mtdna content  (Qiagen)


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    QIAcube
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    For fully automated sample prep using QIAGEN spin column kits Kit contents Robotic workstation for automated purification of DNA RNA or proteins using QIAGEN spin column kits includes1 year warranty on parts and labor Benefits Automation of trusted QIAGEN spin column kits Elimination of manual processing steps Purification of DNA RNA or proteins More free time with affordable automated processing Standardized results and increased productivity
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    Structured Review

    Qiagen mtdna content
    QIAcube
    For fully automated sample prep using QIAGEN spin column kits Kit contents Robotic workstation for automated purification of DNA RNA or proteins using QIAGEN spin column kits includes1 year warranty on parts and labor Benefits Automation of trusted QIAGEN spin column kits Elimination of manual processing steps Purification of DNA RNA or proteins More free time with affordable automated processing Standardized results and increased productivity
    https://www.bioz.com/result/mtdna content/product/Qiagen
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    mtdna content - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice"

    Article Title: Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33930-w

    Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P
    Figure Legend Snippet: Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P

    Techniques Used: Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Mouse Assay

    2) Product Images from "The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer"

    Article Title: The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0137-x

    UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in ( e and g ) (* P
    Figure Legend Snippet: UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in ( e and g ) (* P

    Techniques Used: Over Expression, Western Blot, Transfection, shRNA

    3) Product Images from "Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics"

    Article Title: Molecular Signature of CAID Syndrome: Noncanonical Roles of SGO1 in Regulation of TGF-β Signaling and Epigenomics

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2018.10.011

    Epigenetic profile of CAID patients. ( A ) Heatmap representation of methylation levels in CAID patients vs controls among all tiles at early (p8) and late stage (p14) showed a global hypermethylation pattern. ( B ) Volcano plot of the methylation difference (%) at early (p8) and late stage (p14). Yellow ≥-30% and blue ≥30% methylation differences. ( C ) Tile proportions of differentially methylated regions at early (p8) and late stage (p14). For each group, N = 3 independent biological replicates. P values were corrected for multiple testing using the Benjamini–Hochberg method (q value). ( D ) Methylation percentage of LINE-1 CpG sites assessed by pyrosequencing. LINE-1 CpGs are significantly more methylated in CAID patients than in controls. Error bars signify SD. For each condition, the experiment was performed on N = 3 independent biological replicates in technical replicates. Significance was calculated by 1-way analysis of variance with the Bonferroni post-test ( • P
    Figure Legend Snippet: Epigenetic profile of CAID patients. ( A ) Heatmap representation of methylation levels in CAID patients vs controls among all tiles at early (p8) and late stage (p14) showed a global hypermethylation pattern. ( B ) Volcano plot of the methylation difference (%) at early (p8) and late stage (p14). Yellow ≥-30% and blue ≥30% methylation differences. ( C ) Tile proportions of differentially methylated regions at early (p8) and late stage (p14). For each group, N = 3 independent biological replicates. P values were corrected for multiple testing using the Benjamini–Hochberg method (q value). ( D ) Methylation percentage of LINE-1 CpG sites assessed by pyrosequencing. LINE-1 CpGs are significantly more methylated in CAID patients than in controls. Error bars signify SD. For each condition, the experiment was performed on N = 3 independent biological replicates in technical replicates. Significance was calculated by 1-way analysis of variance with the Bonferroni post-test ( • P

    Techniques Used: Methylation

    4) Product Images from "Reappraisal of the Therapeutic Role of Celecoxib in Cholangiocarcinoma"

    Article Title: Reappraisal of the Therapeutic Role of Celecoxib in Cholangiocarcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069928

    COX-2 expression in rat CCA and apoptosis detection in treated rat CCA. (A) Rat CCA tissue revealed positive cytoplasmic immunoreactivity for COX-2 (×400). (B) Apoptotic DNA fragmentation of rat CCA tissue after treatment with celecoxib (160 mg/kg body weight) for 5 weeks visualised by the DNA laddering assay. No apoptotic DNA fragmentation was observed in the control group treated with DMSO buffer for 5 weeks. (C, D, E) TUNNEL assay for determination of rat CCA apoptosis. The positive cells for TUNNEL assay with brown stains are shown in the positive control (TACS-Nuclease treated tissues ) figure (D). No apoptosis was detected in the control group (C). (E) Apoptosis cells were detected in rat CCA tissues treated with celecoxib (160 mg/kg body weight).
    Figure Legend Snippet: COX-2 expression in rat CCA and apoptosis detection in treated rat CCA. (A) Rat CCA tissue revealed positive cytoplasmic immunoreactivity for COX-2 (×400). (B) Apoptotic DNA fragmentation of rat CCA tissue after treatment with celecoxib (160 mg/kg body weight) for 5 weeks visualised by the DNA laddering assay. No apoptotic DNA fragmentation was observed in the control group treated with DMSO buffer for 5 weeks. (C, D, E) TUNNEL assay for determination of rat CCA apoptosis. The positive cells for TUNNEL assay with brown stains are shown in the positive control (TACS-Nuclease treated tissues ) figure (D). No apoptosis was detected in the control group (C). (E) Apoptosis cells were detected in rat CCA tissues treated with celecoxib (160 mg/kg body weight).

    Techniques Used: Expressing, DNA Laddering, Positive Control

    5) Product Images from "Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification"

    Article Title: Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

    Journal: Infectious Diseases

    doi: 10.4137/IDRT.S32162

    LAMP of DNA isolated from DBS showing real-time amplification in the positive control (genomic Plasmodium DNA) and amplification from DNA isolated from DBS containing 10 3 and 10 6 parasites/µL.
    Figure Legend Snippet: LAMP of DNA isolated from DBS showing real-time amplification in the positive control (genomic Plasmodium DNA) and amplification from DNA isolated from DBS containing 10 3 and 10 6 parasites/µL.

    Techniques Used: Isolation, Amplification, Positive Control

    6) Product Images from "Identification of a novel β-adrenergic octopamine receptor-like gene (βAOR-like) and increased ATP-binding cassette B10 (ABCB10) expression in a Rhipicephalus microplus cell line derived from acaricide-resistant ticks"

    Article Title: Identification of a novel β-adrenergic octopamine receptor-like gene (βAOR-like) and increased ATP-binding cassette B10 (ABCB10) expression in a Rhipicephalus microplus cell line derived from acaricide-resistant ticks

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-016-1708-x

    βAOR PCR in Rhipicephalus cell lines. a Detection of βAOR in genomic DNA (gDNA) and complementary DNA (cDNA) of Rhipicephalus microplus cell lines. The expected amplicon of 183 bp was detected in gDNA and cDNA of cell lines BME/CTVM2 (2), BME/CTVM5 (5), BME/CTVM23 (23), BME/CTVM30 (30) and BmVIII-SCC (SCC). BME/CTVM6 (6) gave an alternative amplicon of ~220 bp from both gDNA and cDNA. Additional amplicons of ~245 bp and ~220 bp were also detected in gDNA of BME/CTVM5. b Detection of βAOR in gDNA and cDNA of other tick cell lines. The expected amplicon was detected in gDNA and cDNA of cell lines RAN/CTVM3 (RAN), REN/CTVM32 (REN), RSE/PILS35 (RSE) and RA243, and only in gDNA of RML-RSE (RML). No βAOR gene or transcript was detected in cell lines AVL/CTVM13 (AVL), HAE/CTVM9 (HAE) or IRE/CTVM19 (IRE). Abbreviations : M, Marker; NTC, Non-template control; −ve, reverse transcription negative control
    Figure Legend Snippet: βAOR PCR in Rhipicephalus cell lines. a Detection of βAOR in genomic DNA (gDNA) and complementary DNA (cDNA) of Rhipicephalus microplus cell lines. The expected amplicon of 183 bp was detected in gDNA and cDNA of cell lines BME/CTVM2 (2), BME/CTVM5 (5), BME/CTVM23 (23), BME/CTVM30 (30) and BmVIII-SCC (SCC). BME/CTVM6 (6) gave an alternative amplicon of ~220 bp from both gDNA and cDNA. Additional amplicons of ~245 bp and ~220 bp were also detected in gDNA of BME/CTVM5. b Detection of βAOR in gDNA and cDNA of other tick cell lines. The expected amplicon was detected in gDNA and cDNA of cell lines RAN/CTVM3 (RAN), REN/CTVM32 (REN), RSE/PILS35 (RSE) and RA243, and only in gDNA of RML-RSE (RML). No βAOR gene or transcript was detected in cell lines AVL/CTVM13 (AVL), HAE/CTVM9 (HAE) or IRE/CTVM19 (IRE). Abbreviations : M, Marker; NTC, Non-template control; −ve, reverse transcription negative control

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

    7) Product Images from "Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function"

