map2325  (Roche)


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    Structured Review

    Roche map2325
    PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, <t>MAP2325,</t> and MAP 16S rRNA
    Map2325, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 16129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System"

    Article Title: Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00441-12

    PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA
    Figure Legend Snippet: PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA

    Techniques Used: Polymerase Chain Reaction, Amplification

    2) Product Images from "ABCC3 Expressed by CD56dim CD16+ NK Cells Predicts Response in Glioblastoma Patients Treated with Combined Chemotherapy and Dendritic Cell Immunotherapy"

    Article Title: ABCC3 Expressed by CD56dim CD16+ NK Cells Predicts Response in Glioblastoma Patients Treated with Combined Chemotherapy and Dendritic Cell Immunotherapy

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20235886

    ( A , B ) Representative western blot analysis performed on enriched ( A ) NK and ( B ) CD8 + T cells from donor PBLs showing that 25 μM of TMZ increased the activation of NRF2 (phosphoSer40) after 30 min of treatment. Vinculin was used as loading control. The immunoblot is representative of three experiments. ( C , D ) Densitometric quantification of p-NRF2 expression in NK cells and CD8 + T cells treated with DMSO or TMZ at two different time points. Data are presented as mean ± SD of three independent experiments at the two different time points ( p = 0.004). ( E ). Flow cytometry stacked histograms showing intracellular staining of p-NRF2 in donor-derived CD56 dim CD16 + ABCC3 + . The DMSO treatment is represented as light grey histogram plot. The activation of NRF2 at 10 and 30 min is displayed in dark grey and black, respectively.
    Figure Legend Snippet: ( A , B ) Representative western blot analysis performed on enriched ( A ) NK and ( B ) CD8 + T cells from donor PBLs showing that 25 μM of TMZ increased the activation of NRF2 (phosphoSer40) after 30 min of treatment. Vinculin was used as loading control. The immunoblot is representative of three experiments. ( C , D ) Densitometric quantification of p-NRF2 expression in NK cells and CD8 + T cells treated with DMSO or TMZ at two different time points. Data are presented as mean ± SD of three independent experiments at the two different time points ( p = 0.004). ( E ). Flow cytometry stacked histograms showing intracellular staining of p-NRF2 in donor-derived CD56 dim CD16 + ABCC3 + . The DMSO treatment is represented as light grey histogram plot. The activation of NRF2 at 10 and 30 min is displayed in dark grey and black, respectively.

    Techniques Used: Western Blot, Activation Assay, Expressing, Flow Cytometry, Cytometry, Staining, Derivative Assay

    DelC genetic variant is related to a better response to chemoimmunotherapy. ( A , B ) Kaplan–Meier survival curves showing the correlation between DelC SNP and a good outcome, expressed as ( A ) PFS and ( B ) OS (DelC patients, n = 14, Wt patients, n = 16). ( C ) Scatter dot plots showing the frequency of NK cells expressing ABCC3 in a total of 23 patients (Wt or DelC), at the time of leukapheresis (L). ( D ) Time course of NK cells expressing ABCC3 measured by flow cytometry in DENDR1 patients carrying DelC compared to Wt. ( E , F ). ( E ) Scatter dot plots showing the frequency of NK cells expressing ABCC3 in 13 healthy donors divided in Wt ( n = 6) and DelC ( n = 7). ( F ) Representative histogram overlays for flow-cytometric analysis of ABCC3 expression on NK cells from healthy donors. The isotype control is represented as white histogram plot. The specific fluorescent signals are shown in light grey for Wt donors and dark grey for DelC donors.
    Figure Legend Snippet: DelC genetic variant is related to a better response to chemoimmunotherapy. ( A , B ) Kaplan–Meier survival curves showing the correlation between DelC SNP and a good outcome, expressed as ( A ) PFS and ( B ) OS (DelC patients, n = 14, Wt patients, n = 16). ( C ) Scatter dot plots showing the frequency of NK cells expressing ABCC3 in a total of 23 patients (Wt or DelC), at the time of leukapheresis (L). ( D ) Time course of NK cells expressing ABCC3 measured by flow cytometry in DENDR1 patients carrying DelC compared to Wt. ( E , F ). ( E ) Scatter dot plots showing the frequency of NK cells expressing ABCC3 in 13 healthy donors divided in Wt ( n = 6) and DelC ( n = 7). ( F ) Representative histogram overlays for flow-cytometric analysis of ABCC3 expression on NK cells from healthy donors. The isotype control is represented as white histogram plot. The specific fluorescent signals are shown in light grey for Wt donors and dark grey for DelC donors.

