red) and Ki67 (brown); counterstained with hematoxylin (blue) (bar = 50 μm). (H) PCR genotyping of mammary fibroblasts derived from Tlk1 F/F and Tlk2 F/F mice with or without Fsp-cre to detect the Flox allele ( Tlk F ; P1/P2 primers), the deletion product formed after Fsp-cre -mediated recombination ( Tlk Δ ; P1/P3 primers) and Fsp-cre. (I) qRT-PCR for Tlk1 or Tlk2 on cDNA derived from fibroblasts isolated from Tlk1 F/F ;Tlk2 F/F mammary glands of control (−) and Fsp-cre positive (+) mice. Expression was normalized to Gapdh. (J) Mammary gland wholemounts from Fsp-cre;Rosa loxP/+ and Rosa loxP/+ (left, inset) mice stained for β-galactosidase. Higher (10×) magnification of a wholemount gland (top-right) and a cross section stained for β-galactosidase and counterstained with nuclear fast red (bottom-right). Str, stromal fibroblasts; lu, lumen; epi, epithelial cells. (K) qRT-PCR for Tlk1 expression (left) on cDNA derived from Tlk1 F/F mammary fibroblasts (MMFs) or mammary epithelial cells (MECs) lacking (−) or containing (+) the Fsp-cre transgene. qRT-PCR for Tlk2 expression (right) on cDNA derived from Tlk2 F/F mammary fibroblasts (MMFs) or mammary epithelial cells (MECs) lacking (−) or containing (+) the Fsp-cre transgene. Expression was normalized to Gapdh. .

    Techniques Used: In Vivo, Plasmid Preparation, Knock-Out, Mouse Assay, Southern Blot, Polymerase Chain Reaction, Staining, Marker, MANN-WHITNEY, Derivative Assay, Quantitative RT-PCR, Isolation, Expressing

    6) Product Images from "Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR"

    Article Title: Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-020-0452-7

    Examples of nonoptimal primer set test results using PCR. Electrophoresis results after PCR (35 cycles) with various nonoptimal primer sets with or without template DNA (SARS-CoV-2 cDNA) and under different T a conditions. a An example of the appearance of short primer dimers in all conditions, performed with the SARS-CoV-2_IBS_RdRP1 primer set. b An example of the appearance of short and long primer dimers under a low T a , with the SARS-CoV-2_IBS_S1 primer set. c An example of the appearance of long primer dimers under a low T a , with the SARS-CoV-2_IBS_E1 primer set. d An example of low-efficiency primers in PCR performed with the CDC_RNAse P primer set. e An example of the appearance of a nonspecific band at a low T a , in PCR performed with the SARS-CoV-2_IBS_N1 primer set. Red asterisks indicate long primer dimers.
    Figure Legend Snippet: Examples of nonoptimal primer set test results using PCR. Electrophoresis results after PCR (35 cycles) with various nonoptimal primer sets with or without template DNA (SARS-CoV-2 cDNA) and under different T a conditions. a An example of the appearance of short primer dimers in all conditions, performed with the SARS-CoV-2_IBS_RdRP1 primer set. b An example of the appearance of short and long primer dimers under a low T a , with the SARS-CoV-2_IBS_S1 primer set. c An example of the appearance of long primer dimers under a low T a , with the SARS-CoV-2_IBS_E1 primer set. d An example of low-efficiency primers in PCR performed with the CDC_RNAse P primer set. e An example of the appearance of a nonspecific band at a low T a , in PCR performed with the SARS-CoV-2_IBS_N1 primer set. Red asterisks indicate long primer dimers.

    Techniques Used: Polymerase Chain Reaction, Electrophoresis

    7) Product Images from "B-Chromosome Ribosomal DNA Is Functional in the Grasshopper Eyprepocnemis plorans"

    Article Title: B-Chromosome Ribosomal DNA Is Functional in the Grasshopper Eyprepocnemis plorans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036600

    Amplification of the ITS2_B region with the ITSA and ITSB primers on genomic (gDNA) and complementary (cDNA) DNA from representative males with 0–3 B chromosomes (upper panel) and females with 0–2 B chromosomes (lower panel). Note the presence of PCR product on gDNA of B-carrying individuals but absence in the case of B-lacking ones. Also note the presence of PCR product on cDNA of only some B-carrying individuals. Ø = Negative control (with no DNA). C + = Positive control (gDNA from 1B male).
    Figure Legend Snippet: Amplification of the ITS2_B region with the ITSA and ITSB primers on genomic (gDNA) and complementary (cDNA) DNA from representative males with 0–3 B chromosomes (upper panel) and females with 0–2 B chromosomes (lower panel). Note the presence of PCR product on gDNA of B-carrying individuals but absence in the case of B-lacking ones. Also note the presence of PCR product on cDNA of only some B-carrying individuals. Ø = Negative control (with no DNA). C + = Positive control (gDNA from 1B male).

    Techniques Used: Amplification, Polymerase Chain Reaction, Negative Control, Positive Control

    8) Product Images from "A birth of bipartite exon by intragenic deletion"

    Article Title: A birth of bipartite exon by intragenic deletion

    Journal: Molecular Genetics & Genomic Medicine

    doi: 10.1002/mgg3.277

    Genomic deletion activating a bipartite COL 4A5 exon in Alport syndrome. (A) Identification of a heterozygous deletion by multiplex ligation‐dependent probe amplification ( MLPA ) analysis of COL 4A5 exons. Exons with signal intensities at ~0.5 are denoted by a horizontal bar. Y ‐axis, normalized MLPA values. Both parents showed a normal MLPA pattern and no evidence for mosaicism (data not shown). (B) RT ‐ PCR of control (C) and patient (P) total RNA samples. M, DNA size marker (nt). PCR products are shown schematically to the right. The new boundary is denoted by a vertical red line, the new exon is in blue and canonical exons are numbered. Amplification primers (arrows) were located in exon 33/34 and exon 42. (C) Sequence chromatogram of the aberrant cDNA product revealing a cryptic exon (blue bar). The L1 homology region (boxed) extends into the 5′ portion of the bipartite exon; stop codon is underlined. (D) PCR product amplified across the deletion breakpoint from control (C) and patient (P) DNA using primers in the indicated introns. (E) Sequence chromatogram of the corresponding fragment. For legend, see panel C. The polypyrimidine tract ( PPT , gray bar) and the branch point adenine ( BP , in yellow) of the new exon were predicted by a support vector machine ( SVM ) algorithm, with a SVM score of 0.81 (Corvelo et al. 2010 ). Conserved dinucleotides at new splice sites are in red boxes. (F) Summary of transposed elements across the centromeric deletion breakpoint ( upper panel ) and the alignment of the L1 ME in COL 4A5 intron 37 with a L1 ME consensus ( lower panel ). Mutation creating the AG dinucleotide in the COL 4A5 L1 ME is in red. (G) Putative interactions between RNA ‐binding proteins and sequence motifs flanking the deletion breakpoint, as predicted by the RBP map (Paz et al. 2014 ).
    Figure Legend Snippet: Genomic deletion activating a bipartite COL 4A5 exon in Alport syndrome. (A) Identification of a heterozygous deletion by multiplex ligation‐dependent probe amplification ( MLPA ) analysis of COL 4A5 exons. Exons with signal intensities at ~0.5 are denoted by a horizontal bar. Y ‐axis, normalized MLPA values. Both parents showed a normal MLPA pattern and no evidence for mosaicism (data not shown). (B) RT ‐ PCR of control (C) and patient (P) total RNA samples. M, DNA size marker (nt). PCR products are shown schematically to the right. The new boundary is denoted by a vertical red line, the new exon is in blue and canonical exons are numbered. Amplification primers (arrows) were located in exon 33/34 and exon 42. (C) Sequence chromatogram of the aberrant cDNA product revealing a cryptic exon (blue bar). The L1 homology region (boxed) extends into the 5′ portion of the bipartite exon; stop codon is underlined. (D) PCR product amplified across the deletion breakpoint from control (C) and patient (P) DNA using primers in the indicated introns. (E) Sequence chromatogram of the corresponding fragment. For legend, see panel C. The polypyrimidine tract ( PPT , gray bar) and the branch point adenine ( BP , in yellow) of the new exon were predicted by a support vector machine ( SVM ) algorithm, with a SVM score of 0.81 (Corvelo et al. 2010 ). Conserved dinucleotides at new splice sites are in red boxes. (F) Summary of transposed elements across the centromeric deletion breakpoint ( upper panel ) and the alignment of the L1 ME in COL 4A5 intron 37 with a L1 ME consensus ( lower panel ). Mutation creating the AG dinucleotide in the COL 4A5 L1 ME is in red. (G) Putative interactions between RNA ‐binding proteins and sequence motifs flanking the deletion breakpoint, as predicted by the RBP map (Paz et al. 2014 ).

    Techniques Used: Multiplex Assay, Ligation, Amplification, Multiplex Ligation-dependent Probe Amplification, Reverse Transcription Polymerase Chain Reaction, Marker, Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Mutagenesis, RNA Binding Assay

    9) Product Images from "Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion"

    Article Title: Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion

    Journal: Neurobiology of Aging

    doi: 10.1016/j.neurobiolaging.2014.01.016

    Analysis of C9orf72 protein levels in postmortem tissue using the custom rabbit polyclonal antibody C9-3721. (A) Immunoblotting of HEK293T lysates detects approximate 48 kDa and 50 kDa endogenous protein species. Knockdown of C9orf72 using a panel of siRNA duplexes (siC91-4) leads reduction of the 48-kDa species (black arrowhead, endo C9). β-actin was used a loading control. (B) Initial immunoblots of postmortem frontal cortex tissue detected variable lower molecular weight (white arrowhead) and approximate 48-kDa protein species (black arrowhead) that obscured the endogenous C9orf72 bands. These suspected GFAP-related products were removed using the pre-absorption method. (C) Immunoblot analysis of HEK293Ts transfected with increasing amounts of a GFAP cDNA construct. C9-3721 showed evidence of cross-reactivity with recombinant GFAP that is removed following pre-absorption using a recombinant GFAP column. (D) Example immunoblots of protein extractions from frontal cortex (11 cases and 6 controls) and cerebellar cortex (12 cases and 6 controls) with recombinant myc-tagged C9orf72 long form blot control (myc-C9 LF, black arrowheads) used for LI-COR quantification. Endogenous C9orf72 long form migrates at approximately 48 kDa (endo C9, white arrowheads). (E) Quantification of C9orf72 protein levels using β-actin as a normalizer for total protein. Cases show a significant reduction in the long form compared with controls in frontal cortex using a 1-tailed t test assuming unequal variance. This was not observed in cerebellar cortex samples. Abbreviations: cDNA, complementary DNA; GFAP, glial fibrillary acidic protein; siRNA, small interfering RNA.
    Figure Legend Snippet: Analysis of C9orf72 protein levels in postmortem tissue using the custom rabbit polyclonal antibody C9-3721. (A) Immunoblotting of HEK293T lysates detects approximate 48 kDa and 50 kDa endogenous protein species. Knockdown of C9orf72 using a panel of siRNA duplexes (siC91-4) leads reduction of the 48-kDa species (black arrowhead, endo C9). β-actin was used a loading control. (B) Initial immunoblots of postmortem frontal cortex tissue detected variable lower molecular weight (white arrowhead) and approximate 48-kDa protein species (black arrowhead) that obscured the endogenous C9orf72 bands. These suspected GFAP-related products were removed using the pre-absorption method. (C) Immunoblot analysis of HEK293Ts transfected with increasing amounts of a GFAP cDNA construct. C9-3721 showed evidence of cross-reactivity with recombinant GFAP that is removed following pre-absorption using a recombinant GFAP column. (D) Example immunoblots of protein extractions from frontal cortex (11 cases and 6 controls) and cerebellar cortex (12 cases and 6 controls) with recombinant myc-tagged C9orf72 long form blot control (myc-C9 LF, black arrowheads) used for LI-COR quantification. Endogenous C9orf72 long form migrates at approximately 48 kDa (endo C9, white arrowheads). (E) Quantification of C9orf72 protein levels using β-actin as a normalizer for total protein. Cases show a significant reduction in the long form compared with controls in frontal cortex using a 1-tailed t test assuming unequal variance. This was not observed in cerebellar cortex samples. Abbreviations: cDNA, complementary DNA; GFAP, glial fibrillary acidic protein; siRNA, small interfering RNA.