    Article Title: Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function

    Journal: Nature Communications

    doi: 10.1038/ncomms12723

    Catecholamine treatment triggers Perilipin 5 interaction with PGC-1α and SIRT1 that promotes PGC-1α deacetylation. ( a ) Immunoprecipitation (IP) of PGC-1α (left) and of Perilipin 5 (right) from nuclear extracts of C2C12 myotubes treated with vehicle or Iso/IBMX for 2 h. ( b ) IP of Perilipin 5 from BAT of wild-type mice treated with β3 adrenergic agonist CL 316,243 (CL) or vehicle. ( c , d ) IP of Perilipin 5 ( c ) or PGC-1α ( d ) from whole-cell lysate of brown adipocytes treated with vehicle or Iso/IBMX for 2 h. IgG control IP and siRNA duplexes for PGC-1α and/or Perilipin 5 were used as IP controls. ( e ) IP from whole-cell extract of C2C12 myoblasts transfected with myc-Perilipin 5 and flag-PGC-1α and treated with Iso/IBMX for 2 h. Non-transfected cells were used as IP control. ( f ) PLA reveals Perilipin 5 interaction with PGC-1α in C2C12 differentiating myoblasts treated with vehicle or Iso/IBMX for 2 h. Red dots indicate proximity of interaction partners within 20–40 nm (left) with quantification using Cell Profiler software (right). ( g ) IP of PGC-1α from whole-cell lysate of brown adypocytes treated with Iso/IBMX for 2 h. Cells were additionally treated with orlistat or vehicle and siATGL or siControl. IgG control and siPGC-1α were used as IP controls. ( h ) IP of PGC-1α from nuclear extracts of MLTC-1 cells transfected with siControl or siPerilipin 5. Non-immune rabbit IgG was used as the IP-negative control.
    Figure Legend Snippet: Catecholamine treatment triggers Perilipin 5 interaction with PGC-1α and SIRT1 that promotes PGC-1α deacetylation. ( a ) Immunoprecipitation (IP) of PGC-1α (left) and of Perilipin 5 (right) from nuclear extracts of C2C12 myotubes treated with vehicle or Iso/IBMX for 2 h. ( b ) IP of Perilipin 5 from BAT of wild-type mice treated with β3 adrenergic agonist CL 316,243 (CL) or vehicle. ( c , d ) IP of Perilipin 5 ( c ) or PGC-1α ( d ) from whole-cell lysate of brown adipocytes treated with vehicle or Iso/IBMX for 2 h. IgG control IP and siRNA duplexes for PGC-1α and/or Perilipin 5 were used as IP controls. ( e ) IP from whole-cell extract of C2C12 myoblasts transfected with myc-Perilipin 5 and flag-PGC-1α and treated with Iso/IBMX for 2 h. Non-transfected cells were used as IP control. ( f ) PLA reveals Perilipin 5 interaction with PGC-1α in C2C12 differentiating myoblasts treated with vehicle or Iso/IBMX for 2 h. Red dots indicate proximity of interaction partners within 20–40 nm (left) with quantification using Cell Profiler software (right). ( g ) IP of PGC-1α from whole-cell lysate of brown adypocytes treated with Iso/IBMX for 2 h. Cells were additionally treated with orlistat or vehicle and siATGL or siControl. IgG control and siPGC-1α were used as IP controls. ( h ) IP of PGC-1α from nuclear extracts of MLTC-1 cells transfected with siControl or siPerilipin 5. Non-immune rabbit IgG was used as the IP-negative control.

    Techniques Used: Pyrolysis Gas Chromatography, Immunoprecipitation, Mouse Assay, Transfection, Proximity Ligation Assay, Software, Negative Control

    Perilipin 5 regulates catecholamine-stimulated SIRT1 deacetylation activity and mitochondrial biogenesis and respiration. ( a ) MLTC-1 cells were transfected with the indicated siRNA duplexes. After 72 h, cells were treated with Iso/IBMX or vehicle for 30 min and SIRT1 deacetylase activity was measured in the cell lysates (left). Western blotting for SIRT1, Perilipin 5 and GAPDH document siRNA specificity (bottom). Intracellular NAD + levels with Iso/IBMX or siPerilipin 5 treatments (right). ( b ) Immunoprecipitation of SIRT1 followed by WB for DBC1 and SIRT1 from MLTC-1 cells transfected with the indicated siRNA duplexes. Cells were treated with vehicle or Iso/IBMX for 30 min before immunoprecipitation. Relative quantification of immunoprecipitated DBC-1 normalized for immunoprecipitated SIRT1 is shown in the histogram. ( c ) PLA for SIRT1-DBC1 complexes (red dots) in MLTC-1 cells transfected with the indicated siRNA duplexes for 48 h followed by treatment with vehicle or Iso/IBMX for 30 min (left) with quantification using Cell Profiler (right). ( d ) mtDNA content analysed by qPCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX for 2 h. ( e ) OCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX. n =3 biological replicates/experimental condition with each biological replicate representing an independent transfection and treatment. Values are expressed as the mean±s.e.m. These data were replicated in a second independent experiment. * P
    Figure Legend Snippet: Perilipin 5 regulates catecholamine-stimulated SIRT1 deacetylation activity and mitochondrial biogenesis and respiration. ( a ) MLTC-1 cells were transfected with the indicated siRNA duplexes. After 72 h, cells were treated with Iso/IBMX or vehicle for 30 min and SIRT1 deacetylase activity was measured in the cell lysates (left). Western blotting for SIRT1, Perilipin 5 and GAPDH document siRNA specificity (bottom). Intracellular NAD + levels with Iso/IBMX or siPerilipin 5 treatments (right). ( b ) Immunoprecipitation of SIRT1 followed by WB for DBC1 and SIRT1 from MLTC-1 cells transfected with the indicated siRNA duplexes. Cells were treated with vehicle or Iso/IBMX for 30 min before immunoprecipitation. Relative quantification of immunoprecipitated DBC-1 normalized for immunoprecipitated SIRT1 is shown in the histogram. ( c ) PLA for SIRT1-DBC1 complexes (red dots) in MLTC-1 cells transfected with the indicated siRNA duplexes for 48 h followed by treatment with vehicle or Iso/IBMX for 30 min (left) with quantification using Cell Profiler (right). ( d ) mtDNA content analysed by qPCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX for 2 h. ( e ) OCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX. n =3 biological replicates/experimental condition with each biological replicate representing an independent transfection and treatment. Values are expressed as the mean±s.e.m. These data were replicated in a second independent experiment. * P

    Techniques Used: Activity Assay, Transfection, Histone Deacetylase Assay, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Real-time Polymerase Chain Reaction

    8) Product Images from "The interactions of dopamine and oxidative damage in the striatum of patients with neurodegenerative diseases"

    Article Title: The interactions of dopamine and oxidative damage in the striatum of patients with neurodegenerative diseases

    Journal: Journal of Neurochemistry

    doi: 10.1111/jnc.14898

    (a) Concentration of dopamine in the caudate and putamen from patients with diseases [Parkinson disease (PD): n = 10, Parkinson disease dementia (PDD): n = 7, dementia with Lewy bodies (DLB): n = 10, Alzheimer's disease (AD): n = 26] and age‐matched controls ( n = 10). Value shown are means ± SEM. (b) Concentration of dopamine vs level of 8‐oxo‐7,8‐dihydro‐2'‐deoxyguanosine (8‐oxo‐dG) in the caudate from diseases brains, significantly association was observed only in AD group ( p = 0.026). (c) Concentration of dopamine vs vesicular monoamine transporter 2 (VMAT2) expression in the putamen from diseases brains, significantly association was observed only in DLB group ( p = 0.050). r s , the Spearman's rank correlation coefficient.
    Figure Legend Snippet: (a) Concentration of dopamine in the caudate and putamen from patients with diseases [Parkinson disease (PD): n = 10, Parkinson disease dementia (PDD): n = 7, dementia with Lewy bodies (DLB): n = 10, Alzheimer's disease (AD): n = 26] and age‐matched controls ( n = 10). Value shown are means ± SEM. (b) Concentration of dopamine vs level of 8‐oxo‐7,8‐dihydro‐2'‐deoxyguanosine (8‐oxo‐dG) in the caudate from diseases brains, significantly association was observed only in AD group ( p = 0.026). (c) Concentration of dopamine vs vesicular monoamine transporter 2 (VMAT2) expression in the putamen from diseases brains, significantly association was observed only in DLB group ( p = 0.050). r s , the Spearman's rank correlation coefficient.

    Techniques Used: Concentration Assay, Expressing

    8‐oxo‐dG levels in the caudate and putamen from patients with diseases [Parkinson disease (PD): n = 10, Parkinson disease dementia (PDD): n = 7, dementia with Lewy bodies (DLB): n = 10, Alzheimer's disease (AD): n = 26] and age‐matched controls ( n = 10). Value shown are means ± SEM as the concentration of 8‐oxo‐7,8‐dihydro‐2'‐deoxyguanosine (8‐oxo‐dG) (pg) per total DNA (µg). A p value of
    Figure Legend Snippet: 8‐oxo‐dG levels in the caudate and putamen from patients with diseases [Parkinson disease (PD): n = 10, Parkinson disease dementia (PDD): n = 7, dementia with Lewy bodies (DLB): n = 10, Alzheimer's disease (AD): n = 26] and age‐matched controls ( n = 10). Value shown are means ± SEM as the concentration of 8‐oxo‐7,8‐dihydro‐2'‐deoxyguanosine (8‐oxo‐dG) (pg) per total DNA (µg). A p value of

    Techniques Used: Concentration Assay

    9) Product Images from "Distinct Role of Sesn2 in Response to UVB-Induced DNA Damage and UVA-Induced Oxidative Stress in Melanocytes †"

    Article Title: Distinct Role of Sesn2 in Response to UVB-Induced DNA Damage and UVA-Induced Oxidative Stress in Melanocytes †

    Journal: Photochemistry and photobiology

    doi: 10.1111/php.12624

    Role of Sesn2 in UVB-induced DNA damage repair. (A) Slot blot analysis of CPD levels in A375 cells stably infected with plu-GFP (control) or plu-Sesn2 at 0, 20 h or 48 h post-UVB irradiation. (B) Quantification of A. (C) Slot blot analysis of CPD levels
    Figure Legend Snippet: Role of Sesn2 in UVB-induced DNA damage repair. (A) Slot blot analysis of CPD levels in A375 cells stably infected with plu-GFP (control) or plu-Sesn2 at 0, 20 h or 48 h post-UVB irradiation. (B) Quantification of A. (C) Slot blot analysis of CPD levels

    Techniques Used: Dot Blot, Stable Transfection, Infection, Irradiation

    10) Product Images from "Rapid pathogen identification using a novel microarray-based assay with purulent meningitis in cerebrospinal fluid"

    Article Title: Rapid pathogen identification using a novel microarray-based assay with purulent meningitis in cerebrospinal fluid

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34051-0

    The specificity and sensitivity of the pathogen probes. ( A ) Microarray hybridized with the probe untarget bacteria and human genome DNA. ( B ) Microarray hybridized with the probe target bacteria. NC means microarray hybridized with ddH 2 O and positive control sequence (show the signal of PC probes). ( C ) Microarray hybridized with the Escherichia coli (ATCC 25922) which diluted for concentration gradient (the original DNA samples were extracted from CSF).
    Figure Legend Snippet: The specificity and sensitivity of the pathogen probes. ( A ) Microarray hybridized with the probe untarget bacteria and human genome DNA. ( B ) Microarray hybridized with the probe target bacteria. NC means microarray hybridized with ddH 2 O and positive control sequence (show the signal of PC probes). ( C ) Microarray hybridized with the Escherichia coli (ATCC 25922) which diluted for concentration gradient (the original DNA samples were extracted from CSF).