    Techniques Used: Variant Assay, Expressing, Flow Cytometry, Cytometry

    ABCC3 expressed by CD56 dim CD16 + NK cells is an indicator of better patient prognosis. ( A – C ). ( A ) Representative dot plots showing that CD56 dim CD16 + NK cells express ( B ) high levels of ABCC3, ( C ) and CD56 dim CD16 + ABCC3 + NK cells express IFN-γ. ( D ) Time course of frequency of NK cells expressing ABCC3 measured by flow cytometry (* p
    Figure Legend Snippet: ABCC3 expressed by CD56 dim CD16 + NK cells is an indicator of better patient prognosis. ( A – C ). ( A ) Representative dot plots showing that CD56 dim CD16 + NK cells express ( B ) high levels of ABCC3, ( C ) and CD56 dim CD16 + ABCC3 + NK cells express IFN-γ. ( D ) Time course of frequency of NK cells expressing ABCC3 measured by flow cytometry (* p

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    3) Product Images from "The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA"

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    Journal: Genetics

    doi: 10.1534/genetics.118.301672

    R-loop enrichment at the FMR1 and C9orf72 genes by DRIP analysis. (A) R-loop enrichments were determined by DRIP-quantitative PCR with the S9.6 antibody in the FMR1 locus (green bars), RPL13A (positive gene control, blue bars), and ERG1 ). DRIP samples were pretreated (light green, light blue, and light red, respectively) or untreated (dark green, dark blue, and dark red, respectively) with RNase H. The addition of RNase H was used as a control for the pulldown assay. DRIP values are presented as percentage of input (paired t -test, * P
    Figure Legend Snippet: R-loop enrichment at the FMR1 and C9orf72 genes by DRIP analysis. (A) R-loop enrichments were determined by DRIP-quantitative PCR with the S9.6 antibody in the FMR1 locus (green bars), RPL13A (positive gene control, blue bars), and ERG1 ). DRIP samples were pretreated (light green, light blue, and light red, respectively) or untreated (dark green, dark blue, and dark red, respectively) with RNase H. The addition of RNase H was used as a control for the pulldown assay. DRIP values are presented as percentage of input (paired t -test, * P

    Techniques Used: Real-time Polymerase Chain Reaction

    4) Product Images from "Evidence for a rapid rate of molecular evolution at the hypervariable and immunogenic Mycobacterium tuberculosis PPE38 gene region"

    Article Title: Evidence for a rapid rate of molecular evolution at the hypervariable and immunogenic Mycobacterium tuberculosis PPE38 gene region

