microsatellite profiles  (Thermo Fisher)


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    Structured Review

    Thermo Fisher microsatellite profiles
    Estimated population structure for 33 Moroccan L. infantum strains as inferred by STRUCTURE software on the basis of data for 14 <t>microsatellite</t> markers. Each of the strains is represented by a single vertical line divided into K colors, where K is the number of populations assumed. Each color represents one population, and the length of the colors segment shows the strain’s estimated proportion of membership in that population.(A) the two main populations derived from the whole dataset which divided strains into MON-1 and non-MON-1 populations. (B) Sub-population analysis of Population 1 (MON-1 group) shows two sub-populations. K represents the true number of populations and sub-populations.
    Microsatellite Profiles, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 11531 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Moroccan Leishmania infantum: Genetic Diversity and Population Structure as Revealed by Multi-Locus Microsatellite Typing"

    Article Title: Moroccan Leishmania infantum: Genetic Diversity and Population Structure as Revealed by Multi-Locus Microsatellite Typing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077778

    Estimated population structure for 33 Moroccan L. infantum strains as inferred by STRUCTURE software on the basis of data for 14 microsatellite markers. Each of the strains is represented by a single vertical line divided into K colors, where K is the number of populations assumed. Each color represents one population, and the length of the colors segment shows the strain’s estimated proportion of membership in that population.(A) the two main populations derived from the whole dataset which divided strains into MON-1 and non-MON-1 populations. (B) Sub-population analysis of Population 1 (MON-1 group) shows two sub-populations. K represents the true number of populations and sub-populations.
    Figure Legend Snippet: Estimated population structure for 33 Moroccan L. infantum strains as inferred by STRUCTURE software on the basis of data for 14 microsatellite markers. Each of the strains is represented by a single vertical line divided into K colors, where K is the number of populations assumed. Each color represents one population, and the length of the colors segment shows the strain’s estimated proportion of membership in that population.(A) the two main populations derived from the whole dataset which divided strains into MON-1 and non-MON-1 populations. (B) Sub-population analysis of Population 1 (MON-1 group) shows two sub-populations. K represents the true number of populations and sub-populations.

    Techniques Used: Software, Derivative Assay

    Unrooted neighbor-joining tree inferred from genetic distances derived from the proportions of alleles shared among 55 Moroccan L. infantum strains based on 14 microsatellites markers. Two main clusters were detected and the “MON-1” cluster was further sub-divided into two sub-clusters. This was in full agreement with the STRUCTURE results ( Figure 2 ). Mixed 1A/1B genotypes were shown in intermediate positions in the tree.
    Figure Legend Snippet: Unrooted neighbor-joining tree inferred from genetic distances derived from the proportions of alleles shared among 55 Moroccan L. infantum strains based on 14 microsatellites markers. Two main clusters were detected and the “MON-1” cluster was further sub-divided into two sub-clusters. This was in full agreement with the STRUCTURE results ( Figure 2 ). Mixed 1A/1B genotypes were shown in intermediate positions in the tree.

    Techniques Used: Derivative Assay

    2) Product Images from "R31C GNRH1 Mutation and Congenital Hypogonadotropic Hypogonadism"

    Article Title: R31C GNRH1 Mutation and Congenital Hypogonadotropic Hypogonadism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069616

    Pedigree of nCHH families carrying c.91C > T (p.R31[R, C]) GNRH1 mutation. In the family 1, the propositus (II.1) has a de novo mutation and his filiation has been confirmed by DNA microsatellites. In the family 2 the mutation was transmitted from the mother (I.2) to her son (II.1). She required medical assistance for procreation. Clinical and demographic data of all patients are reported in Table 1 . Electropherogram represents the heterozygous mutation in the individual II.1 from family 1. In the panel below, pre-pro-GnRH amino acid sequence conservation. Decapeptide is shown in red.
    Figure Legend Snippet: Pedigree of nCHH families carrying c.91C > T (p.R31[R, C]) GNRH1 mutation. In the family 1, the propositus (II.1) has a de novo mutation and his filiation has been confirmed by DNA microsatellites. In the family 2 the mutation was transmitted from the mother (I.2) to her son (II.1). She required medical assistance for procreation. Clinical and demographic data of all patients are reported in Table 1 . Electropherogram represents the heterozygous mutation in the individual II.1 from family 1. In the panel below, pre-pro-GnRH amino acid sequence conservation. Decapeptide is shown in red.

    Techniques Used: Mutagenesis, Sequencing

    3) Product Images from "In-Frame Deletion and Missense Mutations of the C-Terminal Helicase Domain of SMARCA2 in Three Patients with Nicolaides-Baraitser Syndrome"

    Article Title: In-Frame Deletion and Missense Mutations of the C-Terminal Helicase Domain of SMARCA2 in Three Patients with Nicolaides-Baraitser Syndrome

    Journal: Molecular Syndromology

    doi: 10.1159/000337323

    Identified defects in SMARCA2 . A Affymetrix 6.0 Array data of patient 1 and his parents showing an intragenic de novo deletion of 32 kb within the SMARCA2 gene at 9p24.3. The first and last deleted probes as indicated by the array were SNP_A-8705668 (hg18:
    Figure Legend Snippet: Identified defects in SMARCA2 . A Affymetrix 6.0 Array data of patient 1 and his parents showing an intragenic de novo deletion of 32 kb within the SMARCA2 gene at 9p24.3. The first and last deleted probes as indicated by the array were SNP_A-8705668 (hg18:

    Techniques Used:

    Phenotypes of 3 patients with defects in SMARCA2 . A , B Patient 1 at the age of 21 months. C–F Facial gestalt, hand and foot of patient 1 at the age of 3 years and 7 months. G , H Patient 2 at the age of 3 years and 6 months. I–L Facial
    Figure Legend Snippet: Phenotypes of 3 patients with defects in SMARCA2 . A , B Patient 1 at the age of 21 months. C–F Facial gestalt, hand and foot of patient 1 at the age of 3 years and 7 months. G , H Patient 2 at the age of 3 years and 6 months. I–L Facial

    Techniques Used:

    4) Product Images from "Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype–phenotype correlations, codon bias and dominant-negative effects"

    Article Title: Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype–phenotype correlations, codon bias and dominant-negative effects

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv226

    Detection of de novo AP2S1 mutations in families 02/03 and 06/13b. ( A ) DNA sequence analyses of the probands (arrowed) revealed a G-to-T transversion at codon 15, predicted to result in a missense amino acid substitution of Arg to Leu, and loss of a Hh aI restriction endonuclease site. ( B ) Restriction map showing that Hh aI digestion would result in two products of 143 and 252 bp from the wild-type (WT) sequence, but would not affect the mutant (m) sequence. PCR and Hh aI digestion revealed the probands [individual II.4 of family 02/03 ( C ) and individual II.1 of family 06/13b ( D )] to be heterozygous for the Arg15Leu mutation. The absence of the Arg15Leu mutation in the unaffected parents of both probands is consistent with the mutation arising de novo .
    Figure Legend Snippet: Detection of de novo AP2S1 mutations in families 02/03 and 06/13b. ( A ) DNA sequence analyses of the probands (arrowed) revealed a G-to-T transversion at codon 15, predicted to result in a missense amino acid substitution of Arg to Leu, and loss of a Hh aI restriction endonuclease site. ( B ) Restriction map showing that Hh aI digestion would result in two products of 143 and 252 bp from the wild-type (WT) sequence, but would not affect the mutant (m) sequence. PCR and Hh aI digestion revealed the probands [individual II.4 of family 02/03 ( C ) and individual II.1 of family 06/13b ( D )] to be heterozygous for the Arg15Leu mutation. The absence of the Arg15Leu mutation in the unaffected parents of both probands is consistent with the mutation arising de novo .

    Techniques Used: Sequencing, Mutagenesis, Polymerase Chain Reaction

    5) Product Images from "The miRNA Expression Profile of Experimental Autoimmune Encephalomyelitis Reveals Novel Potential Disease Biomarkers"

    Article Title: The miRNA Expression Profile of Experimental Autoimmune Encephalomyelitis Reveals Novel Potential Disease Biomarkers

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123990

    The differential expression of miRNAs in response to the disease-related antigen (MOG 35–55 ). Splenocytes (SPCs) of MOG 35–55 -induced experimental autoimmune encephalomyelitis (MOG-EAE) mice were restimulated for 24 h with MOG 35–55 (25 μg/mL) or in medium alone without MOG 35–55 (Medium control). The total RNA isolated from these SPCs was then used for miRNA expression using GeneChip TM miRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA). The data was analyzed statistically as follows, ( a ) three-dimensional scatter plot of principal component analysis (PCA) displaying the relationship between the test and control groups and ( b ) heatmap and hierarchical clustering of miRNA expression profiles of MOG 35–55 -restimulated splenocytes of EAE mice and that of SPCs in medium alone (baseline). Red color indicates higher expression, whereas blue color indicates reduced expression of miRNAs. ( n = 3 each for MOG 35–55 restimulation and medium control).
    Figure Legend Snippet: The differential expression of miRNAs in response to the disease-related antigen (MOG 35–55 ). Splenocytes (SPCs) of MOG 35–55 -induced experimental autoimmune encephalomyelitis (MOG-EAE) mice were restimulated for 24 h with MOG 35–55 (25 μg/mL) or in medium alone without MOG 35–55 (Medium control). The total RNA isolated from these SPCs was then used for miRNA expression using GeneChip TM miRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA). The data was analyzed statistically as follows, ( a ) three-dimensional scatter plot of principal component analysis (PCA) displaying the relationship between the test and control groups and ( b ) heatmap and hierarchical clustering of miRNA expression profiles of MOG 35–55 -restimulated splenocytes of EAE mice and that of SPCs in medium alone (baseline). Red color indicates higher expression, whereas blue color indicates reduced expression of miRNAs. ( n = 3 each for MOG 35–55 restimulation and medium control).

    Techniques Used: Expressing, Mouse Assay, Isolation

    6) Product Images from "The Patterned Structure of Galactoglucomannan Suggests It May Bind to Cellulose in Seed Mucilage 1The Patterned Structure of Galactoglucomannan Suggests It May Bind to Cellulose in Seed Mucilage 1 [OPEN]"

    Article Title: The Patterned Structure of Galactoglucomannan Suggests It May Bind to Cellulose in Seed Mucilage 1The Patterned Structure of Galactoglucomannan Suggests It May Bind to Cellulose in Seed Mucilage 1 [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.18.00709

    Man26A- and then α-galactosidase-digested products of wild-type adherent mucilage AIR were confirmed as linear. A, Man26A- and then α-galactosidase-digested products of adherent mucilage AIR were labeled with 2-AB and analyzed by MALDI-TOF MS. B and C, (Hex) 4 and (Hex) 6 in A were identified to be linear by MS/MS. Glc and Man assignments are based on the PACE results.
    Figure Legend Snippet: Man26A- and then α-galactosidase-digested products of wild-type adherent mucilage AIR were confirmed as linear. A, Man26A- and then α-galactosidase-digested products of adherent mucilage AIR were labeled with 2-AB and analyzed by MALDI-TOF MS. B and C, (Hex) 4 and (Hex) 6 in A were identified to be linear by MS/MS. Glc and Man assignments are based on the PACE results.

    Techniques Used: Labeling, Mass Spectrometry, Gas Chromatography

    All mannosyl residues on the glucomannan backbone except that on the reducing end were substituted by single α-Gal at C-6. A, Man26A-digested products of adherent mucilage AIR were labeled with 2-AB and analyzed by MALDI-TOF-MS. B and C, (Hex) 5 and (Hex) 8 in A were analyzed by high-energy MALDI-CID.
    Figure Legend Snippet: All mannosyl residues on the glucomannan backbone except that on the reducing end were substituted by single α-Gal at C-6. A, Man26A-digested products of adherent mucilage AIR were labeled with 2-AB and analyzed by MALDI-TOF-MS. B and C, (Hex) 5 and (Hex) 8 in A were analyzed by high-energy MALDI-CID.

    Techniques Used: Labeling, Mass Spectrometry

    7) Product Images from "Automated IS6110-based fingerprinting of Mycobacterium tuberculosis: Reaching unprecedented discriminatory power and versatility"

    Article Title: Automated IS6110-based fingerprinting of Mycobacterium tuberculosis: Reaching unprecedented discriminatory power and versatility

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197913

    Box plot showing the sizes of IS 6110 polymorphic amplicons generated by IS 6110 -5’3’FP using the 11-banded laboratory reference strain genomic DNA digested either by BstU I or Hinc II. The IS 6110 -5’3’FP products were fractionated without being diluted on an ABI PRISM 3100 capillary DNA sequencer (Applied Biosystems Inc., CA, USA). The boxes show the 25% to 75% interquartile range.
    Figure Legend Snippet: Box plot showing the sizes of IS 6110 polymorphic amplicons generated by IS 6110 -5’3’FP using the 11-banded laboratory reference strain genomic DNA digested either by BstU I or Hinc II. The IS 6110 -5’3’FP products were fractionated without being diluted on an ABI PRISM 3100 capillary DNA sequencer (Applied Biosystems Inc., CA, USA). The boxes show the 25% to 75% interquartile range.