    Techniques Used: Western Blot, Molecular Weight, Transfection, Construct, Recombinant, Small Interfering RNA

    10) Product Images from "A novel testis-specific long noncoding RNA, Tesra, activates the Prss42/Tessp-2 gene during mouse spermatogenesis †"

    Article Title: A novel testis-specific long noncoding RNA, Tesra, activates the Prss42/Tessp-2 gene during mouse spermatogenesis †

    Journal: Biology of Reproduction

    doi: 10.1093/biolre/ioy230

    Overexpression of Tesra increases Prss42/Tessp-2 expression and promoter activity. (A) Endogenous expression of Prss/Tessp cluster genes and Tesra in Hepa1–6 cells. qRT-PCR was performed with total RNAs from Hepa1–6 cells. Reverse transcription was done by using the oligo(dT) primer, and the Aip gene was examined as an internal control. Expression levels were normalized to Aip . (B) Successful overexpression of Tesra . Tesra-OE or the control vector was transiently transfected into Hepa1–6 cells, and after selection with G418, total RNA was purified from each sample. Complementary DNA was synthesized with the oligo(dT) primer, and PCR was conducted to detect Tesra and Gapdh expression. The cycle number is shown in parenthesis. A representative result from three experiments is shown. We could not see any difference among the three data sets. (C) Relative expression of Prss42/Tessp-2 mRNA by overexpression of Tesra . qRT-PCR was performed with the cDNAs prepared in (B). Data normalization was done by using Aip as an internal control. Transient overexpression of Tesra significantly increased Prss42/Tessp-2 expression in Hepa1–6 cells. The data are presented as means ± SD from three independent experiments and were analyzed by the Student t test. * P
    Figure Legend Snippet: Overexpression of Tesra increases Prss42/Tessp-2 expression and promoter activity. (A) Endogenous expression of Prss/Tessp cluster genes and Tesra in Hepa1–6 cells. qRT-PCR was performed with total RNAs from Hepa1–6 cells. Reverse transcription was done by using the oligo(dT) primer, and the Aip gene was examined as an internal control. Expression levels were normalized to Aip . (B) Successful overexpression of Tesra . Tesra-OE or the control vector was transiently transfected into Hepa1–6 cells, and after selection with G418, total RNA was purified from each sample. Complementary DNA was synthesized with the oligo(dT) primer, and PCR was conducted to detect Tesra and Gapdh expression. The cycle number is shown in parenthesis. A representative result from three experiments is shown. We could not see any difference among the three data sets. (C) Relative expression of Prss42/Tessp-2 mRNA by overexpression of Tesra . qRT-PCR was performed with the cDNAs prepared in (B). Data normalization was done by using Aip as an internal control. Transient overexpression of Tesra significantly increased Prss42/Tessp-2 expression in Hepa1–6 cells. The data are presented as means ± SD from three independent experiments and were analyzed by the Student t test. * P

    Techniques Used: Over Expression, Expressing, Activity Assay, Quantitative RT-PCR, Plasmid Preparation, Transfection, Selection, Purification, Synthesized, Polymerase Chain Reaction

    11) Product Images from "A Zinc-dependent metalloproteinase in the intracellular adaptation of Brucella abortus in macrophages"

    Article Title: A Zinc-dependent metalloproteinase in the intracellular adaptation of Brucella abortus in macrophages

    Journal: bioRxiv

    doi: 10.1101/2020.04.17.046490

    A Zinc-dependent metalloproteinase of B. abortus is an operon that forms a putative type II Toxin-Antitoxin. Identification of the transcriptional unit (operon) constituted by the ORF BAB1_0270 and a transcriptional regulator in B. abortus 2308 expressed in A) the genomic DNA and B) the cDNA from total RNA. MW: Molecular weight; lane 1: Transcriptional regulator (357 bp); lane 2: BAB1_0270 (549 bp); and lane 3: operon constituted by ORF BAB1_0270-transcriptional regulator (906 bp). C) Hypothetic Type II Toxin-Antitoxin (TA) model for operon constitute by Zn-dependent metalloproteinase and transcriptional regulator. Toxin (Zinc-dependent metalloproteinase) and anti-toxin (transcriptional regulator) are transcribed to mRNA together. Proteases are activated under stress condition, cleaving anti-toxin, which increases the levels of Toxin free, inducing diverse biological functions in bacteria. Predicted promoter at site −35 and −10 binding by RNA polymerase sigma factor rpoD. ATG A: nucleotides share between final part of transcriptional factor and metalloproteinase codified by the BAB1_0270 ORF.
    Figure Legend Snippet: A Zinc-dependent metalloproteinase of B. abortus is an operon that forms a putative type II Toxin-Antitoxin. Identification of the transcriptional unit (operon) constituted by the ORF BAB1_0270 and a transcriptional regulator in B. abortus 2308 expressed in A) the genomic DNA and B) the cDNA from total RNA. MW: Molecular weight; lane 1: Transcriptional regulator (357 bp); lane 2: BAB1_0270 (549 bp); and lane 3: operon constituted by ORF BAB1_0270-transcriptional regulator (906 bp). C) Hypothetic Type II Toxin-Antitoxin (TA) model for operon constitute by Zn-dependent metalloproteinase and transcriptional regulator. Toxin (Zinc-dependent metalloproteinase) and anti-toxin (transcriptional regulator) are transcribed to mRNA together. Proteases are activated under stress condition, cleaving anti-toxin, which increases the levels of Toxin free, inducing diverse biological functions in bacteria. Predicted promoter at site −35 and −10 binding by RNA polymerase sigma factor rpoD. ATG A: nucleotides share between final part of transcriptional factor and metalloproteinase codified by the BAB1_0270 ORF.

    Techniques Used: Molecular Weight, Binding Assay

    12) Product Images from "Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin"

    Article Title: Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082346

    Changes in the percentage of EpCAM-positive cells upon control medium (DMEM containing 10% FBS) with 4 mM BCAA stimulation or 100 nM rapamycin pretreatment and 4 mM BCAA stimulation (A) or liver cirrhosis modified medium (LC) containing 10% FBS stimulation (B) for 72 h in Huh7 by using the target activation protocol of array scan. The rate change of EpCAM-positive cells in 5000 cells with overexpression of caRheb or control plasmid cDNA (pc DNA) in control medium (DMEM containing 10% FBS) with and without 4 mM BCAA stimulation for 24 h in Huh7 (C). The detection of P70S6 kinase phosphorylation, a member of downstream mTOR signaling, in the presence of DMEM, BCAA treatment, pretreatment with rapamycin and BCAA treatment, or LC stimulation for 72 h in Huh7 (A,B). Tukey’s test **p
    Figure Legend Snippet: Changes in the percentage of EpCAM-positive cells upon control medium (DMEM containing 10% FBS) with 4 mM BCAA stimulation or 100 nM rapamycin pretreatment and 4 mM BCAA stimulation (A) or liver cirrhosis modified medium (LC) containing 10% FBS stimulation (B) for 72 h in Huh7 by using the target activation protocol of array scan. The rate change of EpCAM-positive cells in 5000 cells with overexpression of caRheb or control plasmid cDNA (pc DNA) in control medium (DMEM containing 10% FBS) with and without 4 mM BCAA stimulation for 24 h in Huh7 (C). The detection of P70S6 kinase phosphorylation, a member of downstream mTOR signaling, in the presence of DMEM, BCAA treatment, pretreatment with rapamycin and BCAA treatment, or LC stimulation for 72 h in Huh7 (A,B). Tukey’s test **p

    Techniques Used: Modification, Activation Assay, Over Expression, Plasmid Preparation

    Protein expression and phosphorylation in Huh7 cells under Knockdown conditions for 5 days and overexpression conditions for 1 day, and detection of phosphorylation after 4 mM BCAA treatment for 30 min. Rictor or Raptor Knockdown compared to negative control (NC), caRheb compared to control plasmid cDNA (pc DNA), BCAA treatment compared to DMEM (FBS 10%) only (Ctrl) (A). The average tumor volumes and tumorigenesis ratio at the 4 th week in a xenograft model with transplanted cells with negative control, knockdown of Raptor, Rictor for 5 days, or overexpression of control plasmid DNA, caRheb for 1 day (C), and tumorigenesis rate (B). Dunnett's test, *p
    Figure Legend Snippet: Protein expression and phosphorylation in Huh7 cells under Knockdown conditions for 5 days and overexpression conditions for 1 day, and detection of phosphorylation after 4 mM BCAA treatment for 30 min. Rictor or Raptor Knockdown compared to negative control (NC), caRheb compared to control plasmid cDNA (pc DNA), BCAA treatment compared to DMEM (FBS 10%) only (Ctrl) (A). The average tumor volumes and tumorigenesis ratio at the 4 th week in a xenograft model with transplanted cells with negative control, knockdown of Raptor, Rictor for 5 days, or overexpression of control plasmid DNA, caRheb for 1 day (C), and tumorigenesis rate (B). Dunnett's test, *p

    Techniques Used: Expressing, Over Expression, Negative Control, Plasmid Preparation

    13) Product Images from "Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells"

    Article Title: Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells

    Journal: Pflugers Archiv

    doi: 10.1007/s00424-015-1758-5

    Detection of Ano1 in pancreas and pancreatic islets. a RT-PCR of cDNA prepared from mRNA extracted from rat and human tissues. Transcripts of the expected size for Ano1 are observed (rat: 223 bp; human 314 bp). The 300-bp band is shown in the molecular weight marker column (MWM). Positive control: kidney. Negative control (Neg. control): no DNA. The sequencing of PCR products confirmed 100 % identity with the reference sequence for rat Ano1 cDNA complementary of rat Ano1 mRNA. b Western blot of Ano1 in rat islets, from left to right : molecular weight column (MW) showing the 100-, 150-, and 250-kDa bands, 80 μg rat islet lysate, 30 μg rat islet lysate, and 30 μg human thyroid lysate (positive control). Ano1 is detected at 119 kDa. c Immunofluorescence staining of pancreas section . c1 Immunohistochemical labeling (green-fluorescent Tyramide Alexa 488) of Ano1 in a section photomicrograph of rat pancreas. Most of the islet cells and acinar cells (at the level of apical pole) are labeled. c2 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c1. c3 Specificity control: immunohistochemical labeling of Ano1 in a section photomicrograph of rat pancreas. The primary goat Ano1 antibodies (sc-69343) were coincubated in the presence of Ano1 synthetic peptide (ab97423) in a ratio 1:8. The labeling disappears. c4 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c3. Arrows show islets. Scale bar is 50 μm
    Figure Legend Snippet: Detection of Ano1 in pancreas and pancreatic islets. a RT-PCR of cDNA prepared from mRNA extracted from rat and human tissues. Transcripts of the expected size for Ano1 are observed (rat: 223 bp; human 314 bp). The 300-bp band is shown in the molecular weight marker column (MWM). Positive control: kidney. Negative control (Neg. control): no DNA. The sequencing of PCR products confirmed 100 % identity with the reference sequence for rat Ano1 cDNA complementary of rat Ano1 mRNA. b Western blot of Ano1 in rat islets, from left to right : molecular weight column (MW) showing the 100-, 150-, and 250-kDa bands, 80 μg rat islet lysate, 30 μg rat islet lysate, and 30 μg human thyroid lysate (positive control). Ano1 is detected at 119 kDa. c Immunofluorescence staining of pancreas section . c1 Immunohistochemical labeling (green-fluorescent Tyramide Alexa 488) of Ano1 in a section photomicrograph of rat pancreas. Most of the islet cells and acinar cells (at the level of apical pole) are labeled. c2 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c1. c3 Specificity control: immunohistochemical labeling of Ano1 in a section photomicrograph of rat pancreas. The primary goat Ano1 antibodies (sc-69343) were coincubated in the presence of Ano1 synthetic peptide (ab97423) in a ratio 1:8. The labeling disappears. c4 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c3. Arrows show islets. Scale bar is 50 μm