    Journal: BMC Evolutionary Biology

    doi: 10.1186/1471-2148-9-237

    Schematic representations of the PPE38 gene region in the H37 reference strain published sequences . The PPE38 region from the published H37Rv (2a) and H37Ra (2b) sequences are shown. Colour coding as follows: PPE38 pale blue, PPE71 dark blue, MRA_ 2374 pale green, MRA_ 2375 dark green. Locations of the PPE38F/R and PPE38 IntF/R primers are shown. 2a. H37Rv ATCC reference strain (published whole genome sequence) The published H37Rv sequence [ 1 ] represents the RvD7 genotype. Recombination between PPE38 and PPE71 results in a single PPE38/71 gene (Rv 2352c ) and loss of the 2 esx -like genes MRA_ 2374 and MRA_ 2375 . The PPE38F/R primers (black arrows) are predicted to produce an amplicon of 1335 bp from the RvD7 genotype. It is impossible to determine which PPE38/71 gene has been deleted hence the mixture of colours used. The published H37Rv sequence is not representative of the H37Rv ATCC reference strain, most clinical isolates, or the H37Ra whole genome sequence [ 19 ]. This genotype is also seen in strains SAWC 2240 (CAS, F20), SAWC 1748 (Pre-Haarlem, F24), SAWC 1595 (Quebec/S), SAWC 1841 (Haarlem, F4), CPHL_A (WA-1, M. africanum ), T17 (PGG1, EAI), EAS054 (PGG1, EAI), strain C (LCC,
    Figure Legend Snippet: Schematic representations of the PPE38 gene region in the H37 reference strain published sequences . The PPE38 region from the published H37Rv (2a) and H37Ra (2b) sequences are shown. Colour coding as follows: PPE38 pale blue, PPE71 dark blue, MRA_ 2374 pale green, MRA_ 2375 dark green. Locations of the PPE38F/R and PPE38 IntF/R primers are shown. 2a. H37Rv ATCC reference strain (published whole genome sequence) The published H37Rv sequence [ 1 ] represents the RvD7 genotype. Recombination between PPE38 and PPE71 results in a single PPE38/71 gene (Rv 2352c ) and loss of the 2 esx -like genes MRA_ 2374 and MRA_ 2375 . The PPE38F/R primers (black arrows) are predicted to produce an amplicon of 1335 bp from the RvD7 genotype. It is impossible to determine which PPE38/71 gene has been deleted hence the mixture of colours used. The published H37Rv sequence is not representative of the H37Rv ATCC reference strain, most clinical isolates, or the H37Ra whole genome sequence [ 19 ]. This genotype is also seen in strains SAWC 2240 (CAS, F20), SAWC 1748 (Pre-Haarlem, F24), SAWC 1595 (Quebec/S), SAWC 1841 (Haarlem, F4), CPHL_A (WA-1, M. africanum ), T17 (PGG1, EAI), EAS054 (PGG1, EAI), strain C (LCC, "3 bander") and Haarlem (PGG2, F4) [see additional file 1 ]. 2b. H37Rv ATCC reference strain (actual) and H37Ra (published whole genome sequence) This represents the ancestral MTBC genotype that is also seen in M. canettii . It contains the 2 identical PPE38 (MRA_ 2373) and PPE71 (MRA_ 2376 ) genes separated by the 2 esx -like genes MRA_ 2374 and MRA_ 2375 . Gene annotations are as reported for the H37Ra published sequence [ 19 ]. Locations of primers used for PCR and sequence analysis are indicated (black arrows). This is also the true genotype of the ATTC reference strain H37Rv.

    Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction

    5) Product Images from "The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA"

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    Journal: Genetics

    doi: 10.1534/genetics.118.301672

    Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
    Figure Legend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

    Techniques Used: Footprinting, Sequencing, Amplification, Methylation

    Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.
    Figure Legend Snippet: Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

    Techniques Used: Synthesized, Mutagenesis

    6) Product Images from "Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System"

    Article Title: Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00441-12

    PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA
    Figure Legend Snippet: PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA

    Techniques Used: Polymerase Chain Reaction, Amplification

    7) Product Images from "The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA"

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    Journal: Genetics

    doi: 10.1534/genetics.118.301672

    R-loop enrichment at the FMR1 and C9orf72 genes by DRIP analysis. (A) R-loop enrichments were determined by DRIP-quantitative PCR with the S9.6 antibody in the FMR1 locus (green bars), RPL13A (positive gene control, blue bars), and ERG1 ). DRIP samples were pretreated (light green, light blue, and light red, respectively) or untreated (dark green, dark blue, and dark red, respectively) with RNase H. The addition of RNase H was used as a control for the pulldown assay. DRIP values are presented as percentage of input (paired t -test, * P
    Figure Legend Snippet: R-loop enrichment at the FMR1 and C9orf72 genes by DRIP analysis. (A) R-loop enrichments were determined by DRIP-quantitative PCR with the S9.6 antibody in the FMR1 locus (green bars), RPL13A (positive gene control, blue bars), and ERG1 ). DRIP samples were pretreated (light green, light blue, and light red, respectively) or untreated (dark green, dark blue, and dark red, respectively) with RNase H. The addition of RNase H was used as a control for the pulldown assay. DRIP values are presented as percentage of input (paired t -test, * P

    Techniques Used: Real-time Polymerase Chain Reaction

    8) Product Images from "CRF-Like Diuretic Hormone Negatively Affects Both Feeding and Reproduction in the Desert Locust, Schistocerca gregaria"

    Article Title: CRF-Like Diuretic Hormone Negatively Affects Both Feeding and Reproduction in the Desert Locust, Schistocerca gregaria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031425