    Techniques Used: Generated

    8) Product Images from "Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks"

    Article Title: Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.062526099

    Atlas of the chromosome of serotype M18 strain MGAS8232. Arrowheads in the outermost ring depict the position and orientation of all transposase genes or gene fragments identified in the genome. Transposase genes are color coded to represent the transposase family with the closest BLASTP match (red, IS 1239 ; black, IS 904A ; olive, IS 1562 ; gray blue, tnpA ; green, IS 861 ; light blue, Streptococcus salvarius transposase). The middle three rings show the position and orientation of the six RNA operons (light blue, clockwise; pink, counterclockwise) and ORFs (blue, clockwise; yellow, counterclockwise) identified in the genome. The innermost ring shows the location and name of the phage sequences (orange) identified in the genome.
    Figure Legend Snippet: Atlas of the chromosome of serotype M18 strain MGAS8232. Arrowheads in the outermost ring depict the position and orientation of all transposase genes or gene fragments identified in the genome. Transposase genes are color coded to represent the transposase family with the closest BLASTP match (red, IS 1239 ; black, IS 904A ; olive, IS 1562 ; gray blue, tnpA ; green, IS 861 ; light blue, Streptococcus salvarius transposase). The middle three rings show the position and orientation of the six RNA operons (light blue, clockwise; pink, counterclockwise) and ORFs (blue, clockwise; yellow, counterclockwise) identified in the genome. The innermost ring shows the location and name of the phage sequences (orange) identified in the genome.

    Techniques Used:

    Subset of 223 strain-specific ORFs identified by DNA microarray analysis of 36 serotype M18 strains of GAS. All ORFs had a test/control ratio that either was below the threshold value of 0.7 (yellow) or above the threshold of 1.5 (blue) in at least 1 of 36 strains (black, ORFs with test/control values between the threshold ratios). ORFs are sorted in MGAS8232 genomic order (clockwise from oriC ). Strains are numbered 1–36 as they are listed in Table 2. Phage-associated ORFs are identified with a black vertical bar at the phage region. Other ORFs that showed a “gene difference” represent potential transposase genes, transcription regulators, carbohydrate and metal transport genes, and hypothetical genes.
    Figure Legend Snippet: Subset of 223 strain-specific ORFs identified by DNA microarray analysis of 36 serotype M18 strains of GAS. All ORFs had a test/control ratio that either was below the threshold value of 0.7 (yellow) or above the threshold of 1.5 (blue) in at least 1 of 36 strains (black, ORFs with test/control values between the threshold ratios). ORFs are sorted in MGAS8232 genomic order (clockwise from oriC ). Strains are numbered 1–36 as they are listed in Table 2. Phage-associated ORFs are identified with a black vertical bar at the phage region. Other ORFs that showed a “gene difference” represent potential transposase genes, transcription regulators, carbohydrate and metal transport genes, and hypothetical genes.

    Techniques Used: Microarray

    9) Product Images from "Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice"

    Article Title: Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice

    Journal: Scientific Reports

    doi: 10.1038/srep23765

    ( A ) Unrooted phylogenetic tree depicting the functional molecular diversity and evolutionary relationships among 26 rice accessions using 3052 informative gene-based ISM and ILP markers. All these accessions differentiated into five major groups- I: indica (Ia: lowland and Ib: upland/ aus ), II: long-grained aromatics (IIa: traditional and IIb: improved/evolved), III: traditional short-grained aromatics, IV: japonica and V: wild according to their known species/subspecies-specific origination, pedigree relationships and parentage. ( B ) Population genetic structure depicts best possible structure among 26 rice accessions using 3052 informative gene-based ISM and ILP markers. At optimal population number K = 5, these mapped markers grouped rice accessions into five major populations- indica , long- and short-grained aromatics, japonica and wild according to their known parentage and pedigree relationships. The accessions represented by vertical bars along the horizontal axis were categorized into K colour segments based on their estimated membership fraction in each K cluster. Five different colours signify five major population groups that correspond well with the clustering pattern as obtained by phylogenetic tree construction.
    Figure Legend Snippet: ( A ) Unrooted phylogenetic tree depicting the functional molecular diversity and evolutionary relationships among 26 rice accessions using 3052 informative gene-based ISM and ILP markers. All these accessions differentiated into five major groups- I: indica (Ia: lowland and Ib: upland/ aus ), II: long-grained aromatics (IIa: traditional and IIb: improved/evolved), III: traditional short-grained aromatics, IV: japonica and V: wild according to their known species/subspecies-specific origination, pedigree relationships and parentage. ( B ) Population genetic structure depicts best possible structure among 26 rice accessions using 3052 informative gene-based ISM and ILP markers. At optimal population number K = 5, these mapped markers grouped rice accessions into five major populations- indica , long- and short-grained aromatics, japonica and wild according to their known parentage and pedigree relationships. The accessions represented by vertical bars along the horizontal axis were categorized into K colour segments based on their estimated membership fraction in each K cluster. Five different colours signify five major population groups that correspond well with the clustering pattern as obtained by phylogenetic tree construction.

    Techniques Used: Functional Assay, IA

    Proportionate distribution of 778 ILP ( A ) and 3217 ISM ( B ) markers designed from various known cloned genes that are functionally well-characterized for diverse agronomic traits in rice. The ILP (66%) and ISM (64%) markers derived particularly from multiple known cloned genes governing yield-contributing traits were abundant. Values in parentheses indicate the number of ILP and ISM markers. The detail information regarding known cloned gene-derived ISM and ILP markers are mentioned in the Tables S3 and S4.
    Figure Legend Snippet: Proportionate distribution of 778 ILP ( A ) and 3217 ISM ( B ) markers designed from various known cloned genes that are functionally well-characterized for diverse agronomic traits in rice. The ILP (66%) and ISM (64%) markers derived particularly from multiple known cloned genes governing yield-contributing traits were abundant. Values in parentheses indicate the number of ILP and ISM markers. The detail information regarding known cloned gene-derived ISM and ILP markers are mentioned in the Tables S3 and S4.

    Techniques Used: Clone Assay, Derivative Assay

    ( A ) Melting-curve analysis of one representative HAP gene with ILP marker in quantitative RT-PCR assay using the cDNA-pools of seedlings and two seed developmental stages of IR 64 (at least two biological replicates) produces single peak as desired, confirming the efficacy of ILP marker to amplify single gene-specific PCR product of accurate fragment size. ( B ) Hierarchical cluster display illustrated the differential expression profile of four HAP genes with ISM and ILP markers in seedling (SL) and two seed developmental stages (S1–S2: early cell division and S3–S5: late maturation) of one rice accession IR 64. The blue, black and yellow colour scale (mentioned at the top) signify the low, medium and high level of average log signal expression values of genes in various tissues/stages, respectively. The expression values across diverse tissues/development stages of accession were normalized using an endogenous control Actin1 in RT-PCR assay. The differential expression profiling of genes in two seed developmental stages of IR 64 was compared with their respective vegetative seedling tissue by assigning the gene expression in this tissue as a reference calibrator 1. The detail structural and functional annotation four rice HAP genes with markers are mentioned in the Tables S1 and S2. The genes with marker and tissues/stages used for expression profiling are indicated on the top and right side of an expression map, respectively.
    Figure Legend Snippet: ( A ) Melting-curve analysis of one representative HAP gene with ILP marker in quantitative RT-PCR assay using the cDNA-pools of seedlings and two seed developmental stages of IR 64 (at least two biological replicates) produces single peak as desired, confirming the efficacy of ILP marker to amplify single gene-specific PCR product of accurate fragment size. ( B ) Hierarchical cluster display illustrated the differential expression profile of four HAP genes with ISM and ILP markers in seedling (SL) and two seed developmental stages (S1–S2: early cell division and S3–S5: late maturation) of one rice accession IR 64. The blue, black and yellow colour scale (mentioned at the top) signify the low, medium and high level of average log signal expression values of genes in various tissues/stages, respectively. The expression values across diverse tissues/development stages of accession were normalized using an endogenous control Actin1 in RT-PCR assay. The differential expression profiling of genes in two seed developmental stages of IR 64 was compared with their respective vegetative seedling tissue by assigning the gene expression in this tissue as a reference calibrator 1. The detail structural and functional annotation four rice HAP genes with markers are mentioned in the Tables S1 and S2. The genes with marker and tissues/stages used for expression profiling are indicated on the top and right side of an expression map, respectively.

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

    ( A ) A high-density genetic linkage map (IR 64 x Sonasal), generated by anchoring 2785 known cloned/candidate genes-derived ISM and ILP markers on 12 rice chromosomes, is illustrated in a Circos circular ideogram. The outer circle signifies the diverse genetic map length (cM) (spanning 50 cM uniform genetic distance intervals between bins) of 12 chromosomes coded with multiple colours. (B) The integration of genetic and physical maps delineated six candidate genes with ILP markers at six major genomic regions harbouring grain weight QTL mapped on five rice chromosomes 2, 3, 5, 6 and 8. The genetic (cM)/physical (bp) distance and identities of markers mapped on chromosomes are denoted on the right and left side of the chromosomes, respectively. The detail information regarding ISM and ILP markers and major grain weight QTLs are provided in the Tables S1,S2 and Table 2 .
    Figure Legend Snippet: ( A ) A high-density genetic linkage map (IR 64 x Sonasal), generated by anchoring 2785 known cloned/candidate genes-derived ISM and ILP markers on 12 rice chromosomes, is illustrated in a Circos circular ideogram. The outer circle signifies the diverse genetic map length (cM) (spanning 50 cM uniform genetic distance intervals between bins) of 12 chromosomes coded with multiple colours. (B) The integration of genetic and physical maps delineated six candidate genes with ILP markers at six major genomic regions harbouring grain weight QTL mapped on five rice chromosomes 2, 3, 5, 6 and 8. The genetic (cM)/physical (bp) distance and identities of markers mapped on chromosomes are denoted on the right and left side of the chromosomes, respectively. The detail information regarding ISM and ILP markers and major grain weight QTLs are provided in the Tables S1,S2 and Table 2 .

    Techniques Used: Generated, Clone Assay, Derivative Assay

    Snapshot illustrating the features and utilities of different interfaces included in a public web-resource “ Oryza ISM-ILP marker” database. The snapshot was selected from database webpages developed.
    Figure Legend Snippet: Snapshot illustrating the features and utilities of different interfaces included in a public web-resource “ Oryza ISM-ILP marker” database. The snapshot was selected from database webpages developed.

    Techniques Used: Marker

    Genome-wide distribution pattern [number ( A ) and frequency ( B )] of 84634 ISM and 16510 ILP markers designed from the intronic sequences of 20533 and 9816 genes annotated from 12 rice chromosomes. Number in parenthesis specifies rice genes with ISM and ILP markers. ( C ) The relative distribution of 84634 ISM and 16510 ILP markers physically mapped on 12 rice chromosomes are depicted by a Circos circular ideogram. The outermost circle denotes the physical size (Mb) of 12 rice chromosome-pseudomolecules coded with multiple colours. The next circle I signifies the ISM (blue) and ILP (red) markers designed from rice genes, whereas circles II and III indicate the ISM (blue) and ILP (red) markers, respectively, developed from known cloned genes regulating diverse agronomic traits (yield component and stress tolerance traits) in rice.
    Figure Legend Snippet: Genome-wide distribution pattern [number ( A ) and frequency ( B )] of 84634 ISM and 16510 ILP markers designed from the intronic sequences of 20533 and 9816 genes annotated from 12 rice chromosomes. Number in parenthesis specifies rice genes with ISM and ILP markers. ( C ) The relative distribution of 84634 ISM and 16510 ILP markers physically mapped on 12 rice chromosomes are depicted by a Circos circular ideogram. The outermost circle denotes the physical size (Mb) of 12 rice chromosome-pseudomolecules coded with multiple colours. The next circle I signifies the ISM (blue) and ILP (red) markers designed from rice genes, whereas circles II and III indicate the ISM (blue) and ILP (red) markers, respectively, developed from known cloned genes regulating diverse agronomic traits (yield component and stress tolerance traits) in rice.

    Techniques Used: Genome Wide, Clone Assay

    Schematic depicting the key steps followed for successful discovery, large-scale validation and high-throughput genotyping of ISM and ILP markers derived from diverse intronic sequence components of grain weight-regulating known cloned gene as exemplified by GW8 / SPL16 (annotated from rice genome), to be utilized for multi-dimensional genomics-assisted breeding applications in rice. The Forward (F) and Reverse (R) primers designed from the exonic sequences flanking the introns (without any InDels) and intronic-InDels were developed as ISM and ILP markers, respectively. URR: Upstream regulatory region and DRR: downstream regulatory region. The identities of ISM and ILP markers with their detailed information are mentioned in the Tables S1 and S2.
    Figure Legend Snippet: Schematic depicting the key steps followed for successful discovery, large-scale validation and high-throughput genotyping of ISM and ILP markers derived from diverse intronic sequence components of grain weight-regulating known cloned gene as exemplified by GW8 / SPL16 (annotated from rice genome), to be utilized for multi-dimensional genomics-assisted breeding applications in rice. The Forward (F) and Reverse (R) primers designed from the exonic sequences flanking the introns (without any InDels) and intronic-InDels were developed as ISM and ILP markers, respectively. URR: Upstream regulatory region and DRR: downstream regulatory region. The identities of ISM and ILP markers with their detailed information are mentioned in the Tables S1 and S2.