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Positive Control, Negative Control, Sequencing, Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Immunohistochemistry, Labeling

    14) Product Images from "Molecular characteristics of recurrent triple-negative breast cancer"

    Article Title: Molecular characteristics of recurrent triple-negative breast cancer

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2015.4360

    Reverse transcription-quantitative polymerase chain reaction for validation of the gene expression profiles. Subsequent to extracting total RNA from the tumors, complementary DNA was created using a First Strand cDNA Synthesis kit. Gene expression assays were then used to validate the differential mRNA expression levels of various identified genes, including (A) MMP9 and (B) SERPINE1 in the recurrent and non-recurrent triple-negative breast cancer samples. MMP9, matrix metalloproteinase 9.
    Figure Legend Snippet: Reverse transcription-quantitative polymerase chain reaction for validation of the gene expression profiles. Subsequent to extracting total RNA from the tumors, complementary DNA was created using a First Strand cDNA Synthesis kit. Gene expression assays were then used to validate the differential mRNA expression levels of various identified genes, including (A) MMP9 and (B) SERPINE1 in the recurrent and non-recurrent triple-negative breast cancer samples. MMP9, matrix metalloproteinase 9.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    15) Product Images from "The Multicopper Ferroxidase Hephaestin Enhances Intestinal Iron Absorption in Mice"

    Article Title: The Multicopper Ferroxidase Hephaestin Enhances Intestinal Iron Absorption in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098792

    Verification of whole body hephaestin knockout. A. DNA extracted from a panel of homogenized tissues from Heph -/y and WT littermates was amplified by PCR. Top panel, results for the “ Heph knockout” reaction, which yields a strong band at 230 bp if a Heph knockout allele is present. Bottom panel, results from the “ Heph wild-type” reaction, which yields a strong band at 240 bp if wild-type Heph DNA is present and a band at 300 bp if Heph floxed DNA is present. Tissues from two WT and four Heph -/y mice were tested. B. Heph mRNA expression in proximal enterocytes was analyzed by RT-qPCR of cDNA from Heph -/y and WT littermates, and sla mice, at 6-7 weeks of age (N = 7–12 mice per group). The mean ± SD for results from primers targeting an unmodified region downstream of the knockout site are shown at left, and the mean ± SD from primers targeting the knockout region (exon 4-exon 5 junction) are shown at right. Heph mRNA expression results were normalized to the expression of the Hprt housekeeping gene. For each primer set, groups that share at least one letter are not significantly different. C. HEPH protein expression in enterocytes from Heph -/y and WT littermates (N = 6 per group) was examined by SDS-PAGE followed by immunoblotting with the HEPH D4 antibody. Actin expression in the same samples is shown below.
    Figure Legend Snippet: Verification of whole body hephaestin knockout. A. DNA extracted from a panel of homogenized tissues from Heph -/y and WT littermates was amplified by PCR. Top panel, results for the “ Heph knockout” reaction, which yields a strong band at 230 bp if a Heph knockout allele is present. Bottom panel, results from the “ Heph wild-type” reaction, which yields a strong band at 240 bp if wild-type Heph DNA is present and a band at 300 bp if Heph floxed DNA is present. Tissues from two WT and four Heph -/y mice were tested. B. Heph mRNA expression in proximal enterocytes was analyzed by RT-qPCR of cDNA from Heph -/y and WT littermates, and sla mice, at 6-7 weeks of age (N = 7–12 mice per group). The mean ± SD for results from primers targeting an unmodified region downstream of the knockout site are shown at left, and the mean ± SD from primers targeting the knockout region (exon 4-exon 5 junction) are shown at right. Heph mRNA expression results were normalized to the expression of the Hprt housekeeping gene. For each primer set, groups that share at least one letter are not significantly different. C. HEPH protein expression in enterocytes from Heph -/y and WT littermates (N = 6 per group) was examined by SDS-PAGE followed by immunoblotting with the HEPH D4 antibody. Actin expression in the same samples is shown below.

    Techniques Used: Knock-Out, Amplification, Polymerase Chain Reaction, Mouse Assay, Expressing, Quantitative RT-PCR, SDS Page

    16) Product Images from "The erbB2 gene is required for the development of terminally differentiated spinal cord oligodendrocytes"

    Article Title: The erbB2 gene is required for the development of terminally differentiated spinal cord oligodendrocytes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200104025

    Targeted disruption of the murine erbB2 gene. (A) Schematic representation of the 1,260 amino acid erbB2/neu protein (top) and the 13-kb genomic fragment (bottom) used to generate the targeting construct. The shaded 2.8-kb region represents the genomic fragment containing the exon encoding the essential transmembrane domain of the erbB2 receptor protein, as well as flanking sequences encoding parts of the extracellular and cytoplasmic domains, replaced by the neo r cassette. (B) Southern blot analysis of HindIII-restricted genomic DNA probed with a cDNA fragment found outside the targeting construct. (C) Western blot analysis of 10.5 dpc embryos homogenized in SDS-PAGE buffer, resolved by 5% SDS-PAGE, and probed for erbB2.
    Figure Legend Snippet: Targeted disruption of the murine erbB2 gene. (A) Schematic representation of the 1,260 amino acid erbB2/neu protein (top) and the 13-kb genomic fragment (bottom) used to generate the targeting construct. The shaded 2.8-kb region represents the genomic fragment containing the exon encoding the essential transmembrane domain of the erbB2 receptor protein, as well as flanking sequences encoding parts of the extracellular and cytoplasmic domains, replaced by the neo r cassette. (B) Southern blot analysis of HindIII-restricted genomic DNA probed with a cDNA fragment found outside the targeting construct. (C) Western blot analysis of 10.5 dpc embryos homogenized in SDS-PAGE buffer, resolved by 5% SDS-PAGE, and probed for erbB2.

    Techniques Used: Construct, Southern Blot, Western Blot, SDS Page

    17) Product Images from "EMMPRIN Inhibits bFGF-Induced IL-6 Secretion in an Osteoblastic Cell Line, MC3T3-E1"

    Article Title: EMMPRIN Inhibits bFGF-Induced IL-6 Secretion in an Osteoblastic Cell Line, MC3T3-E1

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.20387

    a) MC3T3-E1 cells were cultured with rh bFGF, rh EMMPRIN or a combination of both for 1 h. The medium was exchanged after 1h of stimulation, and the cells were incubated for 24 h. IL-6 concentrations in the culture supernatants were measured with IL-6 ELISA. b) MC3T3-E1 cells were cultured as in a) for 20 min. The medium was exchanged after 20 min of stimulation, and cells were incubated for 3 h. Total RNA was purified, following which, complementary DNA was generated and subjected to real-time PCR. The mean of at least four different experiments are shown. *p
    Figure Legend Snippet: a) MC3T3-E1 cells were cultured with rh bFGF, rh EMMPRIN or a combination of both for 1 h. The medium was exchanged after 1h of stimulation, and the cells were incubated for 24 h. IL-6 concentrations in the culture supernatants were measured with IL-6 ELISA. b) MC3T3-E1 cells were cultured as in a) for 20 min. The medium was exchanged after 20 min of stimulation, and cells were incubated for 3 h. Total RNA was purified, following which, complementary DNA was generated and subjected to real-time PCR. The mean of at least four different experiments are shown. *p

    Techniques Used: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Purification, Generated, Real-time Polymerase Chain Reaction

    18) Product Images from "MicroRNA 200c-3p regulates autophagy via upregulation of endoplasmic reticulum stress in PC-3 cells"

    Article Title: MicroRNA 200c-3p regulates autophagy via upregulation of endoplasmic reticulum stress in PC-3 cells

    Journal: Cancer Cell International

    doi: 10.1186/s12935-017-0500-0

    miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary DNA was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p
    Figure Legend Snippet: miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary DNA was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p

    Techniques Used: Generated, Quantitative RT-PCR, Expressing, Transfection, Incubation, MTT Assay

    19) Product Images from "Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication"

    Article Title: Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-646

    Summary of RNAi depletion experiments. (A) , (B) Semi-quantitive RT-PCR validation of the effects of RNAi knockdown on the Dnmt1 (A) and Dnmt2 (B) , indicating obvious decrease of expression level of Dnmt1 (A) and Dnmt2 (B) . Lane 1 indicate amplification using cDNA from Dnmt1 RNAi eggs (A) and Dnmt2 RNAi eggs (B) , respectively; Lane 2 indicate amplification using cDNA from Non-specific RNAi control (by embryonic microinjection of gfp dsRNA) eggs. gDNA, PCR using genomic DNA as template to control DNA contamination; M, DNA marker DL2000 (TakaRa, Japan). Actin3 is used as the internal control for Semi-quantitive RT-PCR. (C) Hatching rate of the treated eggs with Dnmt1 RNAi and Dnmt2 RNAi, indicating that RNAi knockdown of Dnmt1 significantly reduces hatched eggs compared to control, but not for Dnmt2 . * significant differences as determined by chi-squared test (p
    Figure Legend Snippet: Summary of RNAi depletion experiments. (A) , (B) Semi-quantitive RT-PCR validation of the effects of RNAi knockdown on the Dnmt1 (A) and Dnmt2 (B) , indicating obvious decrease of expression level of Dnmt1 (A) and Dnmt2 (B) . Lane 1 indicate amplification using cDNA from Dnmt1 RNAi eggs (A) and Dnmt2 RNAi eggs (B) , respectively; Lane 2 indicate amplification using cDNA from Non-specific RNAi control (by embryonic microinjection of gfp dsRNA) eggs. gDNA, PCR using genomic DNA as template to control DNA contamination; M, DNA marker DL2000 (TakaRa, Japan). Actin3 is used as the internal control for Semi-quantitive RT-PCR. (C) Hatching rate of the treated eggs with Dnmt1 RNAi and Dnmt2 RNAi, indicating that RNAi knockdown of Dnmt1 significantly reduces hatched eggs compared to control, but not for Dnmt2 . * significant differences as determined by chi-squared test (p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Polymerase Chain Reaction, Marker

    20) Product Images from "A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri."

    Article Title: A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri.