    Sequences of the S. gregaria OMP-DH precursor cDNAs and the corresponding amino acid sequences. The longest of both precursor cDNAs (OMP-DH-L precursor cDNA, GenBank: JN591547) is shown here. Consecutively, the coding sequences for the signal peptide (white characters, highlighted in black), the OMP-peptide (black characters highlighted in light gray) and the CRF-like peptide (white characters, highlighted in dark gray) are represented. Predicted dibasic cleavage sites are denoted in bold. Start and stop codons, polyadenylation signal and poly(A) tail are denoted in bold and dotted underlined. The shorter precursor cDNA ( S. gregaria OMP-DH-S precursor) differs from the longer one in the 5′UTR, a tripeptide insertion/deletion and a silent point mutation in the OMP-encoding sequence. All these differences are denoted in bold italics and single underlined. The 5′UTR sequence of the OMP-DH-S precursor is depicted under the 5′UTR of the other precursor. The primers used in the 3′RAcE-procedure are dashed underlined.
    Figure Legend Snippet: Sequences of the S. gregaria OMP-DH precursor cDNAs and the corresponding amino acid sequences. The longest of both precursor cDNAs (OMP-DH-L precursor cDNA, GenBank: JN591547) is shown here. Consecutively, the coding sequences for the signal peptide (white characters, highlighted in black), the OMP-peptide (black characters highlighted in light gray) and the CRF-like peptide (white characters, highlighted in dark gray) are represented. Predicted dibasic cleavage sites are denoted in bold. Start and stop codons, polyadenylation signal and poly(A) tail are denoted in bold and dotted underlined. The shorter precursor cDNA ( S. gregaria OMP-DH-S precursor) differs from the longer one in the 5′UTR, a tripeptide insertion/deletion and a silent point mutation in the OMP-encoding sequence. All these differences are denoted in bold italics and single underlined. The 5′UTR sequence of the OMP-DH-S precursor is depicted under the 5′UTR of the other precursor. The primers used in the 3′RAcE-procedure are dashed underlined.

    Techniques Used: Mutagenesis, Sequencing

    9) Product Images from "Deregulated FADD expression and phosphorylation in T-cell lymphoblastic lymphoma"

    Article Title: Deregulated FADD expression and phosphorylation in T-cell lymphoblastic lymphoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11370

    FADD and P-FADD levels in T-LBL ( A , B ) Fadd (A) and Ahr (B) mRNA levels were determined in healthy thymuses (CTRL) and T-cell lymphoblastic lymphoma samples (T-LBL) by quantitative RT-PCR. The results were normalized using the 2 −ΔΔC T method, referring Fadd or Ahr expression to those of G6pd and Hprt , and referenced to the control group. ( C – F ) Total FADD protein and S191-P-FADD levels were determined in CTRL and T-LBL groups by Western blot (C, D) and Immunohistochemistry (E, F). Representative images are shown for the WB (C) and IHC (E) experiments. WB images in (C) are cropped in favor of conciseness. The box-and-whisker plot analyses of the ratio [S191-P-FADD/FADD] for all the samples are shown for the WB (D) and IHC (F) experiments, indicating the statistical significance of the comparisons. ( G ) Kernel density plot showing T-LBLs density for S191-P-FADD levels (continuous line) and standard normal distribution of each cluster (dashed line). * P
    Figure Legend Snippet: FADD and P-FADD levels in T-LBL ( A , B ) Fadd (A) and Ahr (B) mRNA levels were determined in healthy thymuses (CTRL) and T-cell lymphoblastic lymphoma samples (T-LBL) by quantitative RT-PCR. The results were normalized using the 2 −ΔΔC T method, referring Fadd or Ahr expression to those of G6pd and Hprt , and referenced to the control group. ( C – F ) Total FADD protein and S191-P-FADD levels were determined in CTRL and T-LBL groups by Western blot (C, D) and Immunohistochemistry (E, F). Representative images are shown for the WB (C) and IHC (E) experiments. WB images in (C) are cropped in favor of conciseness. The box-and-whisker plot analyses of the ratio [S191-P-FADD/FADD] for all the samples are shown for the WB (D) and IHC (F) experiments, indicating the statistical significance of the comparisons. ( G ) Kernel density plot showing T-LBLs density for S191-P-FADD levels (continuous line) and standard normal distribution of each cluster (dashed line). * P

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Whisker Assay

    10) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    11) Product Images from "Mechanical properties of DNA-like polymers"

    Article Title: Mechanical properties of DNA-like polymers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt808

    Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).
    Figure Legend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

    Techniques Used: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

    12) Product Images from "Mechanical properties of DNA-like polymers"

    Article Title: Mechanical properties of DNA-like polymers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt808

    Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).
    Figure Legend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

    Techniques Used: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

    13) Product Images from "A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment"