    Techniques Used: High Throughput Screening Assay, Derivative Assay, Sequencing, Clone Assay

    10) Product Images from "Identification of genetic variants for clinical management of familial colorectal tumors"

    Article Title: Identification of genetic variants for clinical management of familial colorectal tumors

    Journal: BMC Medical Genetics

    doi: 10.1186/s12881-018-0533-9

    Evaluation of variant-induced splicing alterations by using a cell-based minigene assay. a Structure of pCAS2 minigenes used in the splicing reporter assay. The bent arrow indicates the CMV promoter, boxes represent exons, lines in between the boxes indicate introns, and arrows below the exons represent primers used in RT-PCR reactions. The minigenes were generated by inserting a genomic fragment containing the exon of interest together with its flanking intronic sequences into the intron of pCAS2, as described under Materials and Methods. b Analysis of the splicing pattern of pCAS2 minigenes carrying variants identified in this study. Wild-type (WT) and mutant constructs, as indicated, were introduced into HeLa cells and the transcripts of the minigenes were analyzed by RT-PCR 24 h post-transfection. The image shows the results of a representative experiment in which the RT-PCR products were separated on a 2.5% agarose gel stained with EtBr and visualized by exposure to ultraviolet light. M, 100 bp DNA ladder (New England Biolabs). c Quantification of splicing events observed in the minigene splicing assay. The relative levels of exon inclusion indicated under the gel are based on RT-PCR experiments equivalent to those shown in B but performed with a fluorescent forward primer and then separated on an automated sequencer. Quantification results were obtained by using the GeneMapper v5.0 software (Applied Biosystems) and correspond to the average of two independent fluorescent-RT-PCR experiments. d Representative fluorescent RT-PCR experiment. The panel shows superposed peaks corresponding to the WT and mutant products (in blue and red, respectively), as indicated
    Figure Legend Snippet: Evaluation of variant-induced splicing alterations by using a cell-based minigene assay. a Structure of pCAS2 minigenes used in the splicing reporter assay. The bent arrow indicates the CMV promoter, boxes represent exons, lines in between the boxes indicate introns, and arrows below the exons represent primers used in RT-PCR reactions. The minigenes were generated by inserting a genomic fragment containing the exon of interest together with its flanking intronic sequences into the intron of pCAS2, as described under Materials and Methods. b Analysis of the splicing pattern of pCAS2 minigenes carrying variants identified in this study. Wild-type (WT) and mutant constructs, as indicated, were introduced into HeLa cells and the transcripts of the minigenes were analyzed by RT-PCR 24 h post-transfection. The image shows the results of a representative experiment in which the RT-PCR products were separated on a 2.5% agarose gel stained with EtBr and visualized by exposure to ultraviolet light. M, 100 bp DNA ladder (New England Biolabs). c Quantification of splicing events observed in the minigene splicing assay. The relative levels of exon inclusion indicated under the gel are based on RT-PCR experiments equivalent to those shown in B but performed with a fluorescent forward primer and then separated on an automated sequencer. Quantification results were obtained by using the GeneMapper v5.0 software (Applied Biosystems) and correspond to the average of two independent fluorescent-RT-PCR experiments. d Representative fluorescent RT-PCR experiment. The panel shows superposed peaks corresponding to the WT and mutant products (in blue and red, respectively), as indicated

    Techniques Used: Variant Assay, Mini Gene Assay, Reporter Assay, Reverse Transcription Polymerase Chain Reaction, Generated, Mutagenesis, Construct, Transfection, Agarose Gel Electrophoresis, Staining, Splicing Assay, Software

    11) Product Images from "Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1"

    Article Title: Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1

    Journal: Nature Communications

    doi: 10.1038/ncomms15466

    Effects of risk variants on LOXL1 transcriptional activity in vivo. ( a ) Scatter plot of TaqMan-based allelic discrimination of the LOXL1 SNP 13 (rs12441130). The genotypes of hTCF homoyzgous for the risk (C) or non-risk (T) alleles are shown in relation to genomic DNA and pre-mRNA containing cDNA of heterozygous hTCF cell lines ( n =15). Relative abundance of risk allele C over non-risk allele T in heterozygous hTCF cell lines ( n =15); expression level of T allele was set at 100%. ( b ) ChIP assay for RNA polymerase II (Pol II) binding at rs12441130 (SNP13)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using antibodies against Pol II, histone H3 and acetylated histone H3K27Ac (positive controls), and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. Allele-specific ChIP-qPCR analysis for Pol II chromatin binding and histone H3 is shown (left); expression levels of the non-risk allele T were set at 100%. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (top right) and by qPCR (bottom right) with primers specific for the SNP13 region producing a 121 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin).
    Figure Legend Snippet: Effects of risk variants on LOXL1 transcriptional activity in vivo. ( a ) Scatter plot of TaqMan-based allelic discrimination of the LOXL1 SNP 13 (rs12441130). The genotypes of hTCF homoyzgous for the risk (C) or non-risk (T) alleles are shown in relation to genomic DNA and pre-mRNA containing cDNA of heterozygous hTCF cell lines ( n =15). Relative abundance of risk allele C over non-risk allele T in heterozygous hTCF cell lines ( n =15); expression level of T allele was set at 100%. ( b ) ChIP assay for RNA polymerase II (Pol II) binding at rs12441130 (SNP13)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using antibodies against Pol II, histone H3 and acetylated histone H3K27Ac (positive controls), and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. Allele-specific ChIP-qPCR analysis for Pol II chromatin binding and histone H3 is shown (left); expression levels of the non-risk allele T were set at 100%. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (top right) and by qPCR (bottom right) with primers specific for the SNP13 region producing a 121 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin).

    Techniques Used: Activity Assay, In Vivo, Expressing, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    Effects of risk variants on LOXL1 alternative splicing. ( a ) The two LOXL1 transcripts ( LOXL1 , LOXL1-E1A ) seen in the alignment with position of SNPs 11–14; exons are indicated by numbers (E1–E7), the additional exon included in the alternative transcript is indicated as E1A; the location of stop codons is marked by vertical lines. ( b ) Expression levels of transcripts LOXL1-E1A , LOXL1 and LOXL1-AS1 in hTCF cell lines ( n =16) without (control) or after treatment with puromycin (200 ng ml −1 for 10 h) using real-time PCR technology (left); hTCF were either homozygous for the risk ( n =8) or non-risk ( n =8) alleles for SNPs 1–14. PCR from LOXL1 and LOXL1-E1A transcripts without and after puromycin treatment of hTCF ( n =3) using primer pairs spanning exon 1A (right; lane M: DNA marker). ( c ) SNP rs12441130-correlated expression levels of transcripts LOXL1-E1A , LOXL1 and LOXL1-AS1 in hTCF cell lines homozygous for the risk alleles (C/C) ( n =8) or non-risk alleles (T/T) ( n =8) using real-time PCR technology. ( d ) Real-time PCR analysis of UPF1 and LOXL1-E1A in hTCF cell lines ( n =4) transfected with UPF1-specific siRNA or scrambled control siRNA; expression levels were normalized relative to GAPDH (data represent mean values±s.d.; * P
    Figure Legend Snippet: Effects of risk variants on LOXL1 alternative splicing. ( a ) The two LOXL1 transcripts ( LOXL1 , LOXL1-E1A ) seen in the alignment with position of SNPs 11–14; exons are indicated by numbers (E1–E7), the additional exon included in the alternative transcript is indicated as E1A; the location of stop codons is marked by vertical lines. ( b ) Expression levels of transcripts LOXL1-E1A , LOXL1 and LOXL1-AS1 in hTCF cell lines ( n =16) without (control) or after treatment with puromycin (200 ng ml −1 for 10 h) using real-time PCR technology (left); hTCF were either homozygous for the risk ( n =8) or non-risk ( n =8) alleles for SNPs 1–14. PCR from LOXL1 and LOXL1-E1A transcripts without and after puromycin treatment of hTCF ( n =3) using primer pairs spanning exon 1A (right; lane M: DNA marker). ( c ) SNP rs12441130-correlated expression levels of transcripts LOXL1-E1A , LOXL1 and LOXL1-AS1 in hTCF cell lines homozygous for the risk alleles (C/C) ( n =8) or non-risk alleles (T/T) ( n =8) using real-time PCR technology. ( d ) Real-time PCR analysis of UPF1 and LOXL1-E1A in hTCF cell lines ( n =4) transfected with UPF1-specific siRNA or scrambled control siRNA; expression levels were normalized relative to GAPDH (data represent mean values±s.d.; * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Transfection

    12) Product Images from "Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1"

    Article Title: Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1

    Journal: Nature Communications

    doi: 10.1038/ncomms15466

    Allele-specific transcription factor binding. ( a ) Supershift assays with DNA fragments containing the risk sequence of rs11638944 (S12R), nuclear extracts from human trabecular meshwork cells, and specific antibodies against candidate transcription factors liver X receptor (LXR)α/ß, retinoid X receptor (RXR)α, myogenin, nuclear factor (NF)-1, Roaz, zinc finger protein with interaction domain (ZID), nuclear receptor subfamiliy 2 group F member 1 (NR2F1), nuclear factor (NF)κBp50, NFκBp65, peroxisome proliferator-activated receptor (PPAR)α, PPARγ, and hepatocyte nuclear factor 4 (HNF-4)α showed supershifted bands (arrows) with RXRα and ZID antibodies. ( b ) Supershift assays with a specific antibody against RXRα disrupted the DNA–protein complexes (solid arrows) to produce distinct supershifted bands (dotted arrows) in a differential manner between DNA fragments containing the risk (R) alleles and fragments containing the non-risk (N) alleles. Quantitative analyses of the (super)shifted bands relative to the unshifted bands show mean values±s.d. of five independent experiments (* P
    Figure Legend Snippet: Allele-specific transcription factor binding. ( a ) Supershift assays with DNA fragments containing the risk sequence of rs11638944 (S12R), nuclear extracts from human trabecular meshwork cells, and specific antibodies against candidate transcription factors liver X receptor (LXR)α/ß, retinoid X receptor (RXR)α, myogenin, nuclear factor (NF)-1, Roaz, zinc finger protein with interaction domain (ZID), nuclear receptor subfamiliy 2 group F member 1 (NR2F1), nuclear factor (NF)κBp50, NFκBp65, peroxisome proliferator-activated receptor (PPAR)α, PPARγ, and hepatocyte nuclear factor 4 (HNF-4)α showed supershifted bands (arrows) with RXRα and ZID antibodies. ( b ) Supershift assays with a specific antibody against RXRα disrupted the DNA–protein complexes (solid arrows) to produce distinct supershifted bands (dotted arrows) in a differential manner between DNA fragments containing the risk (R) alleles and fragments containing the non-risk (N) alleles. Quantitative analyses of the (super)shifted bands relative to the unshifted bands show mean values±s.d. of five independent experiments (* P

    Techniques Used: Binding Assay, Sequencing

    RXRα influences LOXL1 expression by functioning as a transcriptional repressor. ( a ) ChIP assay for RXRα binding at rs11638944 (SNP12)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using two different antibodies against RXRα (1: D-20, 2: F-1), histone H3 (positive control) and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (left) and by qPCR (right) with primers specific for the SNP12 region producing a 148 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin). ( b ) Dual luciferase reporter assays using reporter plasmids containing risk (12R) and non-risk (12N) genotypes of SNP 12 alone or co-transfected with RXRα-specific siRNA in human Tenon's capsule fibroblasts (hTCF) compared with the basal LOXL1 promoter activity. Results are expressed as the ratio of Firefly luciferase to Renilla luciferase; the transcriptional activity of the empty pGL4.10 vector was set at 100% (data represent mean values±s.d. of three independent experiments; * P
    Figure Legend Snippet: RXRα influences LOXL1 expression by functioning as a transcriptional repressor. ( a ) ChIP assay for RXRα binding at rs11638944 (SNP12)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using two different antibodies against RXRα (1: D-20, 2: F-1), histone H3 (positive control) and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (left) and by qPCR (right) with primers specific for the SNP12 region producing a 148 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin). ( b ) Dual luciferase reporter assays using reporter plasmids containing risk (12R) and non-risk (12N) genotypes of SNP 12 alone or co-transfected with RXRα-specific siRNA in human Tenon's capsule fibroblasts (hTCF) compared with the basal LOXL1 promoter activity. Results are expressed as the ratio of Firefly luciferase to Renilla luciferase; the transcriptional activity of the empty pGL4.10 vector was set at 100% (data represent mean values±s.d. of three independent experiments; * P

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Binding Assay, Positive Control, Negative Control, Immunoprecipitation, Isolation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Luciferase, Transfection, Activity Assay, Plasmid Preparation

    13) Product Images from "Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization"

    Article Title: Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization

    Journal: Science signaling

    doi: 10.1126/scisignal.aaa0441

    OTUB1 phosphorylation at Ser 16 determines its subcellular localization (A) Fixed cell immunofluorescence in U2OS cells transfected with HAOTUB1 and untreated or treated with TDB (10 μM, 4 hours). Individual and merged images show phosphorylated OTUB1 pSer 16 (red), OTUB1 (HA, green) and DAPI (blue). Scale bar, 10 μm. (B) As in (A) in U2OS cells transfected with HA-OTUB1, HA-OTUB1[S16A] or HA-OTUB1[S16E]. Scale bar, 20 μm. (C) As in (A) in U2OS cells 48 hours after RNA interference [ iOTUB1 or iControl ( FOXO4 )] or after treatment with CK2 inhibitors quinalizarin (10 μM, 4 hours) or TDB (10 μM, 4 hours). Scale bar, 5 μm. (D) Western blotting (IB) for the indicated proteins in cytosolic and nuclear fractions from HeLa or HEK293 cells. All data are representative of 3 independent experiments.
    Figure Legend Snippet: OTUB1 phosphorylation at Ser 16 determines its subcellular localization (A) Fixed cell immunofluorescence in U2OS cells transfected with HAOTUB1 and untreated or treated with TDB (10 μM, 4 hours). Individual and merged images show phosphorylated OTUB1 pSer 16 (red), OTUB1 (HA, green) and DAPI (blue). Scale bar, 10 μm. (B) As in (A) in U2OS cells transfected with HA-OTUB1, HA-OTUB1[S16A] or HA-OTUB1[S16E]. Scale bar, 20 μm. (C) As in (A) in U2OS cells 48 hours after RNA interference [ iOTUB1 or iControl ( FOXO4 )] or after treatment with CK2 inhibitors quinalizarin (10 μM, 4 hours) or TDB (10 μM, 4 hours). Scale bar, 5 μm. (D) Western blotting (IB) for the indicated proteins in cytosolic and nuclear fractions from HeLa or HEK293 cells. All data are representative of 3 independent experiments.