    Journal: Horticulture Research

    doi: 10.1038/hortres.2016.22

    RACE PCR to attempt the amplification of FmFLS2-2 . ( a ) Using ‘Nagami’ kumquat cDNA as template a forward primer (RACE3ʹ-f, Supplementary Table 1 ) was used in combination with the universal reverse primer (GeneRacer 3ʹ, Supplementary Table 1 ), which hybridizes to the poly-A tail at the 3ʹ end of the messenger RNA. ( b ) Schematic representation of the theoretical target versus the PCR product obtained. Top: based on the genomic map reported for C. sinensis and C. clementina ( http://citrus.hzau.edu.cn/orange/ and Phytozome.org) the expected 1-kb-long amplicon would be composed of the 3ʹ protein-coding region of FLS2-2 and its corresponding 3′ UTR. Bottom: the actual amplicon, as revealed by sequencing, was a combination of the protein-coding region of FLS2-1 and the 3′ UTR of FLS2-2. cDNA, complementary DNA; UTR, untranslated region.
    Figure Legend Snippet: RACE PCR to attempt the amplification of FmFLS2-2 . ( a ) Using ‘Nagami’ kumquat cDNA as template a forward primer (RACE3ʹ-f, Supplementary Table 1 ) was used in combination with the universal reverse primer (GeneRacer 3ʹ, Supplementary Table 1 ), which hybridizes to the poly-A tail at the 3ʹ end of the messenger RNA. ( b ) Schematic representation of the theoretical target versus the PCR product obtained. Top: based on the genomic map reported for C. sinensis and C. clementina ( http://citrus.hzau.edu.cn/orange/ and Phytozome.org) the expected 1-kb-long amplicon would be composed of the 3ʹ protein-coding region of FLS2-2 and its corresponding 3′ UTR. Bottom: the actual amplicon, as revealed by sequencing, was a combination of the protein-coding region of FLS2-1 and the 3′ UTR of FLS2-2. cDNA, complementary DNA; UTR, untranslated region.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    21) Product Images from "ATP6V0A2 mutations present in two Mexican Mestizo children with an autosomal recessive cutis laxa syndrome type IIA"

    Article Title: ATP6V0A2 mutations present in two Mexican Mestizo children with an autosomal recessive cutis laxa syndrome type IIA

    Journal: Molecular Genetics and Metabolism Reports

    doi: 10.1016/j.ymgmr.2014.04.003

    Sanger sequencing chromatograms showing mutations. A. Patient 1 showing the homozygous mutation in genomic DNA (c.187C > T) and his parents showing it in a heterozygous fashion. B. Patient 2 showing exon 18 skipping in cDNA. C. Patient 2 showing a homozygous mutation in genomic DNA (c.2293C > T) and his parents showing it in a heterozygous fashion. The control sequence is from a healthy individual.
    Figure Legend Snippet: Sanger sequencing chromatograms showing mutations. A. Patient 1 showing the homozygous mutation in genomic DNA (c.187C > T) and his parents showing it in a heterozygous fashion. B. Patient 2 showing exon 18 skipping in cDNA. C. Patient 2 showing a homozygous mutation in genomic DNA (c.2293C > T) and his parents showing it in a heterozygous fashion. The control sequence is from a healthy individual.

    Techniques Used: Sequencing, Mutagenesis

    22) Product Images from "Tough decoy targeting of predominant let-7 miRNA species in adult human hematopoietic cells"

    Article Title: Tough decoy targeting of predominant let-7 miRNA species in adult human hematopoietic cells

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-017-1273-x

    Let - 7a and let - 7b are predominant miRNAs among the let - 7 family members in peripheral blood cells. Levels of mature let - 7 miRNAs in a monocytes (n = 4), b lymphocytes (n = 4), c neutrophils (n = 4) and d reticulocytes (n = 5). Samples were analyzed by RT-qPCR quantitation of copy number per nanogram of complementary DNA (cDNA) (copies/ng cDNA). Mean value ± SD of independent donors for each condition
    Figure Legend Snippet: Let - 7a and let - 7b are predominant miRNAs among the let - 7 family members in peripheral blood cells. Levels of mature let - 7 miRNAs in a monocytes (n = 4), b lymphocytes (n = 4), c neutrophils (n = 4) and d reticulocytes (n = 5). Samples were analyzed by RT-qPCR quantitation of copy number per nanogram of complementary DNA (cDNA) (copies/ng cDNA). Mean value ± SD of independent donors for each condition

    Techniques Used: Quantitative RT-PCR, Quantitation Assay

    23) Product Images from "Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses"

    Article Title: Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1500671

    Mutated peptide vaccination induced T cell responses against intrinsic mutations in A2.DR1 dtg sarcoma and was superior to wt vaccination in controlling in vivo tumor growth (A) Sequencing histogram analysis of sarcoma cell line 39. Chromosomal DNA as well as cDNA of line 39 were used as templates for amplification with gene specific primers ( Kras and Tp53 ). Amplified PCR products were analyzed by Sanger sequencing to detect mutations in Kras and Tp53 genes and mRNAs. (B) Amino acid sequences of the mutated peptides carrying intrinsic mutations used for vaccination. (C) Recall responses against mutated and wt peptides sequences tested in A2.DR1 dtg mice immunized with p53 R176F (mur) and Kras G12C mutated peptides (vaccination regimen: peptides in PBS-based formulations including 50 μg CpG ODN 1668 as an adjuvant, twice on a bi-weekly basis). In vitro recall responses were obtained from two-color cytokine secretion assays (IL-2, IFN-γ) with pan-T cells. Percentages of IFN-γ/IL-2 double positive CD8 + and CD4 + T cells upon in vitro recall against single wt and mutated peptides presented by CD11c + DCs are displayed. Each peptide and control sample was tested in triplicates. Results are plotted as means of triplicate assays ± SEM. (D) Vaccination schedule for tumor challenge experiments. Depot: IFA-based formulation, Boost: boost vaccination containing CpG ODN 1668 and peptides in PBS (water-based formulation). (E) ( D ) with mutated (‘mut’ group: p53 C176F, Kras G12V) peptides. Mice were boosted only once with respective water-based peptide/CpG formulation during the challenge on day 14. n: number of biological replicates; error bars: mean ± SEM. Significances per unpaired two-tailed t-test are shown. (F) tumor cells were subcutaneously administered in 100 µl of Matrigel on the right flank of each animal at day 0. Two groups of 7 A2.DR1 dtg mice each (n = 7), have been vaccinated prior to the challenge according to the vaccination schedule shown in (D) with either mutated (‘mut’ group: p53 C176F, Kras G12V) or wt (‘wt’ group: p53 C176 wt, Kras G12 wt) peptides. Mice were boosted twice with respective water-based peptide/CpG formulations during the challenge. n: number of biological replicates; error bars, mean ± SEM. (G) Number of splenic T reg cells of differentially vaccinated, tumor bearing mice of the experiment shown in (E) compared to non-tumor bearing mice on the day of sacrifice. T reg cells were stained for in whole splenocyte suspensions with fluorescent-labeled mAbs as CD4 + CD3 + CD25 + Foxp3 + living cells in FACS. Significance per unpaired two-tailed t-test are shown. vac: vaccinated, mut: mutated.
    Figure Legend Snippet: Mutated peptide vaccination induced T cell responses against intrinsic mutations in A2.DR1 dtg sarcoma and was superior to wt vaccination in controlling in vivo tumor growth (A) Sequencing histogram analysis of sarcoma cell line 39. Chromosomal DNA as well as cDNA of line 39 were used as templates for amplification with gene specific primers ( Kras and Tp53 ). Amplified PCR products were analyzed by Sanger sequencing to detect mutations in Kras and Tp53 genes and mRNAs. (B) Amino acid sequences of the mutated peptides carrying intrinsic mutations used for vaccination. (C) Recall responses against mutated and wt peptides sequences tested in A2.DR1 dtg mice immunized with p53 R176F (mur) and Kras G12C mutated peptides (vaccination regimen: peptides in PBS-based formulations including 50 μg CpG ODN 1668 as an adjuvant, twice on a bi-weekly basis). In vitro recall responses were obtained from two-color cytokine secretion assays (IL-2, IFN-γ) with pan-T cells. Percentages of IFN-γ/IL-2 double positive CD8 + and CD4 + T cells upon in vitro recall against single wt and mutated peptides presented by CD11c + DCs are displayed. Each peptide and control sample was tested in triplicates. Results are plotted as means of triplicate assays ± SEM. (D) Vaccination schedule for tumor challenge experiments. Depot: IFA-based formulation, Boost: boost vaccination containing CpG ODN 1668 and peptides in PBS (water-based formulation). (E) ( D ) with mutated (‘mut’ group: p53 C176F, Kras G12V) peptides. Mice were boosted only once with respective water-based peptide/CpG formulation during the challenge on day 14. n: number of biological replicates; error bars: mean ± SEM. Significances per unpaired two-tailed t-test are shown. (F) tumor cells were subcutaneously administered in 100 µl of Matrigel on the right flank of each animal at day 0. Two groups of 7 A2.DR1 dtg mice each (n = 7), have been vaccinated prior to the challenge according to the vaccination schedule shown in (D) with either mutated (‘mut’ group: p53 C176F, Kras G12V) or wt (‘wt’ group: p53 C176 wt, Kras G12 wt) peptides. Mice were boosted twice with respective water-based peptide/CpG formulations during the challenge. n: number of biological replicates; error bars, mean ± SEM. (G) Number of splenic T reg cells of differentially vaccinated, tumor bearing mice of the experiment shown in (E) compared to non-tumor bearing mice on the day of sacrifice. T reg cells were stained for in whole splenocyte suspensions with fluorescent-labeled mAbs as CD4 + CD3 + CD25 + Foxp3 + living cells in FACS. Significance per unpaired two-tailed t-test are shown. vac: vaccinated, mut: mutated.

    Techniques Used: In Vivo, Sequencing, Amplification, Polymerase Chain Reaction, Mouse Assay, In Vitro, Immunofluorescence, Two Tailed Test, Staining, Labeling, FACS

    24) Product Images from "Expression of C-X-C chemokine receptor type 7 in otorhinolaryngologic neoplasms"

    Article Title: Expression of C-X-C chemokine receptor type 7 in otorhinolaryngologic neoplasms

    Journal: Singapore Medical Journal

    doi: 10.11622/smedj.2016057

    The standard curve for the CXCR7 set shows the cycle threshold (Ct) values against the genomic DNA copy number. Using serial dilutions of the standard cDNA, the standard curve ranged from 10 5 to 10 8 copies, generated on the basis of the linear relationship between the existing Ct and the logarithm of the copy number.
    Figure Legend Snippet: The standard curve for the CXCR7 set shows the cycle threshold (Ct) values against the genomic DNA copy number. Using serial dilutions of the standard cDNA, the standard curve ranged from 10 5 to 10 8 copies, generated on the basis of the linear relationship between the existing Ct and the logarithm of the copy number.

    Techniques Used: Generated

    25) Product Images from "Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress"

    Article Title: Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress

    Journal: Scientific Reports

    doi: 10.1038/srep26610

    Agarose gel electrophoretogram and gene structure of MiPDAT . ( A ) Agarose gel electrophoretogram of PCR products generated from the full-length cDNA and DNA cloning of MiPDAT . M: DL 2000 DNA standard marker; M1: DNA Marker IV; Lane 1: PCR products of 5′-RACE; Lane 2: PCR products of 3′-RACE; Lanes 3 and 4: PCR products of DNA cloning. ( B ) Schematic illustration of the gene structure of MiPDAT . The green boxes represent exons. A total of 12 introns with lengths of 361 bp, 136 bp, 215 bp, 239 bp, 266 bp, 384 bp, 176 bp, 199 bp, 211 bp, 246 bp, 227 bp and 209 bp are presented as the blue line. Red lines represent the un-translated region (UTR).
    Figure Legend Snippet: Agarose gel electrophoretogram and gene structure of MiPDAT . ( A ) Agarose gel electrophoretogram of PCR products generated from the full-length cDNA and DNA cloning of MiPDAT . M: DL 2000 DNA standard marker; M1: DNA Marker IV; Lane 1: PCR products of 5′-RACE; Lane 2: PCR products of 3′-RACE; Lanes 3 and 4: PCR products of DNA cloning. ( B ) Schematic illustration of the gene structure of MiPDAT . The green boxes represent exons. A total of 12 introns with lengths of 361 bp, 136 bp, 215 bp, 239 bp, 266 bp, 384 bp, 176 bp, 199 bp, 211 bp, 246 bp, 227 bp and 209 bp are presented as the blue line. Red lines represent the un-translated region (UTR).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Generated, Clone Assay, Marker

    26) Product Images from "T cells specific for post-translational modifications escape intrathymic tolerance induction"

    Article Title: T cells specific for post-translational modifications escape intrathymic tolerance induction