    Article Title: A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073566

    Sequence of the exon-intron junctions of the single GJB2 intron. Exonic sequence is shown in capitals, whereas intronic sequence is shown in lowercase italics. The three acceptor splice sites identified in this work appear boxed in bold: KS, known acceptor site; AS1, alternative site 1; AS2, alternative site 2. The ATG start codon appears in bold. Nucleotides affected by the c. -22-2A > C and c.35delG mutations are indicated by arrows. Locations of the primers used for identifying GJB2 splicing products are underlined.
    Figure Legend Snippet: Sequence of the exon-intron junctions of the single GJB2 intron. Exonic sequence is shown in capitals, whereas intronic sequence is shown in lowercase italics. The three acceptor splice sites identified in this work appear boxed in bold: KS, known acceptor site; AS1, alternative site 1; AS2, alternative site 2. The ATG start codon appears in bold. Nucleotides affected by the c. -22-2A > C and c.35delG mutations are indicated by arrows. Locations of the primers used for identifying GJB2 splicing products are underlined.

    Techniques Used: Sequencing

    Relative expression levels of GJB2 transcripts generated by splicing at different acceptor sites. Each panel shows results for a normal-hearing control (genotype wt/wt) and for c. -22-2A > C/c.35delG compound heterozygous subject II:4. A Relative expression of GJB2 transcript generated by splicing from the known site (two-tailed p
    Figure Legend Snippet: Relative expression levels of GJB2 transcripts generated by splicing at different acceptor sites. Each panel shows results for a normal-hearing control (genotype wt/wt) and for c. -22-2A > C/c.35delG compound heterozygous subject II:4. A Relative expression of GJB2 transcript generated by splicing from the known site (two-tailed p

    Techniques Used: Expressing, Generated, Two Tailed Test

    Sequence electropherograms of cloned GJB2 transcripts from c. -22-2A > C/c.35delG compound heterozygous subject II:4 . The stretch of six guanine nucleotides affected by the c.35delG deletion appears boxed in red, while the start ATG codon is boxed in black. A Sequence of the transcript derived from the c.35delG allele, generated by use of the known acceptor site. Note that the stretch of guanine nucleotides contains only five guanines. B Sequence of the transcript derived from the c. -22-2A > C allele generated by use of the alternative acceptor site 1. Note the cytosine nucleotide at c. -22-2, boxed in purple.
    Figure Legend Snippet: Sequence electropherograms of cloned GJB2 transcripts from c. -22-2A > C/c.35delG compound heterozygous subject II:4 . The stretch of six guanine nucleotides affected by the c.35delG deletion appears boxed in red, while the start ATG codon is boxed in black. A Sequence of the transcript derived from the c.35delG allele, generated by use of the known acceptor site. Note that the stretch of guanine nucleotides contains only five guanines. B Sequence of the transcript derived from the c. -22-2A > C allele generated by use of the alternative acceptor site 1. Note the cytosine nucleotide at c. -22-2, boxed in purple.

    Techniques Used: Sequencing, Clone Assay, Derivative Assay, Generated

    Analysis of GJB2 splicing products. Splicing products from transcription of GJB2 were amplified in a fluorescent RT-PCR assay performed with total RNA extracted from saliva samples and were separated by capillary electrophoresis. A GJB2 splicing products from a normal-hearing control (genotype wt/wt). B GJB2 splicing products from c. -22-2A > C/c.35delG compound heterozygous subject II:4.
    Figure Legend Snippet: Analysis of GJB2 splicing products. Splicing products from transcription of GJB2 were amplified in a fluorescent RT-PCR assay performed with total RNA extracted from saliva samples and were separated by capillary electrophoresis. A GJB2 splicing products from a normal-hearing control (genotype wt/wt). B GJB2 splicing products from c. -22-2A > C/c.35delG compound heterozygous subject II:4.

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

    Hearing impairment in family S1599. A Pedigree of family S1599, indicating the GJB2 genotypes of subjects who were analyzed. B Audiograms (air conduction) of affected individuals II:1 and II:6 at ages 50 and 31 years, respectively (circles, right ear; crosses, left ear). The binaural means of the hearing thresholds for air conduction at frequencies 0.5, 1, and 2 kHz for these subjects are 33.3 and 25.8 dB, respectively, corresponding to mild HI (21-40 dB).
    Figure Legend Snippet: Hearing impairment in family S1599. A Pedigree of family S1599, indicating the GJB2 genotypes of subjects who were analyzed. B Audiograms (air conduction) of affected individuals II:1 and II:6 at ages 50 and 31 years, respectively (circles, right ear; crosses, left ear). The binaural means of the hearing thresholds for air conduction at frequencies 0.5, 1, and 2 kHz for these subjects are 33.3 and 25.8 dB, respectively, corresponding to mild HI (21-40 dB).