    Techniques Used: Immunofluorescence, Transfection, Western Blot

    CK2 phosphorylates OTUB1 in vivo (A) Western blotting (IB) of lysates from HEK293 cells that were untreated, treated with TDB (10 μM, 4 hours) or transfected with OTUB1 siRNA and lysed 48 hours later. (B) Western blotting (IB) of lysates from HEK293 cells that were untreated or, treated with TDB (10 μM, 4 hours) or quinalizarin (10 μM, 4 hours). (C) Western blotting (IB) of lysates from HEK293 cells transfected with HA-OTUB1 and FOXO4 siRNA (siControl) or one of two siRNAs against both CK2α and CK2α’ splice variants. A separate culture of cells was treated with TDB (10 μM, 4 hours). (D) Western blotting (IB) of lysates from HEK293 cells transfected with FOXO4 siRNA (control) or CK2α/α’ siRNA alone or reconstituted with N-terminal FLAG-tagged CK2α. A separate culture of cells was treated with TDB (10 μM, 4 hours) prior to lysis. (E) Western blotting (IB) of lysates from HEK293 cells transfected with vectors encoding N-terminal FLAG-tagged CK2α, FLAG-CK2α[D156A] or FLAG-CK2α’, or treated with TDB (10 μM, 4 hours). (F) Western blotting of homogenized lysates from the indicated mouse tissues. All blots are representative of 3 independent experiments.
    Figure Legend Snippet: CK2 phosphorylates OTUB1 in vivo (A) Western blotting (IB) of lysates from HEK293 cells that were untreated, treated with TDB (10 μM, 4 hours) or transfected with OTUB1 siRNA and lysed 48 hours later. (B) Western blotting (IB) of lysates from HEK293 cells that were untreated or, treated with TDB (10 μM, 4 hours) or quinalizarin (10 μM, 4 hours). (C) Western blotting (IB) of lysates from HEK293 cells transfected with HA-OTUB1 and FOXO4 siRNA (siControl) or one of two siRNAs against both CK2α and CK2α’ splice variants. A separate culture of cells was treated with TDB (10 μM, 4 hours). (D) Western blotting (IB) of lysates from HEK293 cells transfected with FOXO4 siRNA (control) or CK2α/α’ siRNA alone or reconstituted with N-terminal FLAG-tagged CK2α. A separate culture of cells was treated with TDB (10 μM, 4 hours) prior to lysis. (E) Western blotting (IB) of lysates from HEK293 cells transfected with vectors encoding N-terminal FLAG-tagged CK2α, FLAG-CK2α[D156A] or FLAG-CK2α’, or treated with TDB (10 μM, 4 hours). (F) Western blotting of homogenized lysates from the indicated mouse tissues. All blots are representative of 3 independent experiments.

    Techniques Used: In Vivo, Western Blot, Transfection, Lysis

    The catalytic activity, ubiquitin- or E2-binding ability of OTUB1 are not altered by OTUB1 Ser 16 phosphorylation (A) A time course of K48-diubiquitin cleavage assay using unphosphorylated or in vitro CK2-phosphorylated OTUB1 (0.5 μM), in the absence (top panel) or presence of UBE2D2 (bottom panel). Coomassie stains of OTUB1, CK2α and UBE2D2 or Western blotting (IB) of ubiquitin and the phosphorylation of OTUB1 at Ser 16 (pS16) are indicated. (B) As in (A), with phosphorylated GST-OTUB1 (5 μM) incubated with K6-, K11-, K27-, K29-, K33-, K48-, K63-, or M1-di-ubiquitin chains for 10 and 60 min in the absence of UBE2D2 and Coomassie stains indicated. (C) Western blotting (IB) of GST (-), GST-OTUB1 (WT), GST-OTUB1-S16A (S16A) or GST-OTUB1-S16E (S16E) incubated in vitro with K63-linked polyubiquitin chains in an interaction assay. (D) Western blotting (IB) of lysates or HA-immunoprecipitates from HEK293 cells transfected with HA-OTUB1 or HA-OTUB1 mutants treated with or without TDB (10 μM, 4 h). All blots are representative of 3 independent experiments.
    Figure Legend Snippet: The catalytic activity, ubiquitin- or E2-binding ability of OTUB1 are not altered by OTUB1 Ser 16 phosphorylation (A) A time course of K48-diubiquitin cleavage assay using unphosphorylated or in vitro CK2-phosphorylated OTUB1 (0.5 μM), in the absence (top panel) or presence of UBE2D2 (bottom panel). Coomassie stains of OTUB1, CK2α and UBE2D2 or Western blotting (IB) of ubiquitin and the phosphorylation of OTUB1 at Ser 16 (pS16) are indicated. (B) As in (A), with phosphorylated GST-OTUB1 (5 μM) incubated with K6-, K11-, K27-, K29-, K33-, K48-, K63-, or M1-di-ubiquitin chains for 10 and 60 min in the absence of UBE2D2 and Coomassie stains indicated. (C) Western blotting (IB) of GST (-), GST-OTUB1 (WT), GST-OTUB1-S16A (S16A) or GST-OTUB1-S16E (S16E) incubated in vitro with K63-linked polyubiquitin chains in an interaction assay. (D) Western blotting (IB) of lysates or HA-immunoprecipitates from HEK293 cells transfected with HA-OTUB1 or HA-OTUB1 mutants treated with or without TDB (10 μM, 4 h). All blots are representative of 3 independent experiments.

    Techniques Used: Activity Assay, Binding Assay, Cleavage Assay, In Vitro, Western Blot, Incubation, Transfection

    14) Product Images from "Clinical and genetic analysis of the first known Asian family with myotonic dystrophy type 2"

    Article Title: Clinical and genetic analysis of the first known Asian family with myotonic dystrophy type 2

    Journal: Journal of Human Genetics

    doi: 10.1038/jhg.2013.133

    Southern blotting analysis of DM2. Closed arrowhead shows the expanded alleles in DM2. M, λDNA/ Hin d III marker; NC, normal control; II1 and II3, Cases 1 and 3 showing an 18.1-kb expanded allele as well as a normal allele (open arrowhead).
    Figure Legend Snippet: Southern blotting analysis of DM2. Closed arrowhead shows the expanded alleles in DM2. M, λDNA/ Hin d III marker; NC, normal control; II1 and II3, Cases 1 and 3 showing an 18.1-kb expanded allele as well as a normal allele (open arrowhead).

    Techniques Used: Southern Blot, Marker

    Pedigree of the sibling cases carrying the DM2 expansion. The parents did not suffer from muscle weakness and died at the ages of 67 and 72 years. The gender of the unaffected siblings and children of the affected cases is obscured to protect privacy.
    Figure Legend Snippet: Pedigree of the sibling cases carrying the DM2 expansion. The parents did not suffer from muscle weakness and died at the ages of 67 and 72 years. The gender of the unaffected siblings and children of the affected cases is obscured to protect privacy.

    Techniques Used:

    Repeat-primed PCR analysis specific for the DM2 expansion. Negative results from normal control (NC) are shown in the upper panel, whereas a characteristic continuous ladder from Case 2 (II2 in Figure 1 ), indicating the CCTG expansion, is detected in the lower panel. A full color version of this figure is available at the Journal of Human Genetics journal online.
    Figure Legend Snippet: Repeat-primed PCR analysis specific for the DM2 expansion. Negative results from normal control (NC) are shown in the upper panel, whereas a characteristic continuous ladder from Case 2 (II2 in Figure 1 ), indicating the CCTG expansion, is detected in the lower panel. A full color version of this figure is available at the Journal of Human Genetics journal online.

    Techniques Used: Polymerase Chain Reaction

    15) Product Images from "A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae"

    Article Title: A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003473

    Nature of the transformation pilus. ( A ) Purified pili visualized by negative stain electron microscopy. Short pilus fragments ranging from 50 to 500 nm were observed. ( B ) SDS-PAGE analysis of the purified fraction. Left lane, Coommassie blue staining. Middle lane, Western blot using anti-ComGC antibody. Right lane, Western blot analysis using anti-FLAG analysis. ( C ) Nano-ESI FT-MS spectrum of purified pili. Peaks corresponding to ComGC are labelled with a green star those corresponding to ComGC-FLAG with a red triangle. ComGC-FLAG is present at approximately 6+/−2% abundance of the native form. The deconvoluted spectrum showing monoisotopic masses of the neutral protein forms is presented in the inset. The measured mass of methylated comGC (10,442.670 Da) compares very well to the calculated theoretical mass (10,442.636 Da) with an error of +3 ppm.
    Figure Legend Snippet: Nature of the transformation pilus. ( A ) Purified pili visualized by negative stain electron microscopy. Short pilus fragments ranging from 50 to 500 nm were observed. ( B ) SDS-PAGE analysis of the purified fraction. Left lane, Coommassie blue staining. Middle lane, Western blot using anti-ComGC antibody. Right lane, Western blot analysis using anti-FLAG analysis. ( C ) Nano-ESI FT-MS spectrum of purified pili. Peaks corresponding to ComGC are labelled with a green star those corresponding to ComGC-FLAG with a red triangle. ComGC-FLAG is present at approximately 6+/−2% abundance of the native form. The deconvoluted spectrum showing monoisotopic masses of the neutral protein forms is presented in the inset. The measured mass of methylated comGC (10,442.670 Da) compares very well to the calculated theoretical mass (10,442.636 Da) with an error of +3 ppm.

    Techniques Used: Transformation Assay, Purification, Staining, Electron Microscopy, SDS Page, Western Blot, Mass Spectrometry, Methylation

    16) Product Images from "ACAPELLA-1K, A Capillary-Based Submicroliter Automated Fluid Handling System for Genome Analysis"

    Article Title: ACAPELLA-1K, A Capillary-Based Submicroliter Automated Fluid Handling System for Genome Analysis

    Journal: Genome Research

    doi:

    ACAPELLA-1K  Hin dIII digestion of λ DNA. Reactions are 2 μl.
    Figure Legend Snippet: ACAPELLA-1K Hin dIII digestion of λ DNA. Reactions are 2 μl.

    Techniques Used:

    17) Product Images from "A molecular scheme for improved characterization of human embryonic stem cell lines"

    Article Title: A molecular scheme for improved characterization of human embryonic stem cell lines

    Journal: BMC Biology

    doi: 10.1186/1741-7007-4-28

    STR analysis for identification . STR profile of human embryonic stem cell lines. Loci analyzed by the PowerPlex 1.2 system (Promega) comprise D5S818, D13S317, D7S820, D16S539, vWA, TH01, TP0X, CSF1P0, and the sex-chromosome marker amelogenin. Data were collected on an ABI 310 genetic analyzer, and analyzed using Genescan 3.1 and Genotyper 2.0 software (all Applied Biosystems).
    Figure Legend Snippet: STR analysis for identification . STR profile of human embryonic stem cell lines. Loci analyzed by the PowerPlex 1.2 system (Promega) comprise D5S818, D13S317, D7S820, D16S539, vWA, TH01, TP0X, CSF1P0, and the sex-chromosome marker amelogenin. Data were collected on an ABI 310 genetic analyzer, and analyzed using Genescan 3.1 and Genotyper 2.0 software (all Applied Biosystems).

    Techniques Used: Marker, Software

    18) Product Images from "Anti-proliferative and gene expression actions of resveratrol in breast cancer cells in vitro"

    Article Title: Anti-proliferative and gene expression actions of resveratrol in breast cancer cells in vitro

    Journal: Oncotarget

    doi:

    Resveratrol-induced gene expression is not affected by culture media pH (A) The most up-regulated genes induced by resveratrol in human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were treated with or without 10 μM resveratrol for 6 h. Total RNA was harvested and hybrided with Affymetrix HG-U133 plus 2.0 microarray platform. 25 top up-regulated genes from 54675 probe sets. N = 2. (B) MDA-MB-231 cells cultured in different pH conditions were treated with 10 μM resveratrol for 6 h. Cells were harvested and total RNA was extracted for qPCR. Expression of 4 most up-regulated genes induced by resveratrol was examined. N = 4. (C) resveratrol-induced 6 p53-dependent genes were also examined. N = 3 (* p
    Figure Legend Snippet: Resveratrol-induced gene expression is not affected by culture media pH (A) The most up-regulated genes induced by resveratrol in human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were treated with or without 10 μM resveratrol for 6 h. Total RNA was harvested and hybrided with Affymetrix HG-U133 plus 2.0 microarray platform. 25 top up-regulated genes from 54675 probe sets. N = 2. (B) MDA-MB-231 cells cultured in different pH conditions were treated with 10 μM resveratrol for 6 h. Cells were harvested and total RNA was extracted for qPCR. Expression of 4 most up-regulated genes induced by resveratrol was examined. N = 4. (C) resveratrol-induced 6 p53-dependent genes were also examined. N = 3 (* p

    Techniques Used: Expressing, Multiple Displacement Amplification, Microarray, Cell Culture, Real-time Polymerase Chain Reaction

    19) Product Images from "Identification of Hedysarum Varieties Using Amplified Fragment Length Polymorphism on a Capillary Electrophoresis System"

    Article Title: Identification of Hedysarum Varieties Using Amplified Fragment Length Polymorphism on a Capillary Electrophoresis System

    Journal: Journal of Biomolecular Techniques : JBT

    doi:

    Electropherograms of three H. boreale AFLP samples run on the Applied Biosystems 3130 xl Genetic Analyzer displayed in the GeneMapper software. The gray bars represent bins assigned to the peaks. The allele calls are shown on the bottom of each plot. The
    Figure Legend Snippet: Electropherograms of three H. boreale AFLP samples run on the Applied Biosystems 3130 xl Genetic Analyzer displayed in the GeneMapper software. The gray bars represent bins assigned to the peaks. The allele calls are shown on the bottom of each plot. The

    Techniques Used: Software

    20) Product Images from "Non-referenced genome assembly from epigenomic short-read data "

    Article Title: Non-referenced genome assembly from epigenomic short-read data

    Journal: Epigenetics

    doi: 10.4161/15592294.2014.969610

    Coverage of assembled regions when mapped to their respective reference genomes. The percentage of genomic features covered by newly assembled contigs by ChIP-seq for H3K9Ac and ER-α, and DNA methylation enrichment. Gene features derived from Ensembl ver. 72 for D. melanogaster and M. musculus , and Ensembl ver. 54 for H. sapiens are composed of CpG islands, exons, introns, whole gene bodies, promoters (the region 3 kb upstream from the transcription start site) and the whole genome. Genomic regions were filtered by 36 bp read mappability. Coverage is shown for ( A ) D. melanogaster H3K9Ac (no CpG islands are annotated), ( B ) H. sapiens ER-α, ( C ) M. musculus DNA methylation.
    Figure Legend Snippet: Coverage of assembled regions when mapped to their respective reference genomes. The percentage of genomic features covered by newly assembled contigs by ChIP-seq for H3K9Ac and ER-α, and DNA methylation enrichment. Gene features derived from Ensembl ver. 72 for D. melanogaster and M. musculus , and Ensembl ver. 54 for H. sapiens are composed of CpG islands, exons, introns, whole gene bodies, promoters (the region 3 kb upstream from the transcription start site) and the whole genome. Genomic regions were filtered by 36 bp read mappability. Coverage is shown for ( A ) D. melanogaster H3K9Ac (no CpG islands are annotated), ( B ) H. sapiens ER-α, ( C ) M. musculus DNA methylation.