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02763-y

    The CII epitope is expressed in thymic stromal cells of mice and humans. a cDNA derived from thymi of MMC.Aire Suf ( n = 5) and MMC.Aire KO ( n = 6) was prepared and expression of CII ( Col2a1 ) was determined by quantitative RT-PCR. Aire-dependent ( Ins2 ) and Aire-independent ( Gad67 ) genes were used as controls. Data were normalized to the expression of cyclophilin A ( Ppia ) and calibrated with one MMC.Aire KO sample. Mean ± SEM is shown. b Qualitative expression analysis of the immunodominant T cell epitope CII 260–270 by RT-PCR on cDNA prepared from whole thymi of 3-week-old mice. As negative control, cDNA was prepared from spleen cells of wild-type mice. Ins2 and Gad67 were used as controls for genes expressed either in the thymus alone or in both thymus and spleen, respectively. Ppia was used as housekeeping gene, whereas no template samples were used as negative controls. c MMC thymus stained for DNA (Hoechst, in blue), keratin 5 (in green), and CII (mAb cocktail, in red). Scale bar in left panel, 100 μm; in right panel, 50 μm. d Human thymus sections from two different subjects stained with Hoechst (in blue), anti-AIRE (in green), and anti-CII (in red). Orange arrows indicate thymic epithelial cells positive for AIRE alone. White arrows indicate thymic epithelial cell positive for both AIRE and CII. Scale bar indicates 20 μm. ( e ) Stain of joint and thymus with Hoechst (blue) and anti-CII (mAb cocktail, red) or ( f ) Hoechst (blue) and anti-PTM CII (T8 mAb, red), from an MMC mouse. Scale bars indicate 100 μm for left panel of e and both panels of f , and 50 μm in right panel of e . p values were calculated using Mann–Whitney U test. ** p
    Figure Legend Snippet: The CII epitope is expressed in thymic stromal cells of mice and humans. a cDNA derived from thymi of MMC.Aire Suf ( n = 5) and MMC.Aire KO ( n = 6) was prepared and expression of CII ( Col2a1 ) was determined by quantitative RT-PCR. Aire-dependent ( Ins2 ) and Aire-independent ( Gad67 ) genes were used as controls. Data were normalized to the expression of cyclophilin A ( Ppia ) and calibrated with one MMC.Aire KO sample. Mean ± SEM is shown. b Qualitative expression analysis of the immunodominant T cell epitope CII 260–270 by RT-PCR on cDNA prepared from whole thymi of 3-week-old mice. As negative control, cDNA was prepared from spleen cells of wild-type mice. Ins2 and Gad67 were used as controls for genes expressed either in the thymus alone or in both thymus and spleen, respectively. Ppia was used as housekeeping gene, whereas no template samples were used as negative controls. c MMC thymus stained for DNA (Hoechst, in blue), keratin 5 (in green), and CII (mAb cocktail, in red). Scale bar in left panel, 100 μm; in right panel, 50 μm. d Human thymus sections from two different subjects stained with Hoechst (in blue), anti-AIRE (in green), and anti-CII (in red). Orange arrows indicate thymic epithelial cells positive for AIRE alone. White arrows indicate thymic epithelial cell positive for both AIRE and CII. Scale bar indicates 20 μm. ( e ) Stain of joint and thymus with Hoechst (blue) and anti-CII (mAb cocktail, red) or ( f ) Hoechst (blue) and anti-PTM CII (T8 mAb, red), from an MMC mouse. Scale bars indicate 100 μm for left panel of e and both panels of f , and 50 μm in right panel of e . p values were calculated using Mann–Whitney U test. ** p

    Techniques Used: Mouse Assay, Derivative Assay, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Negative Control, Staining, MANN-WHITNEY

    27) Product Images from "Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes"

    Article Title: Fragmentation of the large subunit ribosomal RNA gene in oyster mitochondrial genomes

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-485

    Amplification of mtDNA and cDNA LSU rRNA gene regions in Crassostrea virginica . Diagram A presents the amplicon locations in the C. virginica and A. irradians mtDNA genome. Gel image B contains a 233 bp region of the 5'-half LSU amplified from the C. virginica genomic DNA preparation (lane 2) and cDNA preparation (lane 3), as well as a 494 bp region of the 3'-half LSU amplified from the C. virginica genomic DNA preparation (lane 4) and cDNA preparation (lane 5); lane 1 = 100 bp ladder. Gel image C: a 748 bp product (starting in the 5' half and ending in the 3' half) amplified from a continuous LSU template in Argopecten irradians genomic DNA (lane 2) and cDNA (lane 3), but was not amplified from C. virginica genomic DNA (lane 4) or cDNA (lane 5).
    Figure Legend Snippet: Amplification of mtDNA and cDNA LSU rRNA gene regions in Crassostrea virginica . Diagram A presents the amplicon locations in the C. virginica and A. irradians mtDNA genome. Gel image B contains a 233 bp region of the 5'-half LSU amplified from the C. virginica genomic DNA preparation (lane 2) and cDNA preparation (lane 3), as well as a 494 bp region of the 3'-half LSU amplified from the C. virginica genomic DNA preparation (lane 4) and cDNA preparation (lane 5); lane 1 = 100 bp ladder. Gel image C: a 748 bp product (starting in the 5' half and ending in the 3' half) amplified from a continuous LSU template in Argopecten irradians genomic DNA (lane 2) and cDNA (lane 3), but was not amplified from C. virginica genomic DNA (lane 4) or cDNA (lane 5).

    Techniques Used: Amplification

    28) Product Images from "The Drosophila Retinoblastoma Binding Protein 6 Family Member Has Two Isoforms and Is Potentially Involved in Embryonic Patterning"

    Article Title: The Drosophila Retinoblastoma Binding Protein 6 Family Member Has Two Isoforms and Is Potentially Involved in Embryonic Patterning

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms160510242

    Analysis of the putative Snama promoter. ( A ) Schematic representations of the Snama genomic DNA starting at the putative transcriptional start site (TSS), the cDNA expected from rapid amplification of cDNA ends (RACE) and the domain with no name (DWNN) catalytic module. Arrows indicate primers used as specified in the Materials and Methods; E = exon, I = intron ( B ) 5' RACE products using Drosophila mRNA: (i) Amplification using combinations of GeneRacer 5' nested and GeneRacer 5' forward primers together with the RING finger (RF) tail and RF tail internal as reverse primers gives rise to a ~800-bp product (Lanes 2 and 4), and a smaller product of ~650 bp (indicated by an asterisk) was amplified when the GeneRacer 5' nested and GeneRacer 5' forward primers are combined with RF tail reverse primer (Lanes 1 and 3). A positive control showing the amplification of the RACE-ready β-actin cDNA, created from HeLa RNA, results in a ~900-bp fragment, as expected (Lanes 5 and 6). The difference in size between the β-actin cDNA amplification product with GR 5' nested primer (858 bp) and GR 5' primer (872 bp) is only 14 bp; (ii) Cloning and PCR amplification of the 5' RACE products: Lane 1 shows the smaller ~650-bp cloned fragment (asterisk), whilst Lanes 2 and 3 show the larger ~800-bp cloned fragment; ( C ) Sequence of the putative promoter region. The underlined nucleotide is the putative transcriptional start site; ( D ) (i) Schematic representation of fragments (P1–P3) of the promoter region showing base positions relative to the transcription start site (+1); (ii) Dual luciferase assay to determine the maximal promoter sequence. Measurement of luminescence activity of firefly and Renilla luciferase activity indicated that the maximal promoter sequence required to drive Snama transcription was Promoter 2; ( E ) Mobility shift assay of biotin-labelled and unlabeled DNA Snama promoter DNA. Lanes 1–3 indicate the Epstein–Barr nuclear antigen (EBNA) control system, biotin-EBNA control DNA, biotin-EBNA control DNA and EBNA extract and biotin-EBNA control DNA and EBNA extracted with excess unlabeled EBNA DNA, respectively. The biotin-EBNA control DNA shows no shift; the biotin-EBNA control DNA and EBNA extract shows a shift; and the biotin-EBNA control DNA and EBNA extract and excess unlabeled EBNA DNA show minimal shift due to competition. Lanes 4–6 show the unlabeled DNA fragments P1, P2 and P3. Lanes 7–9 represent labelled promoters P1, P2 and P3 and a mobility shift due to the binding of nuclear proteins to the promoters; ( F ) Electromobility shift assay (EMSA) of biotin-labelled protein-DNA complexes separated from crude extracts by streptavidin affinity chromatography. Promoter 0 represents the −231 region. (i) Increasing concentrations of labelled Promoter 2 DNA were used to elute binding proteins from embryonic nuclear extracts; (ii) Four micrograms of promoter DNA as indicated were used in order to select the best promoter to use in (i). Arrows indicate the bound nuclear proteins that were selected for identification by mass spectrometry; ( G ) Schematic representation of the (−673; +49) sequence shows the predicted transcription factor binding sites.
    Figure Legend Snippet: Analysis of the putative Snama promoter. ( A ) Schematic representations of the Snama genomic DNA starting at the putative transcriptional start site (TSS), the cDNA expected from rapid amplification of cDNA ends (RACE) and the domain with no name (DWNN) catalytic module. Arrows indicate primers used as specified in the Materials and Methods; E = exon, I = intron ( B ) 5' RACE products using Drosophila mRNA: (i) Amplification using combinations of GeneRacer 5' nested and GeneRacer 5' forward primers together with the RING finger (RF) tail and RF tail internal as reverse primers gives rise to a ~800-bp product (Lanes 2 and 4), and a smaller product of ~650 bp (indicated by an asterisk) was amplified when the GeneRacer 5' nested and GeneRacer 5' forward primers are combined with RF tail reverse primer (Lanes 1 and 3). A positive control showing the amplification of the RACE-ready β-actin cDNA, created from HeLa RNA, results in a ~900-bp fragment, as expected (Lanes 5 and 6). The difference in size between the β-actin cDNA amplification product with GR 5' nested primer (858 bp) and GR 5' primer (872 bp) is only 14 bp; (ii) Cloning and PCR amplification of the 5' RACE products: Lane 1 shows the smaller ~650-bp cloned fragment (asterisk), whilst Lanes 2 and 3 show the larger ~800-bp cloned fragment; ( C ) Sequence of the putative promoter region. The underlined nucleotide is the putative transcriptional start site; ( D ) (i) Schematic representation of fragments (P1–P3) of the promoter region showing base positions relative to the transcription start site (+1); (ii) Dual luciferase assay to determine the maximal promoter sequence. Measurement of luminescence activity of firefly and Renilla luciferase activity indicated that the maximal promoter sequence required to drive Snama transcription was Promoter 2; ( E ) Mobility shift assay of biotin-labelled and unlabeled DNA Snama promoter DNA. Lanes 1–3 indicate the Epstein–Barr nuclear antigen (EBNA) control system, biotin-EBNA control DNA, biotin-EBNA control DNA and EBNA extract and biotin-EBNA control DNA and EBNA extracted with excess unlabeled EBNA DNA, respectively. The biotin-EBNA control DNA shows no shift; the biotin-EBNA control DNA and EBNA extract shows a shift; and the biotin-EBNA control DNA and EBNA extract and excess unlabeled EBNA DNA show minimal shift due to competition. Lanes 4–6 show the unlabeled DNA fragments P1, P2 and P3. Lanes 7–9 represent labelled promoters P1, P2 and P3 and a mobility shift due to the binding of nuclear proteins to the promoters; ( F ) Electromobility shift assay (EMSA) of biotin-labelled protein-DNA complexes separated from crude extracts by streptavidin affinity chromatography. Promoter 0 represents the −231 region. (i) Increasing concentrations of labelled Promoter 2 DNA were used to elute binding proteins from embryonic nuclear extracts; (ii) Four micrograms of promoter DNA as indicated were used in order to select the best promoter to use in (i). Arrows indicate the bound nuclear proteins that were selected for identification by mass spectrometry; ( G ) Schematic representation of the (−673; +49) sequence shows the predicted transcription factor binding sites.