    Techniques Used: Microarray, Positive Control, Sequencing, Concentration Assay

    11) Product Images from "Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells"

    Article Title: Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-62703-739-6_31

    PCR products of QIAcube-extracted mouse DNA+/−digestion with HaeII. QIAcube extraction apparently results in mostly covalently closed supercoiled mtDNA, which limits primer access. HaeII digestion near the D-Loop (bp ~2,607) greatly increases amplification of the large target. Raw fluorescence values of Lmito ( a ) and Smito ( b ) and hence lesion frequencies ( c ) are affected by digestion. Lesion frequencies represent the decrease in amplification of the large mitochondrial fragment normalized to the small fragment. Data represent the mean+/−SD of two biological samples. Net fluorescence: Picogreen fluorescence of the PCR product minus a “no-template” control
    Figure Legend Snippet: PCR products of QIAcube-extracted mouse DNA+/−digestion with HaeII. QIAcube extraction apparently results in mostly covalently closed supercoiled mtDNA, which limits primer access. HaeII digestion near the D-Loop (bp ~2,607) greatly increases amplification of the large target. Raw fluorescence values of Lmito ( a ) and Smito ( b ) and hence lesion frequencies ( c ) are affected by digestion. Lesion frequencies represent the decrease in amplification of the large mitochondrial fragment normalized to the small fragment. Data represent the mean+/−SD of two biological samples. Net fluorescence: Picogreen fluorescence of the PCR product minus a “no-template” control

    Techniques Used: Polymerase Chain Reaction, Amplification, Fluorescence

    12) Product Images from "The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability"

    Article Title: The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability

    Journal: Nigerian Medical Journal : Journal of the Nigeria Medical Association

    doi: 10.4103/0300-1652.188321

    5 different extraction methods for three sizes, (a) Figure: 110 bp size, (b) 256 and (c) 512bp of amplified β-globin gene primers. 1. Phenol-chloroform method, 2. QIAamp DNA FFPE Tissue Kit method, 3. Proteinase K and xylol method, 4. Heat alkaline method, 5. Heat alkaline plus mineral oil method and 6 is Negative control
    Figure Legend Snippet: 5 different extraction methods for three sizes, (a) Figure: 110 bp size, (b) 256 and (c) 512bp of amplified β-globin gene primers. 1. Phenol-chloroform method, 2. QIAamp DNA FFPE Tissue Kit method, 3. Proteinase K and xylol method, 4. Heat alkaline method, 5. Heat alkaline plus mineral oil method and 6 is Negative control

    Techniques Used: Amplification, Formalin-fixed Paraffin-Embedded, Negative Control

    13) Product Images from "Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities"

    Article Title: Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169877

    Normalised DNA yield (ng/ μ L) from (A) plaque and (B) saliva (mean ± SEM). M: MoBio PowerSoil ® DNA Isolation Kit, Q: QIAamp ® DNA Mini Kit, Z: Zymo Bacterial/Fungal DNA Mini Prep TM , P: Phenol:chloroform-based DNA isolation. DNA extraction methods not linked by the same letter are significantly different to each other (Tukey-Kramer p ≤ 0.05).
    Figure Legend Snippet: Normalised DNA yield (ng/ μ L) from (A) plaque and (B) saliva (mean ± SEM). M: MoBio PowerSoil ® DNA Isolation Kit, Q: QIAamp ® DNA Mini Kit, Z: Zymo Bacterial/Fungal DNA Mini Prep TM , P: Phenol:chloroform-based DNA isolation. DNA extraction methods not linked by the same letter are significantly different to each other (Tukey-Kramer p ≤ 0.05).

    Techniques Used: DNA Extraction

    Taxa plots summarising the relative abundance of taxon-assigned OTUs identified in pooled homogenates for (A) bacterial genera in plaque; (B) fungal genera in plaque; (C) bacterial genera in saliva and (D) fungal genera in saliva. Each bar represents sequencing from three replicates, rarefied to 6000 sequences per sample for bacterial genera and 959 for fungal genera, with replicates that did not meet these criteria excluded. M: MoBio PowerSoil ® DNA Isolation Kit, Q: QIAamp ® DNA Mini Kit, Z: Zymo Bacterial/Fungal DNA Mini Prep TM , P: Phenol:chloroform-based DNA isolation.
    Figure Legend Snippet: Taxa plots summarising the relative abundance of taxon-assigned OTUs identified in pooled homogenates for (A) bacterial genera in plaque; (B) fungal genera in plaque; (C) bacterial genera in saliva and (D) fungal genera in saliva. Each bar represents sequencing from three replicates, rarefied to 6000 sequences per sample for bacterial genera and 959 for fungal genera, with replicates that did not meet these criteria excluded. M: MoBio PowerSoil ® DNA Isolation Kit, Q: QIAamp ® DNA Mini Kit, Z: Zymo Bacterial/Fungal DNA Mini Prep TM , P: Phenol:chloroform-based DNA isolation.

    Techniques Used: Sequencing, DNA Extraction

    14) Product Images from "Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR"

    Article Title: Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169846

    Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns. Genomic and plasmid DNA were isolated from E . coli DH5α and E . coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P
    Figure Legend Snippet: Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns. Genomic and plasmid DNA were isolated from E . coli DH5α and E . coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P

    Techniques Used: Plasmid Preparation, Isolation, Spectrophotometry, Lysis, Standard Deviation

    Quantification of pBR322 plasmid copy number by digital droplet PCR. E . coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A ( Table 1A ). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs , respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs . Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.
    Figure Legend Snippet: Quantification of pBR322 plasmid copy number by digital droplet PCR. E . coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A ( Table 1A ). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs , respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs . Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Isolation, Amplification

    15) Product Images from "Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid"

    Article Title: Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22416-4

    Sample collection, DNA isolation and shotgun metagenomic sequencing. ( A ) (I.) Sample collection: Vitreous body (intraocular body fluid) was collected through vitrectomy from 14 patients with endophthalmitis following cataract surgery (n = 7) and intravitreal injection (n = 7). As control, vitreous was collected from 7 patients without postoperative endophthalmitis during macula hole surgery. Six aliquots (3 sample pairs) were obtained from balanced salt solution (BSS) that is infused into the eye during vitrectomy. Three aliquots were collected from separate BSS bottles (BSS-B) and the second set of aliquots was collected from the vitrectomy surgical system (BSS-S) after it had passed through the vitrectomy infusion line, respectively. The samples were examined using (II.) Cultivation-based analyses and (III.) DNA isolation (2 methods) Metagenomic shotgun sequencing, including the examination of DNA extraction (blank) controls. A total of 62 samples were sequenced using Illumina MiSeq sequencing technology. ( B ) More details to steps (II.) and (III.): (II.) Cultivation-based analyses: Aliquots of the vitreous body fluid and balanced salt solution samples were subjected to cultivation-based analyses separately at the hospital and research laboratories. Obtained isolates were analyzed using mass spectrometry and whole genome sequencing. (III.) DNA isolation Metagenomic shotgun sequencing: Samples were extracted using two DNA isolation procedures: QIAamp DNA Mini Kit (QIA) and QIAamp UCP Pathogen Mini kit (UCP). A DNA extraction (blank) control was included at each round of DNA isolation, i.e. one DNA extraction control for 12–14 samples in total per extraction round (more vitreous samples were extracted than analyzed in this study). To verify the presence of the main microorganisms detected in the metagenomics analysis, the shotgun metagenomics reads were mapped to the genome assemblies of the isolates obtained from the vitreous samples. Not displayed here is the mapping of metagenomic shotgun reads to microbial reference genomes in the database (Provided in Fig. 4 ). As an additional verification, PCR analyses were carried out to detect the presence of the most abundant microorganisms in the vitreous samples using organism-specific primer sets.
    Figure Legend Snippet: Sample collection, DNA isolation and shotgun metagenomic sequencing. ( A ) (I.) Sample collection: Vitreous body (intraocular body fluid) was collected through vitrectomy from 14 patients with endophthalmitis following cataract surgery (n = 7) and intravitreal injection (n = 7). As control, vitreous was collected from 7 patients without postoperative endophthalmitis during macula hole surgery. Six aliquots (3 sample pairs) were obtained from balanced salt solution (BSS) that is infused into the eye during vitrectomy. Three aliquots were collected from separate BSS bottles (BSS-B) and the second set of aliquots was collected from the vitrectomy surgical system (BSS-S) after it had passed through the vitrectomy infusion line, respectively. The samples were examined using (II.) Cultivation-based analyses and (III.) DNA isolation (2 methods) Metagenomic shotgun sequencing, including the examination of DNA extraction (blank) controls. A total of 62 samples were sequenced using Illumina MiSeq sequencing technology. ( B ) More details to steps (II.) and (III.): (II.) Cultivation-based analyses: Aliquots of the vitreous body fluid and balanced salt solution samples were subjected to cultivation-based analyses separately at the hospital and research laboratories. Obtained isolates were analyzed using mass spectrometry and whole genome sequencing. (III.) DNA isolation Metagenomic shotgun sequencing: Samples were extracted using two DNA isolation procedures: QIAamp DNA Mini Kit (QIA) and QIAamp UCP Pathogen Mini kit (UCP). A DNA extraction (blank) control was included at each round of DNA isolation, i.e. one DNA extraction control for 12–14 samples in total per extraction round (more vitreous samples were extracted than analyzed in this study). To verify the presence of the main microorganisms detected in the metagenomics analysis, the shotgun metagenomics reads were mapped to the genome assemblies of the isolates obtained from the vitreous samples. Not displayed here is the mapping of metagenomic shotgun reads to microbial reference genomes in the database (Provided in Fig. 4 ). As an additional verification, PCR analyses were carried out to detect the presence of the most abundant microorganisms in the vitreous samples using organism-specific primer sets.