    Techniques Used: Chromatin Immunoprecipitation, DNA Methylation Assay, Derivative Assay

    21) Product Images from "Expression of Serum Amyloid A in Human Ovarian Epithelial Tumors: Implication for a Role in Ovarian Tumorigenesis"

    Article Title: Expression of Serum Amyloid A in Human Ovarian Epithelial Tumors: Implication for a Role in Ovarian Tumorigenesis

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/jhc.2010.956821

    RT-PCR analysis of ovarian tissues and OVCAR-3 cells. ( A ) RT-PCR analysis of ovarian serous carcinoma. Primers specific for the four known human SAA genes were used, and PCR fragments were analyzed on a 2% agarose gel. Markers of a DNA ladder (100-bp
    Figure Legend Snippet: RT-PCR analysis of ovarian tissues and OVCAR-3 cells. ( A ) RT-PCR analysis of ovarian serous carcinoma. Primers specific for the four known human SAA genes were used, and PCR fragments were analyzed on a 2% agarose gel. Markers of a DNA ladder (100-bp

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    22) Product Images from "Making (anti-) sense out of huntingtin levels in Huntington disease"

    Article Title: Making (anti-) sense out of huntingtin levels in Huntington disease

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-015-0018-7

    Wild-type and mutant HTT mRNA levels in juvenile HD brain. Wild-type and mutant HTT mRNA PCR products were separated by gel electrophoresis. (A) RT-PCR products from brain tissue derived from two juvenile HD patients (HD192 and HC104). CAG repeat sizes for the wild-type and mutant alleles are indicated below each lane. gDNA was used to control for differences in PCR amplification between the wild-type and mutant product due to the CAG repeat expansion. (B) Whisker box plot comparing wild-type and mutant HTT mRNA expression levels, relative to gDNA. Pair wise differences were evaluated using linear mixed model, n = 4.
    Figure Legend Snippet: Wild-type and mutant HTT mRNA levels in juvenile HD brain. Wild-type and mutant HTT mRNA PCR products were separated by gel electrophoresis. (A) RT-PCR products from brain tissue derived from two juvenile HD patients (HD192 and HC104). CAG repeat sizes for the wild-type and mutant alleles are indicated below each lane. gDNA was used to control for differences in PCR amplification between the wild-type and mutant product due to the CAG repeat expansion. (B) Whisker box plot comparing wild-type and mutant HTT mRNA expression levels, relative to gDNA. Pair wise differences were evaluated using linear mixed model, n = 4.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Amplification, Whisker Assay, Expressing

    Wild-type and mutant HTT mRNA levels in adult-onset HD brain tissue. Wild-type and mutant HTT mRNA PCR products were separated on gel electrophoresis by differences in their CAG repeat length. (A) RT-PCR products from brain tissue derived from a control (H121) and 10 HD patients. Allelic CAG repeat sizes are indicated below each lane. gDNA from each sample was taken along to control for differences in PCR amplification efficiencies across the CAG repeat. (B) Whisker boxplot of allelic HTT mRNA expression levels in frontal cortex from 5 HD adult-onset HD patients. (C) Whisker boxplot of allelic HTT mRNA expression levels in middle temporal gyrus from 5 HD adult-onset HD patients. Expression levels relative to gDNA. Pair wise differences were evaluated using linear mixed model, n = 5.
    Figure Legend Snippet: Wild-type and mutant HTT mRNA levels in adult-onset HD brain tissue. Wild-type and mutant HTT mRNA PCR products were separated on gel electrophoresis by differences in their CAG repeat length. (A) RT-PCR products from brain tissue derived from a control (H121) and 10 HD patients. Allelic CAG repeat sizes are indicated below each lane. gDNA from each sample was taken along to control for differences in PCR amplification efficiencies across the CAG repeat. (B) Whisker boxplot of allelic HTT mRNA expression levels in frontal cortex from 5 HD adult-onset HD patients. (C) Whisker boxplot of allelic HTT mRNA expression levels in middle temporal gyrus from 5 HD adult-onset HD patients. Expression levels relative to gDNA. Pair wise differences were evaluated using linear mixed model, n = 5.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Amplification, Whisker Assay, Expressing

    HTT antisense expression in HD patient-derived fibroblasts and brain tissue. Gel electrophoresis of HTTAS amplified using strand- and HTTAS isoform-specific primers. (A) RT-PCR of patient-derived fibroblasts from a control (GM04204), an HD patient (GM02173), an HD patient homozygous for the CAG repeat expansion (GM04857) and a juvenile HD patient (GM05539). (B) RT-PCR of post-mortem brain tissue from a control (H121), an HD patient (HC105), and 2 juvenile HD patients (HD192 and HD86). Allelic CAG repeat sizes below each lane.
    Figure Legend Snippet: HTT antisense expression in HD patient-derived fibroblasts and brain tissue. Gel electrophoresis of HTTAS amplified using strand- and HTTAS isoform-specific primers. (A) RT-PCR of patient-derived fibroblasts from a control (GM04204), an HD patient (GM02173), an HD patient homozygous for the CAG repeat expansion (GM04857) and a juvenile HD patient (GM05539). (B) RT-PCR of post-mortem brain tissue from a control (H121), an HD patient (HC105), and 2 juvenile HD patients (HD192 and HD86). Allelic CAG repeat sizes below each lane.

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

    HTT mRNA quantification after RT-PCR amplification across the CAG repeat compared to SNP-specific quantitative RT-PCR. Whisker boxplots of wild-type versus mutant HTT mRNA expression levels in adult-onset HD post-mortem brain material. (A) Quantification after amplification across the CAG repeat, relative to gDNA. Pair wise differences were evaluated using linear mixed model, n = 10. (B) SNP rs362273-specific quantitative RT-PCR, normalized to β-actin (ACTB). Data were evaluated using a two-tailed student t -test, n = 4.
    Figure Legend Snippet: HTT mRNA quantification after RT-PCR amplification across the CAG repeat compared to SNP-specific quantitative RT-PCR. Whisker boxplots of wild-type versus mutant HTT mRNA expression levels in adult-onset HD post-mortem brain material. (A) Quantification after amplification across the CAG repeat, relative to gDNA. Pair wise differences were evaluated using linear mixed model, n = 10. (B) SNP rs362273-specific quantitative RT-PCR, normalized to β-actin (ACTB). Data were evaluated using a two-tailed student t -test, n = 4.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Whisker Assay, Mutagenesis, Expressing, Two Tailed Test

    Validating RT-PCR amplification across the CAG repeat in HD patient-derived fibroblasts. Wild-type and mutant HTT were separated by gel electrophoresis. (A) Standard curve of wild-type and mutant HTT RT-PCR products with increasing PCR cycles from gDNA derived from post-mortem brain tissue of 7 HD patients. PCR linearity was evaluated by determining the individual linear regression coefficients ( r 2 ) of the band intensities of wild-type and mutant HTT expression versus the number of PCR cycles, n = 7. (B) PCR products from cDNA of 4 HD (GM00305, GM02173, GM04022, GM04855) fibroblasts. CAG repeat sizes for the wild-type (lower band) and mutant alleles (upper band) are indicated below each lane. gDNA was used to examine differences in PCR amplification between the wild-type and mutant product due to the CAG repeat expansion. (C) RT-PCR products with input: cDNA (+RT), cDNA lacking reverse transcriptase (−RT) and gDNA of one control (GM04204). (D) Whisker boxplot of RT-PCR from HD patient-derived fibroblasts, comparing wild-type and mutant HTT mRNA expression levels, relative to gDNA. Line = mean, pair wise differences were evaluated using linear mixed model, n = 4.
    Figure Legend Snippet: Validating RT-PCR amplification across the CAG repeat in HD patient-derived fibroblasts. Wild-type and mutant HTT were separated by gel electrophoresis. (A) Standard curve of wild-type and mutant HTT RT-PCR products with increasing PCR cycles from gDNA derived from post-mortem brain tissue of 7 HD patients. PCR linearity was evaluated by determining the individual linear regression coefficients ( r 2 ) of the band intensities of wild-type and mutant HTT expression versus the number of PCR cycles, n = 7. (B) PCR products from cDNA of 4 HD (GM00305, GM02173, GM04022, GM04855) fibroblasts. CAG repeat sizes for the wild-type (lower band) and mutant alleles (upper band) are indicated below each lane. gDNA was used to examine differences in PCR amplification between the wild-type and mutant product due to the CAG repeat expansion. (C) RT-PCR products with input: cDNA (+RT), cDNA lacking reverse transcriptase (−RT) and gDNA of one control (GM04204). (D) Whisker boxplot of RT-PCR from HD patient-derived fibroblasts, comparing wild-type and mutant HTT mRNA expression levels, relative to gDNA. Line = mean, pair wise differences were evaluated using linear mixed model, n = 4.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Derivative Assay, Mutagenesis, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Expressing, Whisker Assay

    23) Product Images from "Identification of genetic variants for clinical management of familial colorectal tumors"

    Article Title: Identification of genetic variants for clinical management of familial colorectal tumors

    Journal: BMC Medical Genetics

    doi: 10.1186/s12881-018-0533-9

    Evaluation of variant-induced splicing alterations by using a cell-based minigene assay. a Structure of pCAS2 minigenes used in the splicing reporter assay. The bent arrow indicates the CMV promoter, boxes represent exons, lines in between the boxes indicate introns, and arrows below the exons represent primers used in RT-PCR reactions. The minigenes were generated by inserting a genomic fragment containing the exon of interest together with its flanking intronic sequences into the intron of pCAS2, as described under Materials and Methods. b Analysis of the splicing pattern of pCAS2 minigenes carrying variants identified in this study. Wild-type (WT) and mutant constructs, as indicated, were introduced into HeLa cells and the transcripts of the minigenes were analyzed by RT-PCR 24 h post-transfection. The image shows the results of a representative experiment in which the RT-PCR products were separated on a 2.5% agarose gel stained with EtBr and visualized by exposure to ultraviolet light. M, 100 bp DNA ladder (New England Biolabs). c Quantification of splicing events observed in the minigene splicing assay. The relative levels of exon inclusion indicated under the gel are based on RT-PCR experiments equivalent to those shown in B but performed with a fluorescent forward primer and then separated on an automated sequencer. Quantification results were obtained by using the GeneMapper v5.0 software (Applied Biosystems) and correspond to the average of two independent fluorescent-RT-PCR experiments. d Representative fluorescent RT-PCR experiment. The panel shows superposed peaks corresponding to the WT and mutant products (in blue and red, respectively), as indicated
    Figure Legend Snippet: Evaluation of variant-induced splicing alterations by using a cell-based minigene assay. a Structure of pCAS2 minigenes used in the splicing reporter assay. The bent arrow indicates the CMV promoter, boxes represent exons, lines in between the boxes indicate introns, and arrows below the exons represent primers used in RT-PCR reactions. The minigenes were generated by inserting a genomic fragment containing the exon of interest together with its flanking intronic sequences into the intron of pCAS2, as described under Materials and Methods. b Analysis of the splicing pattern of pCAS2 minigenes carrying variants identified in this study. Wild-type (WT) and mutant constructs, as indicated, were introduced into HeLa cells and the transcripts of the minigenes were analyzed by RT-PCR 24 h post-transfection. The image shows the results of a representative experiment in which the RT-PCR products were separated on a 2.5% agarose gel stained with EtBr and visualized by exposure to ultraviolet light. M, 100 bp DNA ladder (New England Biolabs). c Quantification of splicing events observed in the minigene splicing assay. The relative levels of exon inclusion indicated under the gel are based on RT-PCR experiments equivalent to those shown in B but performed with a fluorescent forward primer and then separated on an automated sequencer. Quantification results were obtained by using the GeneMapper v5.0 software (Applied Biosystems) and correspond to the average of two independent fluorescent-RT-PCR experiments. d Representative fluorescent RT-PCR experiment. The panel shows superposed peaks corresponding to the WT and mutant products (in blue and red, respectively), as indicated

    Techniques Used: Variant Assay, Mini Gene Assay, Reporter Assay, Reverse Transcription Polymerase Chain Reaction, Generated, Mutagenesis, Construct, Transfection, Agarose Gel Electrophoresis, Staining, Splicing Assay, Software

    24) Product Images from "Trisomy 21 Alters DNA Methylation in Parent-of-Origin-Dependent and -Independent Manners"

    Article Title: Trisomy 21 Alters DNA Methylation in Parent-of-Origin-Dependent and -Independent Manners

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154108

    5 m CpG statuses at the WRB CGI-2 DMR in a complete androgenetic mole and a human embryonic stem cell line. A consistent unmethylated pattern of CpG sites at the WRB CGI-2 DMR revealed in a sample of an androgenetic complete hydatidiform mole ( A ) contrast with the hypermethylated pattern observed in the representative HUES 3 embryonic cell line ( B ). Electropherograms of the amplimers (see details of the assay in Fig 2 ) generated from either undigested DNA or Hha I-digested DNA. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer.
    Figure Legend Snippet: 5 m CpG statuses at the WRB CGI-2 DMR in a complete androgenetic mole and a human embryonic stem cell line. A consistent unmethylated pattern of CpG sites at the WRB CGI-2 DMR revealed in a sample of an androgenetic complete hydatidiform mole ( A ) contrast with the hypermethylated pattern observed in the representative HUES 3 embryonic cell line ( B ). Electropherograms of the amplimers (see details of the assay in Fig 2 ) generated from either undigested DNA or Hha I-digested DNA. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer.