    Techniques Used: Rapid Amplification of cDNA Ends, Amplification, Positive Control, Clone Assay, Polymerase Chain Reaction, Sequencing, Luciferase, Activity Assay, Mobility Shift, Binding Assay, Electro Mobility Shift Assay, Affinity Chromatography, Mass Spectrometry

    29) Product Images from "CircRNA Expression Profiles and the Potential Role of CircZFP644 in Mice With Severe Acute Pancreatitis via Sponging miR-21-3p"

    Article Title: CircRNA Expression Profiles and the Potential Role of CircZFP644 in Mice With Severe Acute Pancreatitis via Sponging miR-21-3p

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2020.00206

    Characterization and expression analysis of CircZFP644. (A) Schematic diagram of CircZFP644 formed by back-splicing from the mouse ZFP644 gene at chromosome 5. (B) Schematic view illustrating the design of primers for CircZFP644 used in qRT-PCR. (C) The junction sequences of CircZFP644 were validated by Sanger sequencing. (D) Divergent primers detected CircZFP644 in complementary DNA (cDNA), but not in genomic DNA (gDNA). (E) RNA from mouse cardiac tissue was incubated with RNase R or buffer only (Mock). After digestion, the RNAs were purified. The levels of CircZFP644 and GAPDH mRNA analyzed by qRT-PCR after incubation with RNase R or buffer only (Mock). *** p
    Figure Legend Snippet: Characterization and expression analysis of CircZFP644. (A) Schematic diagram of CircZFP644 formed by back-splicing from the mouse ZFP644 gene at chromosome 5. (B) Schematic view illustrating the design of primers for CircZFP644 used in qRT-PCR. (C) The junction sequences of CircZFP644 were validated by Sanger sequencing. (D) Divergent primers detected CircZFP644 in complementary DNA (cDNA), but not in genomic DNA (gDNA). (E) RNA from mouse cardiac tissue was incubated with RNase R or buffer only (Mock). After digestion, the RNAs were purified. The levels of CircZFP644 and GAPDH mRNA analyzed by qRT-PCR after incubation with RNase R or buffer only (Mock). *** p

    Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Incubation, Purification

    30) Product Images from "Circular RNA has_circ_0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation"

    Article Title: Circular RNA has_circ_0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation

    Journal: Scientific Reports

    doi: 10.1038/srep35576

    Characterization of hsa_circ_0067934 in ESCC cells and tissue. ( A ) Two exons form hsa_circ_0067934 by back splicing from chromosomal region 3q26.2. ( B ) Divergent primers detect circular RNAs in cDNA but not genomic DNA (gDNA).
    Figure Legend Snippet: Characterization of hsa_circ_0067934 in ESCC cells and tissue. ( A ) Two exons form hsa_circ_0067934 by back splicing from chromosomal region 3q26.2. ( B ) Divergent primers detect circular RNAs in cDNA but not genomic DNA (gDNA).

    Techniques Used:

    31) Product Images from "Autologous cell lines from circulating colon cancer cells captured from sequential liquid biopsies as model to study therapy-driven tumor changes"

    Article Title: Autologous cell lines from circulating colon cancer cells captured from sequential liquid biopsies as model to study therapy-driven tumor changes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34365-z

    ) was analyzed in the nine colon CTC lines, HT-29 cells (from a primary colorectal tumor), and SW620 cells (from a lymph node metastasis of colon cancer). These transcripts covered different properties of tumor cells: epithelial ( ECAD, CK19, EPCAM ), mesenchymal ( NCAD, VIM, FN1 ), stemness ( CD133, ALDH1 ), angiogenesis ( VEGF ), proto-oncogene ( MET ), osteomimicry ( OPG, BMP7 ), EMT ( SNAIL, TWIST , WNT, DKK1 ), immune system ( IL33 , DEFA6 , CEACAM1 ), apoptosis ( CCND2, BCL11A, TNFRS1B ), DNA repair ( GAL, SMARCA1 ), cell growth ( BST2, SEMA6A ), cell interactions ( ADAMTS6, GJB6, GATA2, TGFB2 ) and energy metabolism ( ABCB1, PTGS2 ). B2M (housekeeping gene) and CD45 (leukocyte marker) were used as reference genes. Leukocyte cDNA was used as negative control. Results were normalized to the expression level of the reference B2M gene. Cell lines were clustered based on their expression profile.
    Figure Legend Snippet: ) was analyzed in the nine colon CTC lines, HT-29 cells (from a primary colorectal tumor), and SW620 cells (from a lymph node metastasis of colon cancer). These transcripts covered different properties of tumor cells: epithelial ( ECAD, CK19, EPCAM ), mesenchymal ( NCAD, VIM, FN1 ), stemness ( CD133, ALDH1 ), angiogenesis ( VEGF ), proto-oncogene ( MET ), osteomimicry ( OPG, BMP7 ), EMT ( SNAIL, TWIST , WNT, DKK1 ), immune system ( IL33 , DEFA6 , CEACAM1 ), apoptosis ( CCND2, BCL11A, TNFRS1B ), DNA repair ( GAL, SMARCA1 ), cell growth ( BST2, SEMA6A ), cell interactions ( ADAMTS6, GJB6, GATA2, TGFB2 ) and energy metabolism ( ABCB1, PTGS2 ). B2M (housekeeping gene) and CD45 (leukocyte marker) were used as reference genes. Leukocyte cDNA was used as negative control. Results were normalized to the expression level of the reference B2M gene. Cell lines were clustered based on their expression profile.

    Techniques Used: Marker, Negative Control, Expressing

    32) Product Images from "Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic"

    Article Title: Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic

    Journal: eNeuro

    doi: 10.1523/ENEURO.0221-16.2016

    Shu maps to the voltage-gated sodium channel gene paralytic. A , Mapping positions of the Shu mutation on the X chromosome. Red triangles and horizontal lines represent pairs of molecularly defined P-element insertions used to estimate the Shu mutation site; red X’s indicate the mutation sites deduced from the recombination rates between the corresponding P-element insertion and Shu . The estimated sites all reside within the para locus (CG9907). Boxes designate annotated genes near the para locus (based on FlyBase). B , DNA sequencing chromatogram identifying a G-to-A transition mutation (arrowheads) in the Shu genome at the position corresponding to the nucleotide 4249 in the para-RE cDNA (FlyBase). This mutation results in a methionine-to-isoleucine substitution at the amino acid position 1327. C , Schematic structural diagram of a Drosophila voltage-gated sodium channel. Arrow indicates the Shu mutation in the transmembrane segment S2 in homology domain III. The para GEFS+ mutation K1330T, which corresponds to a SCN1A mutation K1270T causing GEFS+ in humans ( Sun et al., 2012 ), lies three codons away from that of Shu . Also shown are the para DS mutation S1291R and the para bss1 mutation L1676F. D , Amino acid sequence alignment of Na v channels of different animal species. Note that the methionine residue, which is mutated to isoleucine in Shu , is present in all Na v channels.
    Figure Legend Snippet: Shu maps to the voltage-gated sodium channel gene paralytic. A , Mapping positions of the Shu mutation on the X chromosome. Red triangles and horizontal lines represent pairs of molecularly defined P-element insertions used to estimate the Shu mutation site; red X’s indicate the mutation sites deduced from the recombination rates between the corresponding P-element insertion and Shu . The estimated sites all reside within the para locus (CG9907). Boxes designate annotated genes near the para locus (based on FlyBase). B , DNA sequencing chromatogram identifying a G-to-A transition mutation (arrowheads) in the Shu genome at the position corresponding to the nucleotide 4249 in the para-RE cDNA (FlyBase). This mutation results in a methionine-to-isoleucine substitution at the amino acid position 1327. C , Schematic structural diagram of a Drosophila voltage-gated sodium channel. Arrow indicates the Shu mutation in the transmembrane segment S2 in homology domain III. The para GEFS+ mutation K1330T, which corresponds to a SCN1A mutation K1270T causing GEFS+ in humans ( Sun et al., 2012 ), lies three codons away from that of Shu . Also shown are the para DS mutation S1291R and the para bss1 mutation L1676F. D , Amino acid sequence alignment of Na v channels of different animal species. Note that the methionine residue, which is mutated to isoleucine in Shu , is present in all Na v channels.

    Techniques Used: Mutagenesis, DNA Sequencing, Sequencing

    33) Product Images from "The TPM3‐NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition), The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition"

    Article Title: The TPM3‐NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition), The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition

    Journal: Molecular Oncology

    doi: 10.1016/j.molonc.2014.06.001

    Genomic characterization of TPM3‐NTRK1 fusion in KM12 cell line A) End‐point PCR was performed on cDNA from KM12 cells with F/R primer couples designed to amplify regions in a) 1F/R: NTRK1 cytoplasmic domain, b) 2F/R: NTRK1 extracellular domain, c) 3F/R: TPM3‐NTRK1 rearrangement, d) 4F/R: TPM3 extracellular domain. See Supplementary Table S3 and Supplementary Figure S2 for primers sequence and position. B) Schematic representation of TPM3‐NTRK1 genomic DNA breakpoints in the KM12 cell line. The position of the breakpoints leading to the TPM3‐NTRK1 rearrangement is indicated with respect to genome reference hg19 version. Dark boxes: exons. Light boxes: introns. Striped box: alternative splicing exon in the TPM3 sequence.
    Figure Legend Snippet: Genomic characterization of TPM3‐NTRK1 fusion in KM12 cell line A) End‐point PCR was performed on cDNA from KM12 cells with F/R primer couples designed to amplify regions in a) 1F/R: NTRK1 cytoplasmic domain, b) 2F/R: NTRK1 extracellular domain, c) 3F/R: TPM3‐NTRK1 rearrangement, d) 4F/R: TPM3 extracellular domain. See Supplementary Table S3 and Supplementary Figure S2 for primers sequence and position. B) Schematic representation of TPM3‐NTRK1 genomic DNA breakpoints in the KM12 cell line. The position of the breakpoints leading to the TPM3‐NTRK1 rearrangement is indicated with respect to genome reference hg19 version. Dark boxes: exons. Light boxes: introns. Striped box: alternative splicing exon in the TPM3 sequence.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    34) Product Images from "Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy"

    Article Title: Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.16-19073

    Copy number of mtDNA and its transcription in the peripheral blood from diabetic subjects. ( a ) Copy number was quantified in the genomic DNA by qPCR using CYTB as mtDNA-encoded and β-ACTIN as a nuclear DNA-encoded gene. ( b ) Transcripts of mtDNA-encoded CYTB were quantified in the blood cDNA by qPCR using β-ACTIN as a housekeeping gene. The values are represented as mean ± SD obtained from five to six diabetic patients each with retinopathy (Diab-Ret) or without retinopathy (Diab-No Ret), and 7 to 10 nondiabetic subjects (Norm). * P
    Figure Legend Snippet: Copy number of mtDNA and its transcription in the peripheral blood from diabetic subjects. ( a ) Copy number was quantified in the genomic DNA by qPCR using CYTB as mtDNA-encoded and β-ACTIN as a nuclear DNA-encoded gene. ( b ) Transcripts of mtDNA-encoded CYTB were quantified in the blood cDNA by qPCR using β-ACTIN as a housekeeping gene. The values are represented as mean ± SD obtained from five to six diabetic patients each with retinopathy (Diab-Ret) or without retinopathy (Diab-No Ret), and 7 to 10 nondiabetic subjects (Norm). * P

    Techniques Used: Real-time Polymerase Chain Reaction

    35) Product Images from "Quantitative gene monitoring of microbial tetracycline resistance using magnetic luminescent nanoparticles"

    Article Title: Quantitative gene monitoring of microbial tetracycline resistance using magnetic luminescent nanoparticles

    Journal: Journal of Environmental Monitoring

    doi: 10.1039/c001974g

    Comparison between cDNA gene expression and gDNA quantification using MLNPs-DNA assay. cDNA was generated from RNAs extracted from pooled microcosm samples from weeks 0 through 4. gDNA copies depicted on the graph were obtained by combining and averaging from the gene copy numbers of each week’s sample. 16S rRNA gene copies represent total bacterial abundance. The dotted circle shows the impact of TCS and TCC on the increased antibiotic resistant gene abundance and expression in comparison to the control. The signal and error bars represent average and standard deviations based on triplicate reactions.
    Figure Legend Snippet: Comparison between cDNA gene expression and gDNA quantification using MLNPs-DNA assay. cDNA was generated from RNAs extracted from pooled microcosm samples from weeks 0 through 4. gDNA copies depicted on the graph were obtained by combining and averaging from the gene copy numbers of each week’s sample. 16S rRNA gene copies represent total bacterial abundance. The dotted circle shows the impact of TCS and TCC on the increased antibiotic resistant gene abundance and expression in comparison to the control. The signal and error bars represent average and standard deviations based on triplicate reactions.