    Techniques Used: DNA Extraction, Sequencing, Injection, Shotgun Sequencing, Mass Spectrometry, Polymerase Chain Reaction

    Summary of cultivation-based, metagenomics and whole genome sequence analyses. Bacterial isolates were obtained at the hospital laboratory (1 st cultivation) from vitreous from endophthalmitis patients following cataract surgery (C1-7) and intravitreal injection (I1-7) and the taxonomic affiliation of the isolates were determined by MALDI-TOF mass spectrometry (MS). Vitreous was analyzed through metagenomics at the research laboratory using two DNA isolation methods (QIAamp DNA Mini Kit, QIA; QIAamp UCP Pathogen Mini kit, UCP) and the taxonomic affiliation of reads was determined. The detected amount of human DNA sequences in percent (%) is provided in the first column of the Metagenomics tab. In the horizontal bar charts, the taxonomic identity and relative fraction of microbial reads for the most abundant identified organisms based on the Kraken + Bracken analysis is indicated for both DNA isolation methods. The read counts for the most abundant organism according to the Kraken + Bracken (all reads) and BLASTN (forward read) analyses are indicated to the right. The read counts for the most abundant organisms per sample as determined by Kraken, Bracken and BLASTn analyses are available through figshare at https://figshare.com/s/5feabfad1d8c495bf7a3 . Bacterial isolates for some samples were obtained in a second round of cultivation at the research laboratory (2 nd cultivation) and one representative per colony morphotype per vitreous sample was subjected to MS and whole genome sequencing (WGS). The taxonomic affiliation of isolates was determined through classification of assembled genomes using a k-mer based approach and genomic MLST, and antibiotic resistance genes were identified using ResFinder. Furthermore, metagenomic assemblies were generated from the shotgun metagenomic reads and analyzed with regards to taxonomic affiliation and selected functional characteristics (Supplementary Table S6 ). A video summary is available from figshare at https://figshare.com/s/38fe043f6a8ef1710444 .
    Figure Legend Snippet: Summary of cultivation-based, metagenomics and whole genome sequence analyses. Bacterial isolates were obtained at the hospital laboratory (1 st cultivation) from vitreous from endophthalmitis patients following cataract surgery (C1-7) and intravitreal injection (I1-7) and the taxonomic affiliation of the isolates were determined by MALDI-TOF mass spectrometry (MS). Vitreous was analyzed through metagenomics at the research laboratory using two DNA isolation methods (QIAamp DNA Mini Kit, QIA; QIAamp UCP Pathogen Mini kit, UCP) and the taxonomic affiliation of reads was determined. The detected amount of human DNA sequences in percent (%) is provided in the first column of the Metagenomics tab. In the horizontal bar charts, the taxonomic identity and relative fraction of microbial reads for the most abundant identified organisms based on the Kraken + Bracken analysis is indicated for both DNA isolation methods. The read counts for the most abundant organism according to the Kraken + Bracken (all reads) and BLASTN (forward read) analyses are indicated to the right. The read counts for the most abundant organisms per sample as determined by Kraken, Bracken and BLASTn analyses are available through figshare at https://figshare.com/s/5feabfad1d8c495bf7a3 . Bacterial isolates for some samples were obtained in a second round of cultivation at the research laboratory (2 nd cultivation) and one representative per colony morphotype per vitreous sample was subjected to MS and whole genome sequencing (WGS). The taxonomic affiliation of isolates was determined through classification of assembled genomes using a k-mer based approach and genomic MLST, and antibiotic resistance genes were identified using ResFinder. Furthermore, metagenomic assemblies were generated from the shotgun metagenomic reads and analyzed with regards to taxonomic affiliation and selected functional characteristics (Supplementary Table S6 ). A video summary is available from figshare at https://figshare.com/s/38fe043f6a8ef1710444 .

    Techniques Used: Sequencing, Injection, Mass Spectrometry, DNA Extraction, Generated, Functional Assay

    16) Product Images from "Detection of Clostridium botulinum group III in environmental samples from farms by real-time PCR using four commercial DNA extraction kits"

    Article Title: Detection of Clostridium botulinum group III in environmental samples from farms by real-time PCR using four commercial DNA extraction kits

    Journal: BMC Research Notes

    doi: 10.1186/s13104-018-3549-5

    Number of samples in which C. botulinum group III was detected using real-time PCR according to the DNA extraction kit used and the analyzed matrix. Total: number of samples analyzed per matrix. QA QIAamp ® DNA Mini Kit (QIAGEN Inc., Valencia, CA, USA), QF QIAamp ® Fast DNA Stool Mini Kit (QIAGEN Inc., Valencia, CA, USA), PS PowerSoil ® DNA isolation kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA), NS NucleoSpin ® Soil (Macherey–Nagel, Duren, Germany)
    Figure Legend Snippet: Number of samples in which C. botulinum group III was detected using real-time PCR according to the DNA extraction kit used and the analyzed matrix. Total: number of samples analyzed per matrix. QA QIAamp ® DNA Mini Kit (QIAGEN Inc., Valencia, CA, USA), QF QIAamp ® Fast DNA Stool Mini Kit (QIAGEN Inc., Valencia, CA, USA), PS PowerSoil ® DNA isolation kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA), NS NucleoSpin ® Soil (Macherey–Nagel, Duren, Germany)

    Techniques Used: Real-time Polymerase Chain Reaction, DNA Extraction

    17) Product Images from "Evaluation of three DNA extraction methods from fungal cultures"

    Article Title: Evaluation of three DNA extraction methods from fungal cultures

    Journal: Medical Journal, Armed Forces India

    doi: 10.1016/j.mjafi.2017.07.009

    Gel electrophoresis of amplicons generated from DNA extracted by: (Panel 1a and 1b) Fungi/Yeast Genomic DNA Isolation Kit (Norgen). (Panel 2) Qiagen DNA mini kit (QIAamp, Qiagen). (Panel 3) Phenol chloroform iso-amyl alcohol method. C.alb – Candida albicans , C.trop – Candida tropicalis , C.neo – Cryptococcus neoformans ; T.asa – Trichosporon asahii , Pen – Penicillium spp., Fus – Fusarium spp., Epid – Epidermophyton mentagrophytes , M.gyp – Microsporum gypseum , A.fum – Aspergillus fumigatus , Rhizopus – Rhizopus spp., Fusariium – Fusarium spp., M.gyp – Microsporum gypseum , E.mento – Epidermophyton mentagrophytes , Penicillium – Penicillium spp., NC – negative control, MM – 100 bp molecular marker (500 bp is marked).
    Figure Legend Snippet: Gel electrophoresis of amplicons generated from DNA extracted by: (Panel 1a and 1b) Fungi/Yeast Genomic DNA Isolation Kit (Norgen). (Panel 2) Qiagen DNA mini kit (QIAamp, Qiagen). (Panel 3) Phenol chloroform iso-amyl alcohol method. C.alb – Candida albicans , C.trop – Candida tropicalis , C.neo – Cryptococcus neoformans ; T.asa – Trichosporon asahii , Pen – Penicillium spp., Fus – Fusarium spp., Epid – Epidermophyton mentagrophytes , M.gyp – Microsporum gypseum , A.fum – Aspergillus fumigatus , Rhizopus – Rhizopus spp., Fusariium – Fusarium spp., M.gyp – Microsporum gypseum , E.mento – Epidermophyton mentagrophytes , Penicillium – Penicillium spp., NC – negative control, MM – 100 bp molecular marker (500 bp is marked).

    Techniques Used: Nucleic Acid Electrophoresis, Generated, DNA Extraction, Negative Control, Marker

    18) Product Images from "Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR"

    Article Title: Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    doi: 10.1007/s10096-011-1191-4

    DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles
    Figure Legend Snippet: DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles

    Techniques Used: DNA Extraction

    Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water
    Figure Legend Snippet: Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water

    Techniques Used: Inhibition, DNA Extraction

    DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit
    Figure Legend Snippet: DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit

    Techniques Used: DNA Extraction

    19) Product Images from "A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification"

    Article Title: A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-13-454

    Amplified pfmdr1 gene products after nested PCR. Standard DNA extraction was carried out using the QIAamp DNA mini blood kit (Qiagen, Hilden, Germany). DNA extraction by microwave irradiation was performed using a microwave oven (MDA, model number: MW17M70G-AU, 230 V, 50 HZ, operated at 800 W). 1 μl of condensed droplets after microwave treatment were utilized for the PCR procedures. First lane: DNA ladder; NC: Negative Control; PC1 and PC2: Standard extraction from archived blood sample and pfmdr1 amplicons at expected sizes; PC3 and PC4: Standard extraction from 3D7 P. falciparum parasites in culture and pfmdr1 amplicons at expected sizes; ME1 and ME2: Microwave based extraction from archived blood sample and pfmdr1 amplicons at expected sizes; ME3 and ME4: Microwave based extraction in 3D7 culture parasites and pfmdr1 amplicons at expected sizes; ME5: Microwave based DNA extraction from fresh blood sample and pfmdr1 amplicons at expected sizes.
    Figure Legend Snippet: Amplified pfmdr1 gene products after nested PCR. Standard DNA extraction was carried out using the QIAamp DNA mini blood kit (Qiagen, Hilden, Germany). DNA extraction by microwave irradiation was performed using a microwave oven (MDA, model number: MW17M70G-AU, 230 V, 50 HZ, operated at 800 W). 1 μl of condensed droplets after microwave treatment were utilized for the PCR procedures. First lane: DNA ladder; NC: Negative Control; PC1 and PC2: Standard extraction from archived blood sample and pfmdr1 amplicons at expected sizes; PC3 and PC4: Standard extraction from 3D7 P. falciparum parasites in culture and pfmdr1 amplicons at expected sizes; ME1 and ME2: Microwave based extraction from archived blood sample and pfmdr1 amplicons at expected sizes; ME3 and ME4: Microwave based extraction in 3D7 culture parasites and pfmdr1 amplicons at expected sizes; ME5: Microwave based DNA extraction from fresh blood sample and pfmdr1 amplicons at expected sizes.

    Techniques Used: Amplification, Nested PCR, DNA Extraction, Irradiation, Multiple Displacement Amplification, Polymerase Chain Reaction, Negative Control

    20) Product Images from "Leukocyte telomere length variation due to DNA extraction method"

    Article Title: Leukocyte telomere length variation due to DNA extraction method

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-7-877

    DNA extraction method impacts leukocyte telomere length (T/S ratio). Bars are mean telomere length and error bars are standard deviations. DNA was extracted using the Purelink, QiaAmp and Lahiri and Nurnberger methods, respectively. ** P = 0.003.
    Figure Legend Snippet: DNA extraction method impacts leukocyte telomere length (T/S ratio). Bars are mean telomere length and error bars are standard deviations. DNA was extracted using the Purelink, QiaAmp and Lahiri and Nurnberger methods, respectively. ** P = 0.003.