    Techniques Used: Generated

    Experimental validation of the 5 m CpG statuses at the WRB CGIs. ( A ) A consistent hemimethylated pattern at the WRB CGI-2 revealed in a representative control disomic DNA sample (blood) using the Hha I methylation-sensitive restriction enzyme-based PCR triplex assay developed in this study. Electropherograms of the amplimers generated from either undigested genomic DNA (upper panel) or DNA digested with Hha I (lower panel) genotyped via quantitative fluorescent PCR. The positive amplimer refers to a locus in the ESCO2 gene with constitutively hypomethylated CpG dinucleotides at the target restriction enzyme sites (100% susceptible to Hha I digestion). The negative amplimer refers to a WRB region that lacks Hha I sites, and is, therefore, refractory to enzymatic digestion. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer. In this representative DNA sample, the ratio of 5 m CpG sites at the WRB CGI-2 was 50.6%. In contrast, the assay revealed a consistent unmethylated pattern of CpG sites at the WRB CGI-1 ( B ) and WRB CGI-3 ( C ).
    Figure Legend Snippet: Experimental validation of the 5 m CpG statuses at the WRB CGIs. ( A ) A consistent hemimethylated pattern at the WRB CGI-2 revealed in a representative control disomic DNA sample (blood) using the Hha I methylation-sensitive restriction enzyme-based PCR triplex assay developed in this study. Electropherograms of the amplimers generated from either undigested genomic DNA (upper panel) or DNA digested with Hha I (lower panel) genotyped via quantitative fluorescent PCR. The positive amplimer refers to a locus in the ESCO2 gene with constitutively hypomethylated CpG dinucleotides at the target restriction enzyme sites (100% susceptible to Hha I digestion). The negative amplimer refers to a WRB region that lacks Hha I sites, and is, therefore, refractory to enzymatic digestion. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer. In this representative DNA sample, the ratio of 5 m CpG sites at the WRB CGI-2 was 50.6%. In contrast, the assay revealed a consistent unmethylated pattern of CpG sites at the WRB CGI-1 ( B ) and WRB CGI-3 ( C ).

    Techniques Used: Methylation, Polymerase Chain Reaction, Generated

    25) Product Images from "Proteomic Analyses Using Grifola frondosa Metalloendoprotease Lys-N"

    Article Title: Proteomic Analyses Using Grifola frondosa Metalloendoprotease Lys-N

    Journal: Journal of proteome research

    doi: 10.1021/pr800774h

    Fraction of theoretical fragments ions that are matched to a peak in the recorded spectra for b- and y- type ions intensity for high confidence spectra recorded on either an LTQ ion trap or QSTAR Pulsar i Q-TOF. For the LTQ, high confidence assignments
    Figure Legend Snippet: Fraction of theoretical fragments ions that are matched to a peak in the recorded spectra for b- and y- type ions intensity for high confidence spectra recorded on either an LTQ ion trap or QSTAR Pulsar i Q-TOF. For the LTQ, high confidence assignments

    Techniques Used:

    26) Product Images from "Role of enzymatic activity in muscle damage and cytotoxicity induced by Bothrops asper Asp49 phospholipase A2 myotoxins: are there additional effector mechanisms involved?"

    Article Title: Role of enzymatic activity in muscle damage and cytotoxicity induced by Bothrops asper Asp49 phospholipase A2 myotoxins: are there additional effector mechanisms involved?

    Journal: PeerJ

    doi: 10.7717/peerj.569

    Modification of the Asp49 phospholipase A 2 by p -bromophenacyl bromide ( p -BPB). (A) unmodified enzyme control; (B) p -BPB-treated enzyme; and (C) p -BPB reagent control. The three samples were separated by RP-HPLC in a semi-preparative C 8 column as in Fig. 1B . (D) Comparison of the phospholipase A 2 (PLA 2 ) activities of the control Asp49 enzyme and the p -BPB-treated enzyme on the synthetic monodisperse substrate 4-nitro-3-octanoyloxybenzoic acid. Each point represents mean ± SD of three replicates. (E) Nano-electrospray mass spectrometry confirmation of the covalent incorporation of a single molecule of p -BPB in the modified Asp49 myotoxin. The observed isotope-averaged masses of the modified Asp49 preparation show an increase of 195 ± 3 Da in comparison to the masses of untreated proteins ( Fig. 2D ).
    Figure Legend Snippet: Modification of the Asp49 phospholipase A 2 by p -bromophenacyl bromide ( p -BPB). (A) unmodified enzyme control; (B) p -BPB-treated enzyme; and (C) p -BPB reagent control. The three samples were separated by RP-HPLC in a semi-preparative C 8 column as in Fig. 1B . (D) Comparison of the phospholipase A 2 (PLA 2 ) activities of the control Asp49 enzyme and the p -BPB-treated enzyme on the synthetic monodisperse substrate 4-nitro-3-octanoyloxybenzoic acid. Each point represents mean ± SD of three replicates. (E) Nano-electrospray mass spectrometry confirmation of the covalent incorporation of a single molecule of p -BPB in the modified Asp49 myotoxin. The observed isotope-averaged masses of the modified Asp49 preparation show an increase of 195 ± 3 Da in comparison to the masses of untreated proteins ( Fig. 2D ).

    Techniques Used: Modification, High Performance Liquid Chromatography, Proximity Ligation Assay, Mass Spectrometry

    Analysis of the Asp49 phospholipase A 2 preparation obtained after cation-exchange chromatography and semi-preparative RP-HPLC on C 8 . A Lys49 myotoxin control (A) and the Asp49 peak shown in Fig. 1 (B) were subjected to analytical RP-HPLC on a C 4 column, using a 60 min gradient (see Materials and Methods). For clarity, the gradient line is omitted and the time scale is magnified in the region of interest. The same samples were analyzed by nano-electrospray mass spectrometry in (C) and (D). Insets within dotted lines show the corresponding deconvolutions of the multicharged ion series and calculation of the isotope-averaged observed molecular masses of these samples.
    Figure Legend Snippet: Analysis of the Asp49 phospholipase A 2 preparation obtained after cation-exchange chromatography and semi-preparative RP-HPLC on C 8 . A Lys49 myotoxin control (A) and the Asp49 peak shown in Fig. 1 (B) were subjected to analytical RP-HPLC on a C 4 column, using a 60 min gradient (see Materials and Methods). For clarity, the gradient line is omitted and the time scale is magnified in the region of interest. The same samples were analyzed by nano-electrospray mass spectrometry in (C) and (D). Insets within dotted lines show the corresponding deconvolutions of the multicharged ion series and calculation of the isotope-averaged observed molecular masses of these samples.

    Techniques Used: Chromatography, High Performance Liquid Chromatography, Mass Spectrometry

    27) Product Images from "UNIT 11.10 N-Terminal Sequence Analysis of Proteins and Peptides"

    Article Title: UNIT 11.10 N-Terminal Sequence Analysis of Proteins and Peptides

    Journal: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]

    doi: 10.1002/0471140864.ps1110s08

    (modified to match modified figure) Sequencer sample cartridges. (A) Glass fiber filter (GFF) sample cartridge for the Applied Biosystems Procise sequencer, used to analyze samples in solution. The sample is loaded onto a Polybrene-treated GFF, inserted into the top reaction cartridge block, and the two halves of the cartridge are sealed with a Zitex (Teflon) cartridge seal. (B) Blott cartridge for an Applied Biosystems Procise sequencer, used to analyze proteins bound to PVDF membranes, which are inserted into the semicircular slot in the top of the Blott cartridge.
    Figure Legend Snippet: (modified to match modified figure) Sequencer sample cartridges. (A) Glass fiber filter (GFF) sample cartridge for the Applied Biosystems Procise sequencer, used to analyze samples in solution. The sample is loaded onto a Polybrene-treated GFF, inserted into the top reaction cartridge block, and the two halves of the cartridge are sealed with a Zitex (Teflon) cartridge seal. (B) Blott cartridge for an Applied Biosystems Procise sequencer, used to analyze proteins bound to PVDF membranes, which are inserted into the semicircular slot in the top of the Blott cartridge.

    Techniques Used: Modification, Blocking Assay

    28) Product Images from "Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells"

    Article Title: Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells

    Journal: Genes & Development

    doi: 10.1101/gad.263202.115

    SWI/SNF is required for maximal VEGFR2 expression, nucleosomal remodeling, and expression of other mutant p53-dependent genes. ( A , B ) MDA-468.shp53 cells were grown for 5 d in 2D cell culture in the presence (−Mut p53; black) and absence (+Mut
    Figure Legend Snippet: SWI/SNF is required for maximal VEGFR2 expression, nucleosomal remodeling, and expression of other mutant p53-dependent genes. ( A , B ) MDA-468.shp53 cells were grown for 5 d in 2D cell culture in the presence (−Mut p53; black) and absence (+Mut

    Techniques Used: Expressing, Mutagenesis, Multiple Displacement Amplification, Cell Culture

    The SWI/SNF complex mediates mutant p53-dependent transcription at many mutant p53-responsive genes. ( A ) RNA-seq was performed on two independent replicates of MDA-468.shp53 cells grown for 4 d with either control siRNA, siRNA to deplete mutant p53 (Mut
    Figure Legend Snippet: The SWI/SNF complex mediates mutant p53-dependent transcription at many mutant p53-responsive genes. ( A ) RNA-seq was performed on two independent replicates of MDA-468.shp53 cells grown for 4 d with either control siRNA, siRNA to deplete mutant p53 (Mut

    Techniques Used: Mutagenesis, RNA Sequencing Assay, Multiple Displacement Amplification

    Mutant p53 promotes VEGFR2 expression in breast cancer cells. ( A ) MDA-468.shp53 cells were grown in 3D culture conditions for 8 d with (+DOX) and without (−DOX) doxycycline to induce an shRNA targeting mutant p53. Total VEGFR2 transcript was assayed
    Figure Legend Snippet: Mutant p53 promotes VEGFR2 expression in breast cancer cells. ( A ) MDA-468.shp53 cells were grown in 3D culture conditions for 8 d with (+DOX) and without (−DOX) doxycycline to induce an shRNA targeting mutant p53. Total VEGFR2 transcript was assayed

    Techniques Used: Mutagenesis, Expressing, Multiple Displacement Amplification, shRNA

    Mutant p53 associates with the VEGFR2 promoter and leads to promoter remodeling. MDA-468.shp53 cells were cultured for 8 d in 3D culture in the presence (−Mut p53; black) and absence (+Mut p53; red) of doxycycline (DOX). Cells were treated with
    Figure Legend Snippet: Mutant p53 associates with the VEGFR2 promoter and leads to promoter remodeling. MDA-468.shp53 cells were cultured for 8 d in 3D culture in the presence (−Mut p53; black) and absence (+Mut p53; red) of doxycycline (DOX). Cells were treated with

    Techniques Used: Mutagenesis, Multiple Displacement Amplification, Cell Culture

    Mutant p53 gain of function is mediated by VEGFR2 and may predict response to bevacizumab. ( A ) MDA-231.shp53 cells were engineered to express control vector, VEGFR2, or VEGFR2 tyrosine phosphorylation mutant Y1059F as described in the Materials and Methods
    Figure Legend Snippet: Mutant p53 gain of function is mediated by VEGFR2 and may predict response to bevacizumab. ( A ) MDA-231.shp53 cells were engineered to express control vector, VEGFR2, or VEGFR2 tyrosine phosphorylation mutant Y1059F as described in the Materials and Methods

    Techniques Used: Mutagenesis, Multiple Displacement Amplification, Plasmid Preparation

    Mutant p53 is found in protein complexes with members of the SWI/SNF CRC at the VEGFR2 promoter. Extracts of MDA-468 ( A , C ), SK-BR-3 ( B ), or MDA-231 ( D ) cells were subjected to immunoprecipitation (IP) with anti-p53 antibodies (mAb DO-1; A , B ) or anti-BAF155
    Figure Legend Snippet: Mutant p53 is found in protein complexes with members of the SWI/SNF CRC at the VEGFR2 promoter. Extracts of MDA-468 ( A , C ), SK-BR-3 ( B ), or MDA-231 ( D ) cells were subjected to immunoprecipitation (IP) with anti-p53 antibodies (mAb DO-1; A , B ) or anti-BAF155