    Techniques Used: Expressing, Generated

    36) Product Images from "Cyclooxygenase-2 Expression Is Related to the Epithelial-to-Mesenchymal Transition in Human Colon Cancers"

    Article Title: Cyclooxygenase-2 Expression Is Related to the Epithelial-to-Mesenchymal Transition in Human Colon Cancers

    Journal: Yonsei Medical Journal

    doi: 10.3349/ymj.2009.50.6.818

    Western blot analyses of E-cadherin, COX-2 and Snail in colon cancer cells, and morphology of HCT8 transfected with cDNA for COX-2 or Snail. (A and B) Endogenous expressions of E-cadherin, COX-2, and Snail in colon cancer cells, and ectopic expressions of COX-2 and Snail in HCT8 and SW620. Forty µg of protein was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The bottom represents GAPDH, which was used as a loading control. (C) Ectopic expression of COX-2 or Snail induced a scattered, flattened phenotype with few intercellular contacts in HCT8. COX-2, cyclooxygenase-2; SDS, sodium dodecyl sulfate; cDNA, complementary DNA; GAPDH, glyceraldehydes 3-phosphate dehydrogenase.
    Figure Legend Snippet: Western blot analyses of E-cadherin, COX-2 and Snail in colon cancer cells, and morphology of HCT8 transfected with cDNA for COX-2 or Snail. (A and B) Endogenous expressions of E-cadherin, COX-2, and Snail in colon cancer cells, and ectopic expressions of COX-2 and Snail in HCT8 and SW620. Forty µg of protein was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The bottom represents GAPDH, which was used as a loading control. (C) Ectopic expression of COX-2 or Snail induced a scattered, flattened phenotype with few intercellular contacts in HCT8. COX-2, cyclooxygenase-2; SDS, sodium dodecyl sulfate; cDNA, complementary DNA; GAPDH, glyceraldehydes 3-phosphate dehydrogenase.

    Techniques Used: Western Blot, Transfection, Polyacrylamide Gel Electrophoresis, Expressing

    37) Product Images from "TLR-4-Dependent and -Independent Mechanisms of Fetal Brain Injury in the Setting of Preterm Birth"

    Article Title: TLR-4-Dependent and -Independent Mechanisms of Fetal Brain Injury in the Setting of Preterm Birth

    Journal: Reproductive Sciences

    doi: 10.1177/1933719112438439

    Toll-like receptor 4 (TLR-4) messenger RNA (mRNA) expression at day 2 and day 7 in culture (cortical) as determined by polymerase chain reaction (PCR). Fetal brains harvested at embryonic day 15 (E15) (A) or E18 (B) have low levels of TLR-4 expression at both 2 and 7 days in culture. Intensity increases at higher cycle numbers (indicated on right). Lane M is base marker. Lanes C1 to C3 represent TLR-4 expression in cortical cultures harvested from fetuses exposed to intrauterine saline (controls). Lanes L1 to L3 represent TLR-4 mRNA expression in cortical cultures from fetuses exposed to intrauterine inflammation (lipopolysaccharide [LPS]). Positive controls for TLR-4 mRNA expression were E18 whole fetal brains (E18), postnatal brains from P21 (PND) and from pregnant mouse uterus, which is known to highly express TLR-4. The last lane represents the negative control with water instead of complementary DNA (cDNA).
    Figure Legend Snippet: Toll-like receptor 4 (TLR-4) messenger RNA (mRNA) expression at day 2 and day 7 in culture (cortical) as determined by polymerase chain reaction (PCR). Fetal brains harvested at embryonic day 15 (E15) (A) or E18 (B) have low levels of TLR-4 expression at both 2 and 7 days in culture. Intensity increases at higher cycle numbers (indicated on right). Lane M is base marker. Lanes C1 to C3 represent TLR-4 expression in cortical cultures harvested from fetuses exposed to intrauterine saline (controls). Lanes L1 to L3 represent TLR-4 mRNA expression in cortical cultures from fetuses exposed to intrauterine inflammation (lipopolysaccharide [LPS]). Positive controls for TLR-4 mRNA expression were E18 whole fetal brains (E18), postnatal brains from P21 (PND) and from pregnant mouse uterus, which is known to highly express TLR-4. The last lane represents the negative control with water instead of complementary DNA (cDNA).

    Techniques Used: Expressing, Polymerase Chain Reaction, Marker, Negative Control

    38) Product Images from "Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles"

    Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S42723

    Expression of MOMP at the protein and gene transcript levels. ( A ) Cos- 7 cells were transfected as described in Figure 7 , immunostained (positive MOMP fluorescence cells) and then mounted with DAPI (blue nuclei stain) combined with an anti-fade mounting solution. ( B ) Bright-field visualization of Cos-7 cell monolayer showing the MOMP expressed protein. Red circle (positive MOMP fluorescence cells) shows expression of the MOMP protein. ( C ) Confirmation of expressed MOMP protein by western blot. Cos-7 cells (4 × 10 5 cells/well) were transfected with DMCNP or phCMV1 vector using Lipofectamine and incubated at 37°C for 48 hours. Cell lysates were collected, run on an SDS-PAGE gel, transferred onto a PVDF membrane and probed using anti-MOMP polyclonal antibodies followed by an Alexa fluor 680 secondary antibody. The bound antibody was viewed using LI-COR Odyssey imaging apparatus. ( D ) In vitro expression of MOMP gene transcript. Notes: RNA samples were extracted from mouse thigh muscles and spleens reversed transcribed to cDNA and then subjected to RT-PCR amplification of the MOMP gene transcript using MOMP specific primers. Lane 1 (MW marker), lanes 2 and 7 (DMOMP positive clones), lane 3 (thigh muscle of DMCNP mice), lane 4 (thigh muscle of PBS mice), lane 5 (spleen from DMCNP mice), and lane 6 (spleen from PBS mice). Red rectangle shows positive MOMP gene transcripts. Abbreviations: DMCNP, DMOMP encapsulated in chitosan nanoparticles; DMOMP, DNA of the major outer membrane protein of C. trachomatis ; MOMP, major outer membrane protein of C. trachomatis ; PVDF, polyvinylidene difluoride.
    Figure Legend Snippet: Expression of MOMP at the protein and gene transcript levels. ( A ) Cos- 7 cells were transfected as described in Figure 7 , immunostained (positive MOMP fluorescence cells) and then mounted with DAPI (blue nuclei stain) combined with an anti-fade mounting solution. ( B ) Bright-field visualization of Cos-7 cell monolayer showing the MOMP expressed protein. Red circle (positive MOMP fluorescence cells) shows expression of the MOMP protein. ( C ) Confirmation of expressed MOMP protein by western blot. Cos-7 cells (4 × 10 5 cells/well) were transfected with DMCNP or phCMV1 vector using Lipofectamine and incubated at 37°C for 48 hours. Cell lysates were collected, run on an SDS-PAGE gel, transferred onto a PVDF membrane and probed using anti-MOMP polyclonal antibodies followed by an Alexa fluor 680 secondary antibody. The bound antibody was viewed using LI-COR Odyssey imaging apparatus. ( D ) In vitro expression of MOMP gene transcript. Notes: RNA samples were extracted from mouse thigh muscles and spleens reversed transcribed to cDNA and then subjected to RT-PCR amplification of the MOMP gene transcript using MOMP specific primers. Lane 1 (MW marker), lanes 2 and 7 (DMOMP positive clones), lane 3 (thigh muscle of DMCNP mice), lane 4 (thigh muscle of PBS mice), lane 5 (spleen from DMCNP mice), and lane 6 (spleen from PBS mice). Red rectangle shows positive MOMP gene transcripts. Abbreviations: DMCNP, DMOMP encapsulated in chitosan nanoparticles; DMOMP, DNA of the major outer membrane protein of C. trachomatis ; MOMP, major outer membrane protein of C. trachomatis ; PVDF, polyvinylidene difluoride.

    Techniques Used: Expressing, Transfection, Fluorescence, Staining, Western Blot, Plasmid Preparation, Incubation, SDS Page, Imaging, In Vitro, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Mouse Assay

    39) Product Images from "B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response"

    Article Title: B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response

    Journal: Science Advances

    doi: 10.1126/sciadv.aay2793

    Activated B cell–specific deletion of residual Sirt1 in Aicda cre Sirt1 fl/fl mice augments the class-switched and hypermutated antibody response. ( A and B ) Serum titers of total IgM, IgG1, and IgA (A) and high-affinity NP 4 -binding IgG1 (B) (ELISA; RU, relative units) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates immunized with NP 16 -CGG at days 0 and 21 at different time points, as indicated ( n = 7 mice in each group). Dotted lines link paired littermates. ( C and D ) AFCs secreting IgM and IgG1 or NP 4 -binding IgG1 (ELISPOTs) in the spleen and bone marrow in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates euthanized 28 days after the first NP 16 -CGG injection. Data are one representative of three independent experiments yielding similar results (C) or means ± SEM of three independent experiments (D). ( E ) Fluorescence microscopy analysis of IgA-producing cells in different gut tissues, as indicated, in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates Aicda cre Sirt1 fl/fl mice (one representative of three independent experiments yielding similar results). Scale bars, 50 μm. ( F ) Overall frequency (change/base) and distribution (pie charts) of point mutations in the V 186.2 region of V 186.2 DJ H -Cγ1 complementary DNA (cDNA; pooled data from two mouse pairs) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice injected (intraperitoneally) with NP 16 -CGG at days 0 and 21 and euthanized at day 28. Also depicted by histograms are frequencies of mutations in the framework (FR) and complementarity-determining (CDR) regions (right). P values were calculated by χ 2 test. ( G and H ) Analysis of class-switched IgM − IgD − IgG + B cells, NP 5 -binding IgG1 + B cells, and NP 5 -binding CD38 + IgG1 + memory B cells (flow cytometry) in spleen (G) and quantification of these cells in the NP 16 -CGG immunized mice (H). ( I ) Flow cytometry analysis of B220 low CD138 + plasmablasts/plasma cells in the spleen and bone marrow. ( J ) Quantification of proportion of B220 low CD138 + plasmablasts/plasma cells among total spleen and bone marrow cells. ( K ) Viable (7-AAD − ) B cells in the spleen (flow cytometry) of the immunized mice. ( L and M ) Flow cytometry analysis of proliferating (incorporating BrdU and BrdU + ) B cells (L) and proportions of T (CD3 + ) cells and B (B220 + ) cells (M). ( N ) Spleen germinal center (B220 + GL7 + CD95 + ) B cells in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice as analyzed by flow cytometry 10 days after NP-CGG injection. ( O ) Quantification of the proportion and of B220 + GL-7 + CD95 + germinal center B cells among total spleen B cells, as analyzed by FACS (left), and total numbers of B220 + GL-7 + CD95 + germinal center B cells in each spleen (right). ( P ) Germinal center structure in the spleen (fluorescence microscopy). Data in (G), (I), (K) to (N), and (P) are one representative of three independent experiments yielding similar results. Scale bar, 100 μm. (H), (J), and (O) are means ± SEM of three or four biological independent experiments. * P
    Figure Legend Snippet: Activated B cell–specific deletion of residual Sirt1 in Aicda cre Sirt1 fl/fl mice augments the class-switched and hypermutated antibody response. ( A and B ) Serum titers of total IgM, IgG1, and IgA (A) and high-affinity NP 4 -binding IgG1 (B) (ELISA; RU, relative units) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates immunized with NP 16 -CGG at days 0 and 21 at different time points, as indicated ( n = 7 mice in each group). Dotted lines link paired littermates. ( C and D ) AFCs secreting IgM and IgG1 or NP 4 -binding IgG1 (ELISPOTs) in the spleen and bone marrow in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates euthanized 28 days after the first NP 16 -CGG injection. Data are one representative of three independent experiments yielding similar results (C) or means ± SEM of three independent experiments (D). ( E ) Fluorescence microscopy analysis of IgA-producing cells in different gut tissues, as indicated, in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates Aicda cre Sirt1 fl/fl mice (one representative of three independent experiments yielding similar results). Scale bars, 50 μm. ( F ) Overall frequency (change/base) and distribution (pie charts) of point mutations in the V 186.2 region of V 186.2 DJ H -Cγ1 complementary DNA (cDNA; pooled data from two mouse pairs) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice injected (intraperitoneally) with NP 16 -CGG at days 0 and 21 and euthanized at day 28. Also depicted by histograms are frequencies of mutations in the framework (FR) and complementarity-determining (CDR) regions (right). P values were calculated by χ 2 test. ( G and H ) Analysis of class-switched IgM − IgD − IgG + B cells, NP 5 -binding IgG1 + B cells, and NP 5 -binding CD38 + IgG1 + memory B cells (flow cytometry) in spleen (G) and quantification of these cells in the NP 16 -CGG immunized mice (H). ( I ) Flow cytometry analysis of B220 low CD138 + plasmablasts/plasma cells in the spleen and bone marrow. ( J ) Quantification of proportion of B220 low CD138 + plasmablasts/plasma cells among total spleen and bone marrow cells. ( K ) Viable (7-AAD − ) B cells in the spleen (flow cytometry) of the immunized mice. ( L and M ) Flow cytometry analysis of proliferating (incorporating BrdU and BrdU + ) B cells (L) and proportions of T (CD3 + ) cells and B (B220 + ) cells (M). ( N ) Spleen germinal center (B220 + GL7 + CD95 + ) B cells in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice as analyzed by flow cytometry 10 days after NP-CGG injection. ( O ) Quantification of the proportion and of B220 + GL-7 + CD95 + germinal center B cells among total spleen B cells, as analyzed by FACS (left), and total numbers of B220 + GL-7 + CD95 + germinal center B cells in each spleen (right). ( P ) Germinal center structure in the spleen (fluorescence microscopy). Data in (G), (I), (K) to (N), and (P) are one representative of three independent experiments yielding similar results. Scale bar, 100 μm. (H), (J), and (O) are means ± SEM of three or four biological independent experiments. * P