    Techniques Used:

    14) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    15) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    16) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    17) Product Images from "Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System"

    Article Title: Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00441-12

    PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA
    Figure Legend Snippet: PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA

    Techniques Used: Polymerase Chain Reaction, Amplification

    18) Product Images from "Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System"

    Article Title: Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00441-12

    PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA
    Figure Legend Snippet: PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA

    Techniques Used: Polymerase Chain Reaction, Amplification

    19) Product Images from "The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA"

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    Journal: Genetics

    doi: 10.1534/genetics.118.301672

    Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
    Figure Legend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

    Techniques Used: Footprinting, Sequencing, Amplification, Methylation

    Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.
    Figure Legend Snippet: Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

    Techniques Used: Synthesized, Mutagenesis

    20) Product Images from "Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity"

    Article Title: Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

    Journal: Journal of Clinical Medicine Research

    doi: 10.14740/jocmr2894w

    Pedigrees of the LQTS families and sequence electropherograms. (a) Family 1 segregates the c.781_782delinsTC mutation in the KCNQ1 gene. The proband (II-1) is indicated by the black arrow. (b) Family 2 segregates the c.2437-5C > A mutation in the SCN5A gene. The proband (II-4) is indicated by the black arrow. The location of the mutation in the sequence electropherograms is indicated by a red box.
    Figure Legend Snippet: Pedigrees of the LQTS families and sequence electropherograms. (a) Family 1 segregates the c.781_782delinsTC mutation in the KCNQ1 gene. The proband (II-1) is indicated by the black arrow. (b) Family 2 segregates the c.2437-5C > A mutation in the SCN5A gene. The proband (II-4) is indicated by the black arrow. The location of the mutation in the sequence electropherograms is indicated by a red box.

    Techniques Used: Sequencing, Mutagenesis

    Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

    Diagrammatic representation of the KCNQ1 and SCN5A proteins. (a) The KCNQ1 protein is composed of six transmembrane domains (S1-S6) with a pore region between S5 and S6 (shown in orange). The section encompassed by the two red scissors and colored in light blue is encoded by exon 6 of the KCNQ1 gene. (b) The SCN5A protein is composed of four homologous transmembrane domains (DI-DIV) with six transmembrane segments (S1-S6) in each section. The four sections’ S1-S4 domains form the channel’s voltage-sensing region, and the four S5-S6 domains with the intervening loop region form the central pore region and selective filter. The section encompassed by the two red scissors and colored in light green is encoded by exon 16 of the SCN5A gene.
    Figure Legend Snippet: Diagrammatic representation of the KCNQ1 and SCN5A proteins. (a) The KCNQ1 protein is composed of six transmembrane domains (S1-S6) with a pore region between S5 and S6 (shown in orange). The section encompassed by the two red scissors and colored in light blue is encoded by exon 6 of the KCNQ1 gene. (b) The SCN5A protein is composed of four homologous transmembrane domains (DI-DIV) with six transmembrane segments (S1-S6) in each section. The four sections’ S1-S4 domains form the channel’s voltage-sensing region, and the four S5-S6 domains with the intervening loop region form the central pore region and selective filter. The section encompassed by the two red scissors and colored in light green is encoded by exon 16 of the SCN5A gene.

    Techniques Used:

    Summary of splice variants reported in HGMD-Pro for the KCNQ1 , KCNH2 and SCN5A genes. Blue boxes represent splice variants that have been characterized by in silico prediction studies and transcript studies; green boxes represent splice variants that have been characterized by transcript studies; orange boxes represent variants that have been predicted to have an effect on splicing using in silico prediction programmes (most using one programme described by Xiong et al [ 11 ]); red boxes represent splice variants that have not been characterized at all by transcript studies or in silico prediction programmes. Dashed purple boxes represent the last exonic nucleotide.
    Figure Legend Snippet: Summary of splice variants reported in HGMD-Pro for the KCNQ1 , KCNH2 and SCN5A genes. Blue boxes represent splice variants that have been characterized by in silico prediction studies and transcript studies; green boxes represent splice variants that have been characterized by transcript studies; orange boxes represent variants that have been predicted to have an effect on splicing using in silico prediction programmes (most using one programme described by Xiong et al [ 11 ]); red boxes represent splice variants that have not been characterized at all by transcript studies or in silico prediction programmes. Dashed purple boxes represent the last exonic nucleotide.