    Techniques Used: Mutagenesis, Multiple Displacement Amplification, Immunoprecipitation

    VEGFR2 inhibition phenocopies loss of mutant p53. MDA-231 cells ( A ) and MDA-468 cells ( B ) were transfected with two independent siRNAs to deplete mutant p53 or VEGFR2 and then grown in 3D culture conditions for 8 d. Representative differential interference
    Figure Legend Snippet: VEGFR2 inhibition phenocopies loss of mutant p53. MDA-231 cells ( A ) and MDA-468 cells ( B ) were transfected with two independent siRNAs to deplete mutant p53 or VEGFR2 and then grown in 3D culture conditions for 8 d. Representative differential interference

    Techniques Used: Inhibition, Mutagenesis, Multiple Displacement Amplification, Transfection

    29) Product Images from "Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA)"

    Article Title: Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA)

    Journal: Parasites & Vectors

    doi: 10.1186/1756-3305-4-98

    Electropherogram showing peaks generated by MLPA . Template DNA from individual species is used in the reaction. The MLPA reaction included all the 3 probes designed, Rox 500 as internal molecular standerd. Fragment sizes (bp) correspond to: 198 = C.sinensis , 170 = O.felineus , 152 = O.viverrini
    Figure Legend Snippet: Electropherogram showing peaks generated by MLPA . Template DNA from individual species is used in the reaction. The MLPA reaction included all the 3 probes designed, Rox 500 as internal molecular standerd. Fragment sizes (bp) correspond to: 198 = C.sinensis , 170 = O.felineus , 152 = O.viverrini

    Techniques Used: Generated, Multiplex Ligation-dependent Probe Amplification

    30) Product Images from "Brucella 'HOOF-Prints': strain typing by multi-locus analysis of variable number tandem repeats (VNTRs)"

    Article Title: Brucella 'HOOF-Prints': strain typing by multi-locus analysis of variable number tandem repeats (VNTRs)

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-3-15

    Analysis of allele variability by agarose gel electrophoresis and fluorescent tagged capillary electrophoresis. [A] Amplified fragments from three representative VNTR loci were resolved by electrophoresis in a horizontal gel composed of 3% Metaphor agarose. Sample identifications are given above their respective lanes. Calculated alleles (repeat units) are provided in red under each product. Product sizes range from 143–193 bp (Locus 1); 91–157 bp (Locus-7); and 86 bp (Locus-8). [B] Electropherograms generated from the capillary electrophoresis of fluorescently tagged products amplified from VNTR Locus-1 of the same strains assayed in Panel A. Product sizes (in bp) are shown in boxes below the major peaks. Sizes were calculated by the GeneScan software relative to the GeneScan 500-ROX size markers included in each sample as an internal standard. The vertical axis is the relative peak height of the detected fluorescent products. The first major peak in each electropherogram is the run-through of unincorporated primer.
    Figure Legend Snippet: Analysis of allele variability by agarose gel electrophoresis and fluorescent tagged capillary electrophoresis. [A] Amplified fragments from three representative VNTR loci were resolved by electrophoresis in a horizontal gel composed of 3% Metaphor agarose. Sample identifications are given above their respective lanes. Calculated alleles (repeat units) are provided in red under each product. Product sizes range from 143–193 bp (Locus 1); 91–157 bp (Locus-7); and 86 bp (Locus-8). [B] Electropherograms generated from the capillary electrophoresis of fluorescently tagged products amplified from VNTR Locus-1 of the same strains assayed in Panel A. Product sizes (in bp) are shown in boxes below the major peaks. Sizes were calculated by the GeneScan software relative to the GeneScan 500-ROX size markers included in each sample as an internal standard. The vertical axis is the relative peak height of the detected fluorescent products. The first major peak in each electropherogram is the run-through of unincorporated primer.

    Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, Amplification, Generated, Software

    31) Product Images from "Identification of New Splice Sites Used for Generation of rev Transcripts in Human Immunodeficiency Virus Type 1 Subtype C Primary Isolates"

    Article Title: Identification of New Splice Sites Used for Generation of rev Transcripts in Human Immunodeficiency Virus Type 1 Subtype C Primary Isolates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030574

    GeneMapper analyses of DS RNAs expressed by three HIV-1 subtype C primary isolates in PBMCs. Green peaks represent PCR products and orange peaks represent size standards. Size of PCR product, encoded gene, and exon composition (named as in previous studies [1] , [2] ) predicted according to the size of the PCR product are shown on top or on the side of each peak. Peaks whose sizes do not match HIV-1 transcripts using previously reported splice sites are marked with interrogation signs. For each subtype C virus, three GeneMapper analyses are shown, corresponding to infections using PBMCs from three different donors.
    Figure Legend Snippet: GeneMapper analyses of DS RNAs expressed by three HIV-1 subtype C primary isolates in PBMCs. Green peaks represent PCR products and orange peaks represent size standards. Size of PCR product, encoded gene, and exon composition (named as in previous studies [1] , [2] ) predicted according to the size of the PCR product are shown on top or on the side of each peak. Peaks whose sizes do not match HIV-1 transcripts using previously reported splice sites are marked with interrogation signs. For each subtype C virus, three GeneMapper analyses are shown, corresponding to infections using PBMCs from three different donors.

    Techniques Used: Polymerase Chain Reaction

    32) Product Images from "Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.)"

    Article Title: Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.)

    Journal: TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik

    doi: 10.1007/s00122-011-1550-7

    Electropherograms displaying the patterns of the DNA fragments detected in Chinese Spring and its nulli–tetrasomic lines with the marker system. a Identification of the LMW-GS genes in Chinese Spring with the developed marker system. b Determination of the location of the DNA fragments with the Chinese Spring nulli–tetrasomic lines N1AT1D, N1BT1D and N1DT1B. The horizontal and vertical axes are the same as those described in Fig. 3 b. In Chinese Spring, 15 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 14 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 7 with LMWGS3b and 2 with LMWGS3c). The data collected from three sets of conserved primers indicated that 12 blue peaks (DNA fragments) were detected in N1AT1D, 12 in N1BT1D and 9 in N1DT1B. However, DNA fragment 684 (marked with red arrows ) could be detected in all three nulli–tetrasomic lines. All data provided are representative of four independent sets of PCR amplifications and analyses using the Applied Biosystems 3730 DNA Analyzer
    Figure Legend Snippet: Electropherograms displaying the patterns of the DNA fragments detected in Chinese Spring and its nulli–tetrasomic lines with the marker system. a Identification of the LMW-GS genes in Chinese Spring with the developed marker system. b Determination of the location of the DNA fragments with the Chinese Spring nulli–tetrasomic lines N1AT1D, N1BT1D and N1DT1B. The horizontal and vertical axes are the same as those described in Fig. 3 b. In Chinese Spring, 15 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 14 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 7 with LMWGS3b and 2 with LMWGS3c). The data collected from three sets of conserved primers indicated that 12 blue peaks (DNA fragments) were detected in N1AT1D, 12 in N1BT1D and 9 in N1DT1B. However, DNA fragment 684 (marked with red arrows ) could be detected in all three nulli–tetrasomic lines. All data provided are representative of four independent sets of PCR amplifications and analyses using the Applied Biosystems 3730 DNA Analyzer

    Techniques Used: Marker, Polymerase Chain Reaction

    33) Product Images from "Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.)"

    Article Title: Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.)

    Journal: TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik

    doi: 10.1007/s00122-011-1550-7

    Electropherograms displaying the patterns of the DNA fragments detected in Chinese Spring and its nulli–tetrasomic lines with the marker system. a Identification of the LMW-GS genes in Chinese Spring with the developed marker system. b Determination of the location of the DNA fragments with the Chinese Spring nulli–tetrasomic lines N1AT1D, N1BT1D and N1DT1B. The horizontal and vertical axes are the same as those described in Fig. 3 b. In Chinese Spring, 15 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 14 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 7 with LMWGS3b and 2 with LMWGS3c). The data collected from three sets of conserved primers indicated that 12 blue peaks (DNA fragments) were detected in N1AT1D, 12 in N1BT1D and 9 in N1DT1B. However, DNA fragment 684 (marked with red arrows ) could be detected in all three nulli–tetrasomic lines. All data provided are representative of four independent sets of PCR amplifications and analyses using the Applied Biosystems 3730 DNA Analyzer
    Figure Legend Snippet: Electropherograms displaying the patterns of the DNA fragments detected in Chinese Spring and its nulli–tetrasomic lines with the marker system. a Identification of the LMW-GS genes in Chinese Spring with the developed marker system. b Determination of the location of the DNA fragments with the Chinese Spring nulli–tetrasomic lines N1AT1D, N1BT1D and N1DT1B. The horizontal and vertical axes are the same as those described in Fig. 3 b. In Chinese Spring, 15 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 14 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 7 with LMWGS3b and 2 with LMWGS3c). The data collected from three sets of conserved primers indicated that 12 blue peaks (DNA fragments) were detected in N1AT1D, 12 in N1BT1D and 9 in N1DT1B. However, DNA fragment 684 (marked with red arrows ) could be detected in all three nulli–tetrasomic lines. All data provided are representative of four independent sets of PCR amplifications and analyses using the Applied Biosystems 3730 DNA Analyzer

    Techniques Used: Marker, Polymerase Chain Reaction

    34) Product Images from "Trisomy 21 Alters DNA Methylation in Parent-of-Origin-Dependent and -Independent Manners"

    Article Title: Trisomy 21 Alters DNA Methylation in Parent-of-Origin-Dependent and -Independent Manners

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154108

    5 m CpG statuses at the WRB CGI-2 DMR in a complete androgenetic mole and a human embryonic stem cell line. A consistent unmethylated pattern of CpG sites at the WRB CGI-2 DMR revealed in a sample of an androgenetic complete hydatidiform mole ( A ) contrast with the hypermethylated pattern observed in the representative HUES 3 embryonic cell line ( B ). Electropherograms of the amplimers (see details of the assay in Fig 2 ) generated from either undigested DNA or Hha I-digested DNA. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer.
    Figure Legend Snippet: 5 m CpG statuses at the WRB CGI-2 DMR in a complete androgenetic mole and a human embryonic stem cell line. A consistent unmethylated pattern of CpG sites at the WRB CGI-2 DMR revealed in a sample of an androgenetic complete hydatidiform mole ( A ) contrast with the hypermethylated pattern observed in the representative HUES 3 embryonic cell line ( B ). Electropherograms of the amplimers (see details of the assay in Fig 2 ) generated from either undigested DNA or Hha I-digested DNA. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer.

    Techniques Used: Generated

    Experimental validation of the 5 m CpG statuses at the WRB CGIs. ( A ) A consistent hemimethylated pattern at the WRB CGI-2 revealed in a representative control disomic DNA sample (blood) using the Hha I methylation-sensitive restriction enzyme-based PCR triplex assay developed in this study. Electropherograms of the amplimers generated from either undigested genomic DNA (upper panel) or DNA digested with Hha I (lower panel) genotyped via quantitative fluorescent PCR. The positive amplimer refers to a locus in the ESCO2 gene with constitutively hypomethylated CpG dinucleotides at the target restriction enzyme sites (100% susceptible to Hha I digestion). The negative amplimer refers to a WRB region that lacks Hha I sites, and is, therefore, refractory to enzymatic digestion. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer. In this representative DNA sample, the ratio of 5 m CpG sites at the WRB CGI-2 was 50.6%. In contrast, the assay revealed a consistent unmethylated pattern of CpG sites at the WRB CGI-1 ( B ) and WRB CGI-3 ( C ).
    Figure Legend Snippet: Experimental validation of the 5 m CpG statuses at the WRB CGIs. ( A ) A consistent hemimethylated pattern at the WRB CGI-2 revealed in a representative control disomic DNA sample (blood) using the Hha I methylation-sensitive restriction enzyme-based PCR triplex assay developed in this study. Electropherograms of the amplimers generated from either undigested genomic DNA (upper panel) or DNA digested with Hha I (lower panel) genotyped via quantitative fluorescent PCR. The positive amplimer refers to a locus in the ESCO2 gene with constitutively hypomethylated CpG dinucleotides at the target restriction enzyme sites (100% susceptible to Hha I digestion). The negative amplimer refers to a WRB region that lacks Hha I sites, and is, therefore, refractory to enzymatic digestion. The numbers in the upper boxes correspond to the amplimer lengths in base pairs while those in the lower boxes refer to the areas under the peak of the amplimer. In this representative DNA sample, the ratio of 5 m CpG sites at the WRB CGI-2 was 50.6%. In contrast, the assay revealed a consistent unmethylated pattern of CpG sites at the WRB CGI-1 ( B ) and WRB CGI-3 ( C ).