    Techniques Used: DNA Extraction

    21) Product Images from "Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells"

    Article Title: Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10701

    Plots of CNA profiles along the whole genome (x axis) ( A ) CNA profile of unamplified DNA from unfixed cells. ( B – G ) plots of CNAs in single SK-BR-3 cells, obtained from EDTA-preserved blood. ( H , I ) CNA profiles of individual CTCs, obtained from EDTA-preserved blood of the same breast cancer patient. WGA kits: (B, E) Ampli1; (C, F, H, I) PicoPlex; (D, G) REPLI-g.
    Figure Legend Snippet: Plots of CNA profiles along the whole genome (x axis) ( A ) CNA profile of unamplified DNA from unfixed cells. ( B – G ) plots of CNAs in single SK-BR-3 cells, obtained from EDTA-preserved blood. ( H , I ) CNA profiles of individual CTCs, obtained from EDTA-preserved blood of the same breast cancer patient. WGA kits: (B, E) Ampli1; (C, F, H, I) PicoPlex; (D, G) REPLI-g.

    Techniques Used: Whole Genome Amplification

    Distribution of identified known SNPs between datasets ( A ) Known SNPs identified in single cells, amplified with Ampli1, PicoPlex, and REPLI-g WGA kits and obtained from EDTA-preserved blood in comparison to unamplified DNA. ( B ) Known SNPs identified in single cells, amplified with Ampli1 or PicoPlex and obtained from EDTA- and CellSave-preserved blood in comparison to unamplified DNA from unfixed cells. ( C ) Known SNPs identified in single CTCs, amplified with PicoPlex in comparison to each other.
    Figure Legend Snippet: Distribution of identified known SNPs between datasets ( A ) Known SNPs identified in single cells, amplified with Ampli1, PicoPlex, and REPLI-g WGA kits and obtained from EDTA-preserved blood in comparison to unamplified DNA. ( B ) Known SNPs identified in single cells, amplified with Ampli1 or PicoPlex and obtained from EDTA- and CellSave-preserved blood in comparison to unamplified DNA from unfixed cells. ( C ) Known SNPs identified in single CTCs, amplified with PicoPlex in comparison to each other.

    Techniques Used: Amplification, Whole Genome Amplification

    22) Product Images from "The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer"

    Article Title: The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0137-x

    DNMT1 maintains CHFR gene expression via promoter DNA methylation, and CHFR participates in UBC13 regulating paclitaxel sensitivity a Detection of methylation status at the promoter region of the CHFR gene in A2780, SKOV3, and 3AO cells with DNMT1-knockdown by Bisulfite sequencing. Ten lines with circles represent the same sequence of ten clones from one sample. CpG sites are shown as filled circles (methylated) or unfilled circles (unmethylated). The lower panel shows the summary data. * P
    Figure Legend Snippet: DNMT1 maintains CHFR gene expression via promoter DNA methylation, and CHFR participates in UBC13 regulating paclitaxel sensitivity a Detection of methylation status at the promoter region of the CHFR gene in A2780, SKOV3, and 3AO cells with DNMT1-knockdown by Bisulfite sequencing. Ten lines with circles represent the same sequence of ten clones from one sample. CpG sites are shown as filled circles (methylated) or unfilled circles (unmethylated). The lower panel shows the summary data. * P

    Techniques Used: Expressing, DNA Methylation Assay, Methylation, Methylation Sequencing, Sequencing, Clone Assay

    Paclitaxel exposure reduces UBC13 and CHFR, increases DNMT1 and Aurora A Western blotting of the indicated proteins in a A2780 and b SKOV3 cells treated with 2, 5, and 10 nM paclitaxel for the indicated times. Cells without paclitaxel treatment were assigned to be the blank control (0 h). One of three representative results is shown. c Working model: UBC13 regulates paclitaxel resistance via DNMT1/CHFR/Aurora A in ovarian cancer cells. UBC13 reduces DNMT1 through ubiquitination, results in DNA hypomethylation of the CHFR promoter, and decreases Aurora A. Reduction of UBC13 increases stability of DNMT1, leads to hypermethylation of the CHFR promoter, elevates Aurora A levels, and prompts paclitaxel resistance in ovarian cancer cells. Ub: ubiquitin
    Figure Legend Snippet: Paclitaxel exposure reduces UBC13 and CHFR, increases DNMT1 and Aurora A Western blotting of the indicated proteins in a A2780 and b SKOV3 cells treated with 2, 5, and 10 nM paclitaxel for the indicated times. Cells without paclitaxel treatment were assigned to be the blank control (0 h). One of three representative results is shown. c Working model: UBC13 regulates paclitaxel resistance via DNMT1/CHFR/Aurora A in ovarian cancer cells. UBC13 reduces DNMT1 through ubiquitination, results in DNA hypomethylation of the CHFR promoter, and decreases Aurora A. Reduction of UBC13 increases stability of DNMT1, leads to hypermethylation of the CHFR promoter, elevates Aurora A levels, and prompts paclitaxel resistance in ovarian cancer cells. Ub: ubiquitin

    Techniques Used: Western Blot

    Differential expressions of proteins between paclitaxel sensitive and resistant ovarian cancer cells by proteomic analysis a Analysis of SKOV3 and SKOV3-TR30 samples by DIGE. Fluorescent (left) and monochrome (right) images of the DIGE Gel. Proteins extracted from SKOV3 (SK) and SKOV3-TR30 (ST) were labeled with Cy3 and Cy5, respectively. The labeled samples were initially separated in the first dimension ( x -axis) on a non-linear gradient pH 3-10, then separated in the second dimension ( y -axis) on a 12% polyacrylamide gel. The circled spots were identified by MALDI-TOF/TOF MS/MS analysis shown in the supplementary material Table S1 – S3 . b Three-dimensional volumetric models of UBC13 in SKOV3 (top) and SKOV3-TR30 (bottom) for DIGE spots. c The expression levels of UBC13 shown on DIGE were calculated by DeCyder analysis and presented as standardized log abundance. SK presents SKOV3 and ST presents SKOV3-TR30. d Western blotting of UBC13 in A2780 vs. A2780-TR and SKOV3 vs. SKOV3-TR30 cells, respectively. One of three representative results is shown in ( d )
    Figure Legend Snippet: Differential expressions of proteins between paclitaxel sensitive and resistant ovarian cancer cells by proteomic analysis a Analysis of SKOV3 and SKOV3-TR30 samples by DIGE. Fluorescent (left) and monochrome (right) images of the DIGE Gel. Proteins extracted from SKOV3 (SK) and SKOV3-TR30 (ST) were labeled with Cy3 and Cy5, respectively. The labeled samples were initially separated in the first dimension ( x -axis) on a non-linear gradient pH 3-10, then separated in the second dimension ( y -axis) on a 12% polyacrylamide gel. The circled spots were identified by MALDI-TOF/TOF MS/MS analysis shown in the supplementary material Table S1 – S3 . b Three-dimensional volumetric models of UBC13 in SKOV3 (top) and SKOV3-TR30 (bottom) for DIGE spots. c The expression levels of UBC13 shown on DIGE were calculated by DeCyder analysis and presented as standardized log abundance. SK presents SKOV3 and ST presents SKOV3-TR30. d Western blotting of UBC13 in A2780 vs. A2780-TR and SKOV3 vs. SKOV3-TR30 cells, respectively. One of three representative results is shown in ( d )

    Techniques Used: Labeling, Mass Spectrometry, Expressing, Western Blot

    UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in ( e and g ) (* P
    Figure Legend Snippet: UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in ( e and g ) (* P

    Techniques Used: Over Expression, Western Blot, Transfection, shRNA

    Paclitaxel induces UBC13 down-regulation, and UBC13 modulates the paclitaxel sensitivity through the DNMT1, CHFR, and Aurora A pathway a A2780 and b SKOV3 cells were treated with 5, 10, 20, and 30 nM paclitaxel for 24 h. Western blotting was performed with the indicated antibodies. Cell viability assays in A2780 and SKOV3 cells with c UBC13-knockdown and d UBC13-overexpression that were treated with paclitaxel at the indicated concentrations. Results are shown as means ± SEM for at least three separate experiments (* P
    Figure Legend Snippet: Paclitaxel induces UBC13 down-regulation, and UBC13 modulates the paclitaxel sensitivity through the DNMT1, CHFR, and Aurora A pathway a A2780 and b SKOV3 cells were treated with 5, 10, 20, and 30 nM paclitaxel for 24 h. Western blotting was performed with the indicated antibodies. Cell viability assays in A2780 and SKOV3 cells with c UBC13-knockdown and d UBC13-overexpression that were treated with paclitaxel at the indicated concentrations. Results are shown as means ± SEM for at least three separate experiments (* P

    Techniques Used: Western Blot, Over Expression

    23) Product Images from "Survivin as a potential therapeutic target of acetylsalicylic acid in pituitary adenomas"

    Article Title: Survivin as a potential therapeutic target of acetylsalicylic acid in pituitary adenomas

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25650

    Effect of ASA treatment on pituitary adenoma cells (A)  Cell proliferation in RC-4 B/C cells but not GH3 cells decreased after 2.5 and 5 mM ASA treatment  (B)  Cell cycle analysis using flow cytometry in RC-4 B/C cells showed decrease in S phase and increase in G2/M phase population upon ASA treatment.  (C)  Survivin mRNA and protein expression decreased after ASA treatment.  * : p=0.013;  **** : p
    Figure Legend Snippet: Effect of ASA treatment on pituitary adenoma cells (A) Cell proliferation in RC-4 B/C cells but not GH3 cells decreased after 2.5 and 5 mM ASA treatment (B) Cell cycle analysis using flow cytometry in RC-4 B/C cells showed decrease in S phase and increase in G2/M phase population upon ASA treatment. (C) Survivin mRNA and protein expression decreased after ASA treatment. * : p=0.013; **** : p