    Techniques Used: In Silico

    21) Product Images from "Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity"

    Article Title: Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

    Journal: Journal of Clinical Medicine Research

    doi: 10.14740/jocmr2894w

    Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

    22) Product Images from "Deregulated FADD expression and phosphorylation in T-cell lymphoblastic lymphoma"

    Article Title: Deregulated FADD expression and phosphorylation in T-cell lymphoblastic lymphoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11370

    FADD and P-FADD levels in T-LBL ( A , B ) Fadd (A) and Ahr (B) mRNA levels were determined in healthy thymuses (CTRL) and T-cell lymphoblastic lymphoma samples (T-LBL) by quantitative RT-PCR. The results were normalized using the 2 −ΔΔC T method, referring Fadd or Ahr expression to those of G6pd and Hprt , and referenced to the control group. ( C – F ) Total FADD protein and S191-P-FADD levels were determined in CTRL and T-LBL groups by Western blot (C, D) and Immunohistochemistry (E, F). Representative images are shown for the WB (C) and IHC (E) experiments. WB images in (C) are cropped in favor of conciseness. The box-and-whisker plot analyses of the ratio [S191-P-FADD/FADD] for all the samples are shown for the WB (D) and IHC (F) experiments, indicating the statistical significance of the comparisons. ( G ) Kernel density plot showing T-LBLs density for S191-P-FADD levels (continuous line) and standard normal distribution of each cluster (dashed line). * P
    Figure Legend Snippet: FADD and P-FADD levels in T-LBL ( A , B ) Fadd (A) and Ahr (B) mRNA levels were determined in healthy thymuses (CTRL) and T-cell lymphoblastic lymphoma samples (T-LBL) by quantitative RT-PCR. The results were normalized using the 2 −ΔΔC T method, referring Fadd or Ahr expression to those of G6pd and Hprt , and referenced to the control group. ( C – F ) Total FADD protein and S191-P-FADD levels were determined in CTRL and T-LBL groups by Western blot (C, D) and Immunohistochemistry (E, F). Representative images are shown for the WB (C) and IHC (E) experiments. WB images in (C) are cropped in favor of conciseness. The box-and-whisker plot analyses of the ratio [S191-P-FADD/FADD] for all the samples are shown for the WB (D) and IHC (F) experiments, indicating the statistical significance of the comparisons. ( G ) Kernel density plot showing T-LBLs density for S191-P-FADD levels (continuous line) and standard normal distribution of each cluster (dashed line). * P

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Whisker Assay

    23) Product Images from "Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity"

    Article Title: Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

    Journal: Journal of Clinical Medicine Research

    doi: 10.14740/jocmr2894w

    Predicted outcomes for carriers of the c.2437-5C > A mutation in the SCN5A gene, together with transcript analysis. (a) Diagrammatic representation of the genomic location of the c.2437-5C > A mutation in the SCN5A gene in relation to the other exons (indicated by the red arrow; top), and the amplicon sizes of the expected PCR products for an unaffected individual (bottom left), and the shortened amplicon sizes for the carriers of the mutation if exon 16 were spliced out (bottom right). The diagram only shows a partial representation of the SCN5A genomic/mRNA sequence instead of all 16 exons. (b) The 2% agarose gel showing the results of PCR amplification of cDNA from the proband’s sister (II-3) and an unrelated control. The sequence electropherogram of the higher molecular weight amplicon (green box) shows the junction between exons 15 and 16 (gray dashed line). The sequence electropherogram of the lower molecular weight amplicon (blue box) shows the in-frame fusion of exons 15 and 17 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.2437-5C > A mutation in the SCN5A gene, together with transcript analysis. (a) Diagrammatic representation of the genomic location of the c.2437-5C > A mutation in the SCN5A gene in relation to the other exons (indicated by the red arrow; top), and the amplicon sizes of the expected PCR products for an unaffected individual (bottom left), and the shortened amplicon sizes for the carriers of the mutation if exon 16 were spliced out (bottom right). The diagram only shows a partial representation of the SCN5A genomic/mRNA sequence instead of all 16 exons. (b) The 2% agarose gel showing the results of PCR amplification of cDNA from the proband’s sister (II-3) and an unrelated control. The sequence electropherogram of the higher molecular weight amplicon (green box) shows the junction between exons 15 and 16 (gray dashed line). The sequence electropherogram of the lower molecular weight amplicon (blue box) shows the in-frame fusion of exons 15 and 17 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Molecular Weight

    Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

    24) Product Images from "Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System"