    Techniques Used: Methylation, Polymerase Chain Reaction, Generated

    Maternal-of-origin-dependent imprinted methylation marks at the WRB CGI-2 DMR. 5 m CpG-sensitive restriction endonuclease sites at the WRB CGI-2 DMR are differentially methylated on the maternal allele versus the paternal allele in a manner consistent with imprinting. Electropherograms of the genotype profiles in a control nuclear family, informative for the rs2244352 (C > A) SNP neighboring the WRB CGI-2 DMR. In the child, the maternal-derived C allele is 100% resistant to Hha I digestion (fully methylated), whereas the paternal-derived A allele is 100% susceptible to Hha I digestion (i.e., unmethylated). The parental allele-specific methylation statuses were validated using the McrBC restriction endonuclease that cleaves methylated DNA and, therefore, the unmethylated paternal allele, but not the maternally methylated allele, remains undigested.
    Figure Legend Snippet: Maternal-of-origin-dependent imprinted methylation marks at the WRB CGI-2 DMR. 5 m CpG-sensitive restriction endonuclease sites at the WRB CGI-2 DMR are differentially methylated on the maternal allele versus the paternal allele in a manner consistent with imprinting. Electropherograms of the genotype profiles in a control nuclear family, informative for the rs2244352 (C > A) SNP neighboring the WRB CGI-2 DMR. In the child, the maternal-derived C allele is 100% resistant to Hha I digestion (fully methylated), whereas the paternal-derived A allele is 100% susceptible to Hha I digestion (i.e., unmethylated). The parental allele-specific methylation statuses were validated using the McrBC restriction endonuclease that cleaves methylated DNA and, therefore, the unmethylated paternal allele, but not the maternally methylated allele, remains undigested.

    Techniques Used: Methylation, Derivative Assay

    35) Product Images from "Role of 5-HTTLPR Polymorphism in the Development of the Inward/Outward Personality Organization: A Genetic Association Study"

    Article Title: Role of 5-HTTLPR Polymorphism in the Development of the Inward/Outward Personality Organization: A Genetic Association Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082192

    Electropherogram of the 5-HTTLPR polymorphism typed by capillary electrophoresis in four different samples. From top to bottom: L-allele homozygosity; S/L heterozygosity; L-allele heterozygosity and intermediate form; S-allele homozygosity.
    Figure Legend Snippet: Electropherogram of the 5-HTTLPR polymorphism typed by capillary electrophoresis in four different samples. From top to bottom: L-allele homozygosity; S/L heterozygosity; L-allele heterozygosity and intermediate form; S-allele homozygosity.

    Techniques Used: Electrophoresis

    36) Product Images from "Sequence variants identification at the KCNQ1OT1:TSS differentially Methylated region in isolated omphalocele cases"

    Article Title: Sequence variants identification at the KCNQ1OT1:TSS differentially Methylated region in isolated omphalocele cases

    Journal: BMC Medical Genetics

    doi: 10.1186/s12881-017-0470-z

    CDKN1C sequence variation in C19 case. In a , the pedigree of C19 is depicted and the proband is indicated by an arrow. As shown, the CDKN1C sequence variation (rs547284149) was inherited from the mother, was also present in the C19 maternal aunt but absent in her daughter who did not show omphalocele. As displayed in. b , this sequence variation determined a non-homogeneous methylation pattern, with a decrease in methylation at the fourth CpG site analyzed by pyrosequencing. The sequence variation and its maternal origin were confirmed by sanger sequencing of KCNQ1OT1 :TSS-DMR: we found a heterozygous non-coding variant G > A at nucleotide 687 (NR_002728.3) ( c )
    Figure Legend Snippet: CDKN1C sequence variation in C19 case. In a , the pedigree of C19 is depicted and the proband is indicated by an arrow. As shown, the CDKN1C sequence variation (rs547284149) was inherited from the mother, was also present in the C19 maternal aunt but absent in her daughter who did not show omphalocele. As displayed in. b , this sequence variation determined a non-homogeneous methylation pattern, with a decrease in methylation at the fourth CpG site analyzed by pyrosequencing. The sequence variation and its maternal origin were confirmed by sanger sequencing of KCNQ1OT1 :TSS-DMR: we found a heterozygous non-coding variant G > A at nucleotide 687 (NR_002728.3) ( c )

    Techniques Used: Sequencing, Methylation, Variant Assay

    Epigenetic and genetic regulation of the 11p15.5 imprinted locus. As shown in ( a) , the 11p15.5 imprinted locus presents with 2 domains (Domain 1 and Domain 2) differentially regulated by H19/IGF2- IG and KCNQ1OT1 :TSS DMRs, respectively. Transcribed genes are depicted in green, silenced genes in red. On the maternal allele, the effect of the enhancer promotes the expression of H19, the most proximal gene, but not of IGF2 . This is the consequence of the CTCF binding to H19/IGF2- IG DMR located between the two genes. On the paternal allele, methylation of this DMR prevents the binding of CTCF, allowing the expression of IGF2 and the silencing of H19 . At KCNQ1OT1 :TSS DMR the opposite methylation pattern is observed. This DMR is methylated on the maternal allele and not on the paternal one. Thus, maternal repression of KCNQ10T allows the expression of CDKN1C and KCNQ1, while the expression of the KCNQ10T on the paternal allele inhibits the transcription of the flanking genes CDKN1C and KCNQ1. CDKN1C gene and protein structure are depicted in ( b) . White striped boxes identify the 5′- and 3′-UTRs and the black portion represents the coding sequence of the gene. CDKN1C encodes for P57 protein (alias KIP2), composed of the three functional domains depicted with different colors. Black arrows indicate previously identified truncating mutations inside the PAPA domain; red arrow points out the susceptibility variant p.A209_P210del identified in this paper
    Figure Legend Snippet: Epigenetic and genetic regulation of the 11p15.5 imprinted locus. As shown in ( a) , the 11p15.5 imprinted locus presents with 2 domains (Domain 1 and Domain 2) differentially regulated by H19/IGF2- IG and KCNQ1OT1 :TSS DMRs, respectively. Transcribed genes are depicted in green, silenced genes in red. On the maternal allele, the effect of the enhancer promotes the expression of H19, the most proximal gene, but not of IGF2 . This is the consequence of the CTCF binding to H19/IGF2- IG DMR located between the two genes. On the paternal allele, methylation of this DMR prevents the binding of CTCF, allowing the expression of IGF2 and the silencing of H19 . At KCNQ1OT1 :TSS DMR the opposite methylation pattern is observed. This DMR is methylated on the maternal allele and not on the paternal one. Thus, maternal repression of KCNQ10T allows the expression of CDKN1C and KCNQ1, while the expression of the KCNQ10T on the paternal allele inhibits the transcription of the flanking genes CDKN1C and KCNQ1. CDKN1C gene and protein structure are depicted in ( b) . White striped boxes identify the 5′- and 3′-UTRs and the black portion represents the coding sequence of the gene. CDKN1C encodes for P57 protein (alias KIP2), composed of the three functional domains depicted with different colors. Black arrows indicate previously identified truncating mutations inside the PAPA domain; red arrow points out the susceptibility variant p.A209_P210del identified in this paper

    Techniques Used: Expressing, Binding Assay, Methylation, Sequencing, Functional Assay, Variant Assay

    37) Product Images from "The miRNA Expression Profile of Experimental Autoimmune Encephalomyelitis Reveals Novel Potential Disease Biomarkers"

    Article Title: The miRNA Expression Profile of Experimental Autoimmune Encephalomyelitis Reveals Novel Potential Disease Biomarkers

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123990

    The differential expression of miRNAs in response to the disease-related antigen (MOG 35–55 ). Splenocytes (SPCs) of MOG 35–55 -induced experimental autoimmune encephalomyelitis (MOG-EAE) mice were restimulated for 24 h with MOG 35–55 (25 μg/mL) or in medium alone without MOG 35–55 (Medium control). The total RNA isolated from these SPCs was then used for miRNA expression using GeneChip TM miRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA). The data was analyzed statistically as follows, ( a ) three-dimensional scatter plot of principal component analysis (PCA) displaying the relationship between the test and control groups and ( b ) heatmap and hierarchical clustering of miRNA expression profiles of MOG 35–55 -restimulated splenocytes of EAE mice and that of SPCs in medium alone (baseline). Red color indicates higher expression, whereas blue color indicates reduced expression of miRNAs. ( n = 3 each for MOG 35–55 restimulation and medium control).
    Figure Legend Snippet: The differential expression of miRNAs in response to the disease-related antigen (MOG 35–55 ). Splenocytes (SPCs) of MOG 35–55 -induced experimental autoimmune encephalomyelitis (MOG-EAE) mice were restimulated for 24 h with MOG 35–55 (25 μg/mL) or in medium alone without MOG 35–55 (Medium control). The total RNA isolated from these SPCs was then used for miRNA expression using GeneChip TM miRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA). The data was analyzed statistically as follows, ( a ) three-dimensional scatter plot of principal component analysis (PCA) displaying the relationship between the test and control groups and ( b ) heatmap and hierarchical clustering of miRNA expression profiles of MOG 35–55 -restimulated splenocytes of EAE mice and that of SPCs in medium alone (baseline). Red color indicates higher expression, whereas blue color indicates reduced expression of miRNAs. ( n = 3 each for MOG 35–55 restimulation and medium control).

    Techniques Used: Expressing, Mouse Assay, Isolation

    38) Product Images from "Long-term rearrangement of retinal structures in a novel mutation of X-linked retinoschisis"

    Article Title: Long-term rearrangement of retinal structures in a novel mutation of X-linked retinoschisis

    Journal: Biomedical Reports

    doi: 10.3892/br.2017.954

    Family pedigree of a Caucasian family with (left) XLRS (left) and (right) p.Ile212Asn electropherograms. The first and second proband are named A and B, respectively, whereas their sister is named C. XLRS, X-linked juvenile retinoschisis.
    Figure Legend Snippet: Family pedigree of a Caucasian family with (left) XLRS (left) and (right) p.Ile212Asn electropherograms. The first and second proband are named A and B, respectively, whereas their sister is named C. XLRS, X-linked juvenile retinoschisis.

    Techniques Used:

    39) Product Images from "Biased T-cell receptor repertoires in patients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome)"

    Article Title: Biased T-cell receptor repertoires in patients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome)

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2003.02134.x

    (a) TCRBV CDR3 spectratyping histograms in a representative patient (#9); (b) Diversity of the TCRBV repertoire analysed by CDR3 spectratyping in patients #1 to #9. The extent of TCRBV repertoire perturbation is represented as the per cent difference between the patient's CDR3 distribution and the corresponding control distribution. A value of perturbation greater than the sum of the s.d.s relative to each CDR3 length found in normal blood donors was considered abnormal. The bars on the left of the doublets represent CD4 + cells, and the bars on the right CD8 + cells. Colours denote the following: grey, abnormal pattern in CD4 + T cells; black, abnormal pattern in CD8 + T cells; white, normal pattern. The BV nomenclature adopted is from Wei et al ].
    Figure Legend Snippet: (a) TCRBV CDR3 spectratyping histograms in a representative patient (#9); (b) Diversity of the TCRBV repertoire analysed by CDR3 spectratyping in patients #1 to #9. The extent of TCRBV repertoire perturbation is represented as the per cent difference between the patient's CDR3 distribution and the corresponding control distribution. A value of perturbation greater than the sum of the s.d.s relative to each CDR3 length found in normal blood donors was considered abnormal. The bars on the left of the doublets represent CD4 + cells, and the bars on the right CD8 + cells. Colours denote the following: grey, abnormal pattern in CD4 + T cells; black, abnormal pattern in CD8 + T cells; white, normal pattern. The BV nomenclature adopted is from Wei et al ].

    Techniques Used:

    Sequence analysis of TCRB V(D)J coding joints from two representative TCRBV families (14 and 21) of patient #9. CDR3 sequences detected more than once in each patient's sample are presented. The repertoire variability within TCRBV14, that showed a gaussian-like CDR3 profile, was 93% versus 96% in the normal (not shown) while that of TCRBV21 was 23% versus 98% in the normal (not shown). The sequence data are available from EMBL/GenBank/DDBJ
    Figure Legend Snippet: Sequence analysis of TCRB V(D)J coding joints from two representative TCRBV families (14 and 21) of patient #9. CDR3 sequences detected more than once in each patient's sample are presented. The repertoire variability within TCRBV14, that showed a gaussian-like CDR3 profile, was 93% versus 96% in the normal (not shown) while that of TCRBV21 was 23% versus 98% in the normal (not shown). The sequence data are available from EMBL/GenBank/DDBJ

    Techniques Used: Sequencing

    40) Product Images from "The LuxR-Type Regulator VpsT Negatively Controls the Transcription of rpoS, Encoding the General Stress Response Regulator, in Vibrio cholerae Biofilms"

    Article Title: The LuxR-Type Regulator VpsT Negatively Controls the Transcription of rpoS, Encoding the General Stress Response Regulator, in Vibrio cholerae Biofilms

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00993-13

    Repression of rpoS transcription by c-di-GMP results in enhanced susceptibility to environmental stressors. V. cholerae C7258 was transformed with plasmid pCMW75 encoding an IPTG-inducible DGC from V. harveyi . Survival in the presence of 2 M NaCl (A),
    Figure Legend Snippet: Repression of rpoS transcription by c-di-GMP results in enhanced susceptibility to environmental stressors. V. cholerae C7258 was transformed with plasmid pCMW75 encoding an IPTG-inducible DGC from V. harveyi . Survival in the presence of 2 M NaCl (A),

    Techniques Used: Transformation Assay, Plasmid Preparation

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    Software:

    Article Title: Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1
    Article Snippet: .. Primers surrounding the 14 intronic SNPs, rs8023330, rs1550436, rs2165241, rs28588430, rs28617339, rs4886778, rs8027022, rs2028386, rs4337252, rs12440667, rs12905253, rs11638944, rs12441130 and rs11631579, and the two exonic SNPs, rs1048661 and rs3825942 of the LOXL1 gene were designed using Primer3 software ( http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi/ ) and supplied from Thermo Scientific (Schwerte, Germany) ( ). .. Purified PCR fragments were sequenced using Big Dye Termination chemistry v.3.1 (Applied Biosystems, ABI, Weiterstadt, Germany) on an automated capillary sequencer (ABI 3730 Genetic Analyzer).

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