    Techniques Used: Cell Cycle Assay, Flow Cytometry, Cytometry, Expressing

    Effect of survivin inhibition (A)  Survivin silencing by siRNA on RC-4 B/C cells.  (B)  Survivin inhibition by YM155, a small molecule inhibitor in RC-4 B/C cells. The effect of YM155  (C)  and survivin siRNA  (D)  on pituitary adenoma cell viability, proliferation and cell cycle phases of RC-4 B/C cells (see details in the text).  (E)  Decrease of CCNA2, CDK2 mRNA and CCNA2, total CDK2 and p-CDK2 protein abundance following ASA treatment in RC-4 B/C cells.  Left:  mRNA,  middle  and  right : western blot and densitometry.  (F)  YM155 and survivin siRNA transfection did not significantly alter CCNA2 and CDK2 expression in RC-4 B/C cells.  (G)  Survivin overexpression did not reduce the viability of RC-4 B/C cells.  * : p
    Figure Legend Snippet: Effect of survivin inhibition (A) Survivin silencing by siRNA on RC-4 B/C cells. (B) Survivin inhibition by YM155, a small molecule inhibitor in RC-4 B/C cells. The effect of YM155 (C) and survivin siRNA (D) on pituitary adenoma cell viability, proliferation and cell cycle phases of RC-4 B/C cells (see details in the text). (E) Decrease of CCNA2, CDK2 mRNA and CCNA2, total CDK2 and p-CDK2 protein abundance following ASA treatment in RC-4 B/C cells. Left: mRNA, middle and right : western blot and densitometry. (F) YM155 and survivin siRNA transfection did not significantly alter CCNA2 and CDK2 expression in RC-4 B/C cells. (G) Survivin overexpression did not reduce the viability of RC-4 B/C cells. * : p

    Techniques Used: Inhibition, Western Blot, Transfection, Expressing, Over Expression

    24) Product Images from "An LSC epigenetic signature is largely mutation independent and implicates the HOXA cluster in AML pathogenesis"

    Article Title: An LSC epigenetic signature is largely mutation independent and implicates the HOXA cluster in AML pathogenesis

    Journal: Nature Communications

    doi: 10.1038/ncomms9489

    The LSC epigenetic signature is associated with overall survival in human AML. ( a ) TCGA samples were classified as LSC like or Blast like based on DNA methylation alone by generating methylation profiles of the LSC and Blast populations, and then calculating scores of each sample based on the probability of being closer to either LSC or Blast. Kaplan–Meier survival analysis was then applied to these groups as indicated. Statistical significance was determined by the log-rank test ( n =192; 93 LSC-like and 99 Blast-like patients). ( b – e ) Expression of the LSC epigenetic signature genes was combined to create an LSC score, which was then calculated in AML samples. The first principal component of genes in the LSC signature was computed, and patients were stratified as ‘high' or ‘low' relative to its median value in four independent cohorts including TCGA ( n =182; 91 high-score and 91 low-score patients), ( b ) Metzeler et al . ( n =163; 81 high-score and 82 low-score patients) ( c ), Wouters et al . ( n =262; 131 high-score and 131 low-score patients) ( d ) and Wilson et al . ( n =169, 84 high-score and 85 low-score patients) ( e ). In each cohort, patients were classified into high and low groups based on the median value. Kaplan–Meier survival analysis was then applied to these groups as indicated. Statistical significance was determined by the log-rank test.
    Figure Legend Snippet: The LSC epigenetic signature is associated with overall survival in human AML. ( a ) TCGA samples were classified as LSC like or Blast like based on DNA methylation alone by generating methylation profiles of the LSC and Blast populations, and then calculating scores of each sample based on the probability of being closer to either LSC or Blast. Kaplan–Meier survival analysis was then applied to these groups as indicated. Statistical significance was determined by the log-rank test ( n =192; 93 LSC-like and 99 Blast-like patients). ( b – e ) Expression of the LSC epigenetic signature genes was combined to create an LSC score, which was then calculated in AML samples. The first principal component of genes in the LSC signature was computed, and patients were stratified as ‘high' or ‘low' relative to its median value in four independent cohorts including TCGA ( n =182; 91 high-score and 91 low-score patients), ( b ) Metzeler et al . ( n =163; 81 high-score and 82 low-score patients) ( c ), Wouters et al . ( n =262; 131 high-score and 131 low-score patients) ( d ) and Wilson et al . ( n =169, 84 high-score and 85 low-score patients) ( e ). In each cohort, patients were classified into high and low groups based on the median value. Kaplan–Meier survival analysis was then applied to these groups as indicated. Statistical significance was determined by the log-rank test.

    Techniques Used: DNA Methylation Assay, Methylation, Expressing

    Epigenetic signatures define subgroups of AML LSC reflecting the cell of origin. ( a ) A total of 216 DMRs identified from all possible pairwise comparisons among six HSPCs were used to cluster all normal HSPCs with all AML subpopulations. The primary AML subpopulations form two major clusters: L-MPP like and GMP like. LSC subpopulations are indicated in bold. ( b ) Clustering analysis of TCGA AML samples with normal human HSPC using the 216 DMRs shows that the L-MPP-like and GMP-like clusters are observed in this cohort as well. ( c ) TCGA samples were classified according to normal HSPC populations based on DNA methylation alone by generating methylation profiles of all the normal HSPC, and then calculating scores of each sample based on the closest population. The normal progenitor cell identity for all the TCGA samples is indicated. ( d ) The L-MPP-like and GMP-like TCGA samples were grouped according to their FAB classification. NA, not classified.
    Figure Legend Snippet: Epigenetic signatures define subgroups of AML LSC reflecting the cell of origin. ( a ) A total of 216 DMRs identified from all possible pairwise comparisons among six HSPCs were used to cluster all normal HSPCs with all AML subpopulations. The primary AML subpopulations form two major clusters: L-MPP like and GMP like. LSC subpopulations are indicated in bold. ( b ) Clustering analysis of TCGA AML samples with normal human HSPC using the 216 DMRs shows that the L-MPP-like and GMP-like clusters are observed in this cohort as well. ( c ) TCGA samples were classified according to normal HSPC populations based on DNA methylation alone by generating methylation profiles of all the normal HSPC, and then calculating scores of each sample based on the closest population. The normal progenitor cell identity for all the TCGA samples is indicated. ( d ) The L-MPP-like and GMP-like TCGA samples were grouped according to their FAB classification. NA, not classified.

    Techniques Used: DNA Methylation Assay, Methylation

    25) Product Images from "Distinct MicroRNA Expression Signatures of Porcine Induced Pluripotent Stem Cells under Mouse and Human ESC Culture Conditions"

    Article Title: Distinct MicroRNA Expression Signatures of Porcine Induced Pluripotent Stem Cells under Mouse and Human ESC Culture Conditions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158655

    The piPSCs-enriched miRNA cluster promotes the reprogramming of porcine cells. (A) The DIANA miPath program predict the pathways regulated by the miR-106a-363 cluster. Heatmap of the miR-106a-363 cluster versus other pathways, where the miRNAs are clustered together when they exhibited similar pathway-targeting patterns, and the pathways are clustered together by related miRNAs. (B) The DIANA miPath program predicted the pathways regulated by the miR-302 cluster. (C) The PCR region used to clone the genomic locus of the miR-106a-363 cluster and putative miR-302 cluster. (D) Amplification of the genomic region containing the miR-106a-363 cluster and putative miRNA-302 cluster. An approximately 1.5 kb range harboring the whole miRNA-106a-363 cluster and a 2 kb range containing the putative porcine miR-302 cluster were amplified. The marker indicates the DNA ladder. (E) AP staining of the piPSCs colonies. (F) Statistical analysis of AP positive colonies. Mean values ± SD are shown. ***P-value
    Figure Legend Snippet: The piPSCs-enriched miRNA cluster promotes the reprogramming of porcine cells. (A) The DIANA miPath program predict the pathways regulated by the miR-106a-363 cluster. Heatmap of the miR-106a-363 cluster versus other pathways, where the miRNAs are clustered together when they exhibited similar pathway-targeting patterns, and the pathways are clustered together by related miRNAs. (B) The DIANA miPath program predicted the pathways regulated by the miR-302 cluster. (C) The PCR region used to clone the genomic locus of the miR-106a-363 cluster and putative miR-302 cluster. (D) Amplification of the genomic region containing the miR-106a-363 cluster and putative miRNA-302 cluster. An approximately 1.5 kb range harboring the whole miRNA-106a-363 cluster and a 2 kb range containing the putative porcine miR-302 cluster were amplified. The marker indicates the DNA ladder. (E) AP staining of the piPSCs colonies. (F) Statistical analysis of AP positive colonies. Mean values ± SD are shown. ***P-value

    Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Staining

    26) Product Images from "DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis"

    Article Title: DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis

    Journal: Biopreservation and Biobanking

    doi: 10.1089/bio.2015.0064

    DNA integrity observation by electrophoresis in agarose gel. DNA samples were analyzed by loading 50 ng of DNA on a 0.8% agarose gel. The Lambda-pUC Mix Marker 4 (MW) was also separated as size reference. Four representative samples of DNA for frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C) , and saliva (D) are shown.
    Figure Legend Snippet: DNA integrity observation by electrophoresis in agarose gel. DNA samples were analyzed by loading 50 ng of DNA on a 0.8% agarose gel. The Lambda-pUC Mix Marker 4 (MW) was also separated as size reference. Four representative samples of DNA for frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C) , and saliva (D) are shown.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Marker, Formalin-fixed Paraffin-Embedded

    DNA purity 260/230 ratio. Absorbance at 260 and 230 nm was measured for each DNA sample isolated from frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C), and saliva (D) , and 260/230 ratio obtained was represented versus its corresponding DNA concentration based on spectrophotometric measurement. The trendline for each group of samples is shown. FFPE, formalin-fixed paraffin-embedded; OCT, optimal cutting temperature.
    Figure Legend Snippet: DNA purity 260/230 ratio. Absorbance at 260 and 230 nm was measured for each DNA sample isolated from frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C), and saliva (D) , and 260/230 ratio obtained was represented versus its corresponding DNA concentration based on spectrophotometric measurement. The trendline for each group of samples is shown. FFPE, formalin-fixed paraffin-embedded; OCT, optimal cutting temperature.

    Techniques Used: Isolation, Formalin-fixed Paraffin-Embedded, Concentration Assay

    DNA performance for real-time PCR assay. Fifteen randomized DNA samples isolated from frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C) , and saliva (D) were amplified by real-time PCR for the GAPDH ( left ) and RPLP0 ( right ) genes. The average C T value and standard deviation for each type of source were calculated. PCR, polymerase chain reaction.
    Figure Legend Snippet: DNA performance for real-time PCR assay. Fifteen randomized DNA samples isolated from frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C) , and saliva (D) were amplified by real-time PCR for the GAPDH ( left ) and RPLP0 ( right ) genes. The average C T value and standard deviation for each type of source were calculated. PCR, polymerase chain reaction.