    Article Title: Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00441-12

    PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA
    Figure Legend Snippet: PCR assays to test the presence or absence of four ORFs located in three long deletions previously identified in S strains. Panels A, B, C, D, and E show the amplified products corresponding to the MAP1485, MAP1487, MAP1738, MAP2325, and MAP 16S rRNA

    Techniques Used: Polymerase Chain Reaction, Amplification

    25) Product Images from "Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity"

    Article Title: Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

    Journal: Journal of Clinical Medicine Research

    doi: 10.14740/jocmr2894w

    Predicted outcomes for carriers of the c.2437-5C > A mutation in the SCN5A gene, together with transcript analysis. (a) Diagrammatic representation of the genomic location of the c.2437-5C > A mutation in the SCN5A gene in relation to the other exons (indicated by the red arrow; top), and the amplicon sizes of the expected PCR products for an unaffected individual (bottom left), and the shortened amplicon sizes for the carriers of the mutation if exon 16 were spliced out (bottom right). The diagram only shows a partial representation of the SCN5A genomic/mRNA sequence instead of all 16 exons. (b) The 2% agarose gel showing the results of PCR amplification of cDNA from the proband’s sister (II-3) and an unrelated control. The sequence electropherogram of the higher molecular weight amplicon (green box) shows the junction between exons 15 and 16 (gray dashed line). The sequence electropherogram of the lower molecular weight amplicon (blue box) shows the in-frame fusion of exons 15 and 17 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.2437-5C > A mutation in the SCN5A gene, together with transcript analysis. (a) Diagrammatic representation of the genomic location of the c.2437-5C > A mutation in the SCN5A gene in relation to the other exons (indicated by the red arrow; top), and the amplicon sizes of the expected PCR products for an unaffected individual (bottom left), and the shortened amplicon sizes for the carriers of the mutation if exon 16 were spliced out (bottom right). The diagram only shows a partial representation of the SCN5A genomic/mRNA sequence instead of all 16 exons. (b) The 2% agarose gel showing the results of PCR amplification of cDNA from the proband’s sister (II-3) and an unrelated control. The sequence electropherogram of the higher molecular weight amplicon (green box) shows the junction between exons 15 and 16 (gray dashed line). The sequence electropherogram of the lower molecular weight amplicon (blue box) shows the in-frame fusion of exons 15 and 17 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Molecular Weight

    Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

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    Article Snippet: .. PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche). .. PCR conditions were an initial denaturation of 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 52–64 °C for 30 s, and 72 °C for 30 s. PCR products were purified with the Qiaquick PCR purification kit (Qiagen) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems).

    Article Title: Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution
    Article Snippet: .. PCR amplification of 16S rRNA genes was conducted using the KAPA2G™ Robust HotStart ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, USA) in a total volume of 25 µl containing inner primer pairs (50 nM each) and the barcoded outer primer mixture (3%) from the PCR Barcoding Kit (SQK-PBK004; Oxford Nanopore Technologies, Oxford, UK). .. Amplification was performed with the following PCR conditions: initial denaturation at 95 °C for 3 min, 5 cycles of 95 °C for 15 sec, 55 °C for 15 sec, and 72 °C for 30 sec, 30 cycles of 95 °C for 15 sec, 62 °C for 15 sec, and 72 °C for 30 sec, followed by a final extension at 72 °C for 1 min. Amplified DNA was purified using AMPure® XP (Beckman Coulter) and quantified by a NanoDrop® 1000 (Thermo Fischer Scientific, Waltham, MA, USA).

    CTG Assay:

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

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    Roche gc rich solution
    Bisulfite footprinting by deep-sequencing across the <t>FMR1</t> repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate <t>(G-rich;</t> left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
    Gc Rich Solution, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gc rich solution/product/Roche
    Average 89 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    gc rich solution - by Bioz Stars, 2020-09
    89/100 stars
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    85
    Roche gc rich solution buffer
    Bisulfite footprinting by deep-sequencing across the <t>FMR1</t> repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate <t>(G-rich;</t> left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
    Gc Rich Solution Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gc rich solution buffer/product/Roche
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gc rich solution buffer - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    Image Search Results


    Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

    Journal: Genetics

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    doi: 10.1534/genetics.118.301672

    Figure Lengend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

    Article Snippet: For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche).

    Techniques: Footprinting, Sequencing, Amplification, Methylation

    Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

    Journal: Genetics

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    doi: 10.1534/genetics.118.301672

    Figure Lengend Snippet: Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

    Article Snippet: For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche).

    Techniques: Synthesized, Mutagenesis