    Techniques Used: Mouse Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Injection, Fluorescence, Microscopy, Flow Cytometry, FACS

    40) Product Images from "Persistence of Anti-Desmoglein 3 IgG+ B-Cell Clones in Pemphigus Patients Over Years"

    Article Title: Persistence of Anti-Desmoglein 3 IgG+ B-Cell Clones in Pemphigus Patients Over Years

    Journal: The Journal of investigative dermatology

    doi: 10.1038/jid.2014.291

    PCR-strategy to identify patient-specific V H -CDR3s found by APD at one time point but not at another (a) PCR-template with forward primer from either CDR1 or CDR2 (F1, F2) and reverse primer from CDR3 (R). (b) PV3 clone III, isolated by APD in 2006, was not found by APD in PV3a (2012), but was identified by PCR (lane T(PV3a)). (c) PV1 clone II was found by APD in 2002, but only by PCR at later times (lanes T1(PV1a) and T2(PV1b)). Deduced amino acid (aa)-sequences from sequencing of PCR products shown below the gels (retrieved); compared to that of the corresponding APD-isolated clone (expected). Presumed somatic mutations (or PCR errors) are in red, V H -CDR2/3 regions in bolded font, primer-covered regions are underlined. Std, 200 bp standard; nC1, negative control (water); nC2, negative control (V H -cDNA from an unrelated PV patient); pC1, positive control (monoclonal phagemid DNA of clone); pC2, positive control (polyclonal plasmid DNA from APD-library from which clone of interest was identified by panning).
    Figure Legend Snippet: PCR-strategy to identify patient-specific V H -CDR3s found by APD at one time point but not at another (a) PCR-template with forward primer from either CDR1 or CDR2 (F1, F2) and reverse primer from CDR3 (R). (b) PV3 clone III, isolated by APD in 2006, was not found by APD in PV3a (2012), but was identified by PCR (lane T(PV3a)). (c) PV1 clone II was found by APD in 2002, but only by PCR at later times (lanes T1(PV1a) and T2(PV1b)). Deduced amino acid (aa)-sequences from sequencing of PCR products shown below the gels (retrieved); compared to that of the corresponding APD-isolated clone (expected). Presumed somatic mutations (or PCR errors) are in red, V H -CDR2/3 regions in bolded font, primer-covered regions are underlined. Std, 200 bp standard; nC1, negative control (water); nC2, negative control (V H -cDNA from an unrelated PV patient); pC1, positive control (monoclonal phagemid DNA of clone); pC2, positive control (polyclonal plasmid DNA from APD-library from which clone of interest was identified by panning).

    Techniques Used: Polymerase Chain Reaction, Isolation, Sequencing, Negative Control, Positive Control, Plasmid Preparation

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    Thermo Fisher verso cdna kit
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    mRNA expression of N-extended AChE, AChE-S, and AChE-R isoforms following treatment of Y79 cells with glucose for 1 h. Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml of glucose) and then with 3.5 mg/ml glucose for different time intervals. Total RNA was extracted and cDNA was prepared for real-time PCR procedure as described under Materials and Methods. Results are presented as fold of control cells cultured in starvation media. Values are means ± SEM, ( N = 4). * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Acetylcholinesterase (AChE) is an important link in the apoptotic pathway induced by hyperglycemia in Y79 retinoblastoma cell line

    doi: 10.3389/fnmol.2012.00069

    Figure Lengend Snippet: mRNA expression of N-extended AChE, AChE-S, and AChE-R isoforms following treatment of Y79 cells with glucose for 1 h. Y79 cells were pre-treated for 16–24 h in starvation medium (1% FBS and 1 mg/ml of glucose) and then with 3.5 mg/ml glucose for different time intervals. Total RNA was extracted and cDNA was prepared for real-time PCR procedure as described under Materials and Methods. Results are presented as fold of control cells cultured in starvation media. Values are means ± SEM, ( N = 4). * p

    Article Snippet: RNA samples (0.2 μg) were used for cDNA synthesis prepared by the Verso™ cDNA Kit (Thermo SCIENTIFIC, UK).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture

    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

    Hoxc9 and Hoxc13 genome-wide binding profiles differ from other posterior Hox TFs. (A) ChIP-seq heatmap showing binding comparisons of Hoxc9 and Hoxc13 in differentiating MNs, at Day 3. Sites bound by both indicated Hox TFs noted as “c9 = c13” sites. Preferentially bound sites by Hoxc9 or Hoxc13 noted as “c9 > c13” or “c13 > c9“. (B-C) Principal Component Analysis (PCA) of the ChIP-seq datasets reveals similarities in the binding patterns of Hox TFs.

    Journal: bioRxiv

    Article Title: Hox binding specificity is directed by DNA sequence preferences and differential abilities to engage inaccessible chromatin

    doi: 10.1101/2019.12.29.890335

    Figure Lengend Snippet: Hoxc9 and Hoxc13 genome-wide binding profiles differ from other posterior Hox TFs. (A) ChIP-seq heatmap showing binding comparisons of Hoxc9 and Hoxc13 in differentiating MNs, at Day 3. Sites bound by both indicated Hox TFs noted as “c9 = c13” sites. Preferentially bound sites by Hoxc9 or Hoxc13 noted as “c9 > c13” or “c13 > c9“. (B-C) Principal Component Analysis (PCA) of the ChIP-seq datasets reveals similarities in the binding patterns of Hox TFs.

    Article Snippet: Hoxa9, Hoxd9, and Hoxc13 cDNA was amplified using Phusion polymerase (Thermo Scientific) from pCAGGS mHoxA9 (JD-114), pCAGGS mHoxD9 (JD-237) and hHoxc13 cDNA (Dharmacon, Accession: BC090850 ), respectively.

    Techniques: Genome Wide, Binding Assay, Chromatin Immunoprecipitation

    Hoxc9 and Hoxc13 have a higher preference for inaccessible chromatin than Hoxc10. (A) ChIP-seq heatmap of the top 10,000 Hoxc13 sites plotted within a 1kb window around the peak center. Selected enriched motifs discovered via MEME-ChIP indicated on the right. (B) ChIP-seq heatmap showing binding comparisons of Hoxc9, Hoxc10 and Hoxc13 in differentiating MNs, at Day 3. Sites bound by all three Hox TFs noted as “c9 = c10 = c13” sites. Preferentially bound sites by Hoxc9, Hoxc13, Hoxc9 and Hoxc10 or Hoxc9 and Hoxc13 noted as “c9 > c10, c13”, “c13 > c9, c10”, “c9, c10 > c13” and “c9, c13 > c10”, respectively. (C) The distribution of MN progenitor ATAC-seq read density at the top 10,000 Hoxc9, Hoxc10 and Hoxc13 sites at Day 3. Data is ordered based on normalized read density (tags per million per site) and divided into quartiles. The box displays the central 50% (quartile 2 and quartile 3) of the data, while the top and bottom whiskers represent the top 25% and bottom 25% (the top and the bottom quartiles) of the data, respectively. (D) ATAC-seq heatmap displaying the accessibility in MN progenitors (before Hox induction) at the indicated Hox binding categories. (E) Metagene plots of accessibility (ATAC-seq reads) in MN progenitors displaying the prior accessibility (before Hox induction) at the indicated binding categories. Normalized read density represents tags per million per 1000 sites.

    Journal: bioRxiv

    Article Title: Hox binding specificity is directed by DNA sequence preferences and differential abilities to engage inaccessible chromatin

    doi: 10.1101/2019.12.29.890335

    Figure Lengend Snippet: Hoxc9 and Hoxc13 have a higher preference for inaccessible chromatin than Hoxc10. (A) ChIP-seq heatmap of the top 10,000 Hoxc13 sites plotted within a 1kb window around the peak center. Selected enriched motifs discovered via MEME-ChIP indicated on the right. (B) ChIP-seq heatmap showing binding comparisons of Hoxc9, Hoxc10 and Hoxc13 in differentiating MNs, at Day 3. Sites bound by all three Hox TFs noted as “c9 = c10 = c13” sites. Preferentially bound sites by Hoxc9, Hoxc13, Hoxc9 and Hoxc10 or Hoxc9 and Hoxc13 noted as “c9 > c10, c13”, “c13 > c9, c10”, “c9, c10 > c13” and “c9, c13 > c10”, respectively. (C) The distribution of MN progenitor ATAC-seq read density at the top 10,000 Hoxc9, Hoxc10 and Hoxc13 sites at Day 3. Data is ordered based on normalized read density (tags per million per site) and divided into quartiles. The box displays the central 50% (quartile 2 and quartile 3) of the data, while the top and bottom whiskers represent the top 25% and bottom 25% (the top and the bottom quartiles) of the data, respectively. (D) ATAC-seq heatmap displaying the accessibility in MN progenitors (before Hox induction) at the indicated Hox binding categories. (E) Metagene plots of accessibility (ATAC-seq reads) in MN progenitors displaying the prior accessibility (before Hox induction) at the indicated binding categories. Normalized read density represents tags per million per 1000 sites.

    Article Snippet: Hoxa9, Hoxd9, and Hoxc13 cDNA was amplified using Phusion polymerase (Thermo Scientific) from pCAGGS mHoxA9 (JD-114), pCAGGS mHoxD9 (JD-237) and hHoxc13 cDNA (Dharmacon, Accession: BC090850 ), respectively.

    Techniques: Chromatin Immunoprecipitation, Binding Assay