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Formalin-fixed Paraffin-Embedded, Amplification, Standard Deviation, Polymerase Chain Reaction

    PicoGreen ® /A260 yield ratio. PicoGreen DNA quantification and DNA quantification using Lambert–Beer law from 260 nm absorbance were performed for each DNA sample isolated from frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C) , and saliva (D) . Absolute yields in micrograms of DNA obtained for both methods were calculated. The average ratio and standard deviation of the ratio between absolute yield using PicoGreen and spectrophotometry for each type of source are represented.
    Figure Legend Snippet: PicoGreen ® /A260 yield ratio. PicoGreen DNA quantification and DNA quantification using Lambert–Beer law from 260 nm absorbance were performed for each DNA sample isolated from frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C) , and saliva (D) . Absolute yields in micrograms of DNA obtained for both methods were calculated. The average ratio and standard deviation of the ratio between absolute yield using PicoGreen and spectrophotometry for each type of source are represented.

    Techniques Used: Isolation, Formalin-fixed Paraffin-Embedded, Standard Deviation, Spectrophotometry

    DNA quality for PCR analysis. Fifteen randomized DNA samples isolated from frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C) , and saliva (D) were amplified by PCR for the ACVR2B , ZFX , AF4 , and GAPDH genes. PCR products of 5049, 1137, 400, and 87 bp, respectively, were analyzed by agarose gel electrophoresis. Four representative samples for each group are shown.
    Figure Legend Snippet: DNA quality for PCR analysis. Fifteen randomized DNA samples isolated from frozen tissue in OCT (A) , FFPE tissue (B) , frozen blood (C) , and saliva (D) were amplified by PCR for the ACVR2B , ZFX , AF4 , and GAPDH genes. PCR products of 5049, 1137, 400, and 87 bp, respectively, were analyzed by agarose gel electrophoresis. Four representative samples for each group are shown.

    Techniques Used: Polymerase Chain Reaction, Isolation, Formalin-fixed Paraffin-Embedded, Amplification, Agarose Gel Electrophoresis

    27) Product Images from "Epigenetic Methylation of Parathyroid CaR and VDR Promoters in Experimental Secondary Hyperparathyroidism"

    Article Title: Epigenetic Methylation of Parathyroid CaR and VDR Promoters in Experimental Secondary Hyperparathyroidism

    Journal: International Journal of Nephrology

    doi: 10.1155/2012/123576

    Methylation detection of CaR and VDR promoter regions in parathyroid glands. Analysis of the CaR and VDR promoter regions by PCR melting curve analysis after bisulfite treatment of genomic DNA. CaR is covered by PCR products CaR #1-2, and VDR is covered by PCR products VDR #1–4. Commercial low- and high-methylated rat DNA is analyzed in all of the PCR reactions shown in the “Methylation control” panel in green and red, respectively; it is also included in the “Sham,” “Uremia,” and “ in vitro ” panels. Control of methylation detection verified high levels of methylation in H19 and VDR downstream gene regions.
    Figure Legend Snippet: Methylation detection of CaR and VDR promoter regions in parathyroid glands. Analysis of the CaR and VDR promoter regions by PCR melting curve analysis after bisulfite treatment of genomic DNA. CaR is covered by PCR products CaR #1-2, and VDR is covered by PCR products VDR #1–4. Commercial low- and high-methylated rat DNA is analyzed in all of the PCR reactions shown in the “Methylation control” panel in green and red, respectively; it is also included in the “Sham,” “Uremia,” and “ in vitro ” panels. Control of methylation detection verified high levels of methylation in H19 and VDR downstream gene regions.

    Techniques Used: Methylation, Polymerase Chain Reaction, In Vitro

    28) Product Images from "Epidemiological and molecular investigation of resurgent cutaneous leishmaniasis in Sudan"

    Article Title: Epidemiological and molecular investigation of resurgent cutaneous leishmaniasis in Sudan

    Journal: International Journal of Infectious Diseases

    doi: 10.1016/j.ijid.2019.08.018

    PCR-RFLP identification of Leishmania species from cutaneous leishmaniasis lesions. (A) DNA extracted from ATL buffer, CL05 and CL06 produced the RFLP pattern characteristic of Leishmania major , with band sizes as described in the text. (B) DNA extracted from culture of the isolate from CL08 (MHOM/SD/2017/ELOBIED) also gave the pattern typical of L. major ; PCR-RFLP of L. tropica was included as control. The L. major identification was verified by DNA sequencing (see text).
    Figure Legend Snippet: PCR-RFLP identification of Leishmania species from cutaneous leishmaniasis lesions. (A) DNA extracted from ATL buffer, CL05 and CL06 produced the RFLP pattern characteristic of Leishmania major , with band sizes as described in the text. (B) DNA extracted from culture of the isolate from CL08 (MHOM/SD/2017/ELOBIED) also gave the pattern typical of L. major ; PCR-RFLP of L. tropica was included as control. The L. major identification was verified by DNA sequencing (see text).

    Techniques Used: Polymerase Chain Reaction, Produced, DNA Sequencing

    29) Product Images from "High-density array analysis of DNA methylation in Tamoxifen-resistant breast cancer cell lines"

    Article Title: High-density array analysis of DNA methylation in Tamoxifen-resistant breast cancer cell lines

    Journal: Epigenetics

    doi: 10.4161/epi.27111

    Figure 2. Scatter plots indicate genome-wide methylation changes in Tamoxifen-resistant lines compared with the parental. TMX2–11 ( A ), TMX2–28 ( B ) and MCF-7 treated with 10 −10 M E 2 for 14 d ( D ) were compared with the parental
    Figure Legend Snippet: Figure 2. Scatter plots indicate genome-wide methylation changes in Tamoxifen-resistant lines compared with the parental. TMX2–11 ( A ), TMX2–28 ( B ) and MCF-7 treated with 10 −10 M E 2 for 14 d ( D ) were compared with the parental

    Techniques Used: Genome Wide, Methylation

    Figure 3. Location of aberrantly methylated CpG sites shared between TMX2–11 and TMX2–28. Functional genomic location of all CpG sites on the BeadChip ( A ) hypermethylated ( B ) and hypomethylated ( C ) CpG sites. Neighborhood location
    Figure Legend Snippet: Figure 3. Location of aberrantly methylated CpG sites shared between TMX2–11 and TMX2–28. Functional genomic location of all CpG sites on the BeadChip ( A ) hypermethylated ( B ) and hypomethylated ( C ) CpG sites. Neighborhood location

    Techniques Used: Methylation, Functional Assay

    30) Product Images from "The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer"

    Article Title: The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0137-x

    DNMT1 maintains CHFR gene expression via promoter DNA methylation, and CHFR participates in UBC13 regulating paclitaxel sensitivity a Detection of methylation status at the promoter region of the CHFR gene in A2780, SKOV3, and 3AO cells with DNMT1-knockdown by Bisulfite sequencing. Ten lines with circles represent the same sequence of ten clones from one sample. CpG sites are shown as filled circles (methylated) or unfilled circles (unmethylated). The lower panel shows the summary data. * P
    Figure Legend Snippet: DNMT1 maintains CHFR gene expression via promoter DNA methylation, and CHFR participates in UBC13 regulating paclitaxel sensitivity a Detection of methylation status at the promoter region of the CHFR gene in A2780, SKOV3, and 3AO cells with DNMT1-knockdown by Bisulfite sequencing. Ten lines with circles represent the same sequence of ten clones from one sample. CpG sites are shown as filled circles (methylated) or unfilled circles (unmethylated). The lower panel shows the summary data. * P

    Techniques Used: Expressing, DNA Methylation Assay, Methylation, Methylation Sequencing, Sequencing, Clone Assay

    31) Product Images from "Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR"

    Article Title: Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    doi: 10.1007/s10096-011-1191-4

    DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles
    Figure Legend Snippet: DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles

    Techniques Used: DNA Extraction

    Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water
    Figure Legend Snippet: Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water

    Techniques Used: Inhibition, DNA Extraction

    DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit
    Figure Legend Snippet: DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit

    Techniques Used: DNA Extraction

    32) Product Images from "Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR"

    Article Title: Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    doi: 10.1007/s10096-011-1191-4

    DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles
    Figure Legend Snippet: DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles

    Techniques Used: DNA Extraction

    Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water
    Figure Legend Snippet: Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water

    Techniques Used: Inhibition, DNA Extraction

    DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit
    Figure Legend Snippet: DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit

    Techniques Used: DNA Extraction

    33) Product Images from "Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR"

    Article Title: Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    doi: 10.1007/s10096-011-1191-4

    DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles
    Figure Legend Snippet: DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles

    Techniques Used: DNA Extraction

    Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water
    Figure Legend Snippet: Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water

    Techniques Used: Inhibition, DNA Extraction

    DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit
    Figure Legend Snippet: DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit

    Techniques Used: DNA Extraction

    34) Product Images from "Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams"

    Article Title: Development of a rapid PCR protocol to detect Vibrio parahaemolyticus in clams

    Journal: Journal of Food Science and Technology

    doi: 10.1007/s13197-017-2986-9

    ), and subsequent molecular, biochemical and cultural analyses. DNA was extracted from T0, T3 and T6 samples by QIAamp DNA Mini Kit (Qiagen); for V. parahaemolyticus ATCC 43996 the extraction was carried out by InstaGene Matrix (Bio-Rad). ASPW alkaline saline peptone water, TCBS thiosulfate citrate bile salts sucrose
    Figure Legend Snippet: ), and subsequent molecular, biochemical and cultural analyses. DNA was extracted from T0, T3 and T6 samples by QIAamp DNA Mini Kit (Qiagen); for V. parahaemolyticus ATCC 43996 the extraction was carried out by InstaGene Matrix (Bio-Rad). ASPW alkaline saline peptone water, TCBS thiosulfate citrate bile salts sucrose

    Techniques Used:

    35) Product Images from "Development of Sensitive Detection of Cryptosporidium and Giardia from Surface Water in Iran"

    Article Title: Development of Sensitive Detection of Cryptosporidium and Giardia from Surface Water in Iran

    Journal: Iranian Journal of Parasitology

    doi:

    1.5% agarose gel electrophoresis of PCR product. Lane 1: Giardia positive control by Phenol-chloroform extraction; Lanes 2 and 3: DNA extraction by Phenol-chloroform of surface water samples; Lanes 4 and 5: DNA extraction by QIAamp DNA mini kit of surface water samples
    Figure Legend Snippet: 1.5% agarose gel electrophoresis of PCR product. Lane 1: Giardia positive control by Phenol-chloroform extraction; Lanes 2 and 3: DNA extraction by Phenol-chloroform of surface water samples; Lanes 4 and 5: DNA extraction by QIAamp DNA mini kit of surface water samples

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Positive Control, DNA Extraction

    36) Product Images from "Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR"

    Article Title: Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169846

    Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns. Genomic and plasmid DNA were isolated from E . coli DH5α and E . coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P
    Figure Legend Snippet: Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns. Genomic and plasmid DNA were isolated from E . coli DH5α and E . coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P

    Techniques Used: Plasmid Preparation, Isolation, Spectrophotometry, Lysis, Standard Deviation

    Quantification of pBR322 plasmid copy number by digital droplet PCR. E . coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A ( Table 1A ). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs , respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs . Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.
    Figure Legend Snippet: Quantification of pBR322 plasmid copy number by digital droplet PCR. E . coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A ( Table 1A ). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs , respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs . Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Isolation, Amplification

    37) Product Images from "Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities"

    Article Title: Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169877

    Normalised DNA yield (ng/ μ L) from (A) plaque and (B) saliva (mean ± SEM). M: MoBio PowerSoil ® DNA Isolation Kit, Q: QIAamp ® DNA Mini Kit, Z: Zymo Bacterial/Fungal DNA Mini Prep TM , P: Phenol:chloroform-based DNA isolation. DNA extraction methods not linked by the same letter are significantly different to each other (Tukey-Kramer p ≤ 0.05).
    Figure Legend Snippet: Normalised DNA yield (ng/ μ L) from (A) plaque and (B) saliva (mean ± SEM). M: MoBio PowerSoil ® DNA Isolation Kit, Q: QIAamp ® DNA Mini Kit, Z: Zymo Bacterial/Fungal DNA Mini Prep TM , P: Phenol:chloroform-based DNA isolation. DNA extraction methods not linked by the same letter are significantly different to each other (Tukey-Kramer p ≤ 0.05).

    Techniques Used: DNA Extraction

    Taxa plots summarising the relative abundance of taxon-assigned OTUs identified in pooled homogenates for (A) bacterial genera in plaque; (B) fungal genera in plaque; (C) bacterial genera in saliva and (D) fungal genera in saliva. Each bar represents sequencing from three replicates, rarefied to 6000 sequences per sample for bacterial genera and 959 for fungal genera, with replicates that did not meet these criteria excluded. M: MoBio PowerSoil ® DNA Isolation Kit, Q: QIAamp ® DNA Mini Kit, Z: Zymo Bacterial/Fungal DNA Mini Prep TM , P: Phenol:chloroform-based DNA isolation.
    Figure Legend Snippet: Taxa plots summarising the relative abundance of taxon-assigned OTUs identified in pooled homogenates for (A) bacterial genera in plaque; (B) fungal genera in plaque; (C) bacterial genera in saliva and (D) fungal genera in saliva. Each bar represents sequencing from three replicates, rarefied to 6000 sequences per sample for bacterial genera and 959 for fungal genera, with replicates that did not meet these criteria excluded. M: MoBio PowerSoil ® DNA Isolation Kit, Q: QIAamp ® DNA Mini Kit, Z: Zymo Bacterial/Fungal DNA Mini Prep TM , P: Phenol:chloroform-based DNA isolation.

    Techniques Used: Sequencing, DNA Extraction

    38) Product Images from "Comparison of Two PCR Assays for Trichomonas vaginalis"

    Article Title: Comparison of Two PCR Assays for Trichomonas vaginalis

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2019.57.1.27

    Two PCR assay systems were used with DNA isolated from the urine of the 4 patients by 2 DNA extraction methods. (A) All 4 urine samples gave positive bands in HY-PCR using DNA extracted with Chelex ® 100. (B, C) PCR with Seeplex ® ACE Detection Kit. (B) Only one of the 4 samples gave a positive band with DNA isolated with Chelex ® 100, and (C) none gave positive band with DNA extracted with QIAamp ® DNA Mini Kit. Arrowhead, T. vaginalis .
    Figure Legend Snippet: Two PCR assay systems were used with DNA isolated from the urine of the 4 patients by 2 DNA extraction methods. (A) All 4 urine samples gave positive bands in HY-PCR using DNA extracted with Chelex ® 100. (B, C) PCR with Seeplex ® ACE Detection Kit. (B) Only one of the 4 samples gave a positive band with DNA isolated with Chelex ® 100, and (C) none gave positive band with DNA extracted with QIAamp ® DNA Mini Kit. Arrowhead, T. vaginalis .

    Techniques Used: Polymerase Chain Reaction, Isolation, DNA Extraction

    Sensitivity of HY-PCR tested with varying numbers of trophozoites (1, 10, 100) of T. vaginalis . (A) Chelex ® 100, (B) QIAamp ® DNA Mini Kit were used for DNA extraction. The PCR product formed a 318 bp band. N, Negative control (DW); 1, 10, 100, number of trichomonads used.
    Figure Legend Snippet: Sensitivity of HY-PCR tested with varying numbers of trophozoites (1, 10, 100) of T. vaginalis . (A) Chelex ® 100, (B) QIAamp ® DNA Mini Kit were used for DNA extraction. The PCR product formed a 318 bp band. N, Negative control (DW); 1, 10, 100, number of trichomonads used.

    Techniques Used: Polymerase Chain Reaction, DNA Extraction, Negative Control

    Comparison between Chelex ® 100 (A) and QIAamp ® DNA Mini Kit (B) as DNA extraction methods for PCR using a Seeplex ® ACE Detection Kit. T. vaginalis trophozoites (1, 10, 100) were added to the urine of a normal male and incubated overnight before DNA extraction. N, negative control (DW); M, marker; arrowhead, T. vaginalis product.
    Figure Legend Snippet: Comparison between Chelex ® 100 (A) and QIAamp ® DNA Mini Kit (B) as DNA extraction methods for PCR using a Seeplex ® ACE Detection Kit. T. vaginalis trophozoites (1, 10, 100) were added to the urine of a normal male and incubated overnight before DNA extraction. N, negative control (DW); M, marker; arrowhead, T. vaginalis product.

    Techniques Used: DNA Extraction, Polymerase Chain Reaction, Incubation, Negative Control, Marker

    39) Product Images from "Calibration-free assays on standard real-time PCR devices"

    Article Title: Calibration-free assays on standard real-time PCR devices

    Journal: Scientific Reports

    doi: 10.1038/srep44854

    Experimental verification of a 16-compartment digital assay with comparison to the performance of qPCR assays. ( A ) The graph shows the results for the same amount of reference DNA suspended in different elution buffers and quantified with conventional qPCR and with the synergistic PCR algorithm. Tests performed on Applied Biosystems 7500 Fast RT System on the IVD Cytomegalovirus PCR kit (GeneProof) according to the prescription. Elution buffers: (1) water, (2) AE elution buffer QIAamp DNA Mini Kit (Quiagen), (3) MBL5 NucleoMag Blood 200 uL (MACHEREY-NAGEL), (4) MagJET Whole Blood Genomic DNA Kit (Thermo Scientific). The gray line shows the expected distribution of results for Real-Time assay. ( B ) The graph shows the result of 24 runs of the synergistic assay, each on 16 partitions of the amplification mix. We conducted two series of 12 assays on the two elution buffers (1 and 4) that provided the largest difference in the result of the conventional qPCR analysis. The gray line shows the expected distribution of results from the synergistic assay used in the experiment, which should provide 60% precision of assessment. This distribution was also verified using 10,000 Monte-Carlo simulations.
    Figure Legend Snippet: Experimental verification of a 16-compartment digital assay with comparison to the performance of qPCR assays. ( A ) The graph shows the results for the same amount of reference DNA suspended in different elution buffers and quantified with conventional qPCR and with the synergistic PCR algorithm. Tests performed on Applied Biosystems 7500 Fast RT System on the IVD Cytomegalovirus PCR kit (GeneProof) according to the prescription. Elution buffers: (1) water, (2) AE elution buffer QIAamp DNA Mini Kit (Quiagen), (3) MBL5 NucleoMag Blood 200 uL (MACHEREY-NAGEL), (4) MagJET Whole Blood Genomic DNA Kit (Thermo Scientific). The gray line shows the expected distribution of results for Real-Time assay. ( B ) The graph shows the result of 24 runs of the synergistic assay, each on 16 partitions of the amplification mix. We conducted two series of 12 assays on the two elution buffers (1 and 4) that provided the largest difference in the result of the conventional qPCR analysis. The gray line shows the expected distribution of results from the synergistic assay used in the experiment, which should provide 60% precision of assessment. This distribution was also verified using 10,000 Monte-Carlo simulations.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification

    40) Product Images from "Calibration-free assays on standard real-time PCR devices"

    Article Title: Calibration-free assays on standard real-time PCR devices

    Journal: Scientific Reports

    doi: 10.1038/srep44854

    Experimental verification of a 16-compartment digital assay with comparison to the performance of qPCR assays. ( A ) The graph shows the results for the same amount of reference DNA suspended in different elution buffers and quantified with conventional qPCR and with the synergistic PCR algorithm. Tests performed on Applied Biosystems 7500 Fast RT System on the IVD Cytomegalovirus PCR kit (GeneProof) according to the prescription. Elution buffers: (1) water, (2) AE elution buffer QIAamp DNA Mini Kit (Quiagen), (3) MBL5 NucleoMag Blood 200 uL (MACHEREY-NAGEL), (4) MagJET Whole Blood Genomic DNA Kit (Thermo Scientific). The gray line shows the expected distribution of results for Real-Time assay. ( B ) The graph shows the result of 24 runs of the synergistic assay, each on 16 partitions of the amplification mix. We conducted two series of 12 assays on the two elution buffers (1 and 4) that provided the largest difference in the result of the conventional qPCR analysis. The gray line shows the expected distribution of results from the synergistic assay used in the experiment, which should provide 60% precision of assessment. This distribution was also verified using 10,000 Monte-Carlo simulations.
    Figure Legend Snippet: Experimental verification of a 16-compartment digital assay with comparison to the performance of qPCR assays. ( A ) The graph shows the results for the same amount of reference DNA suspended in different elution buffers and quantified with conventional qPCR and with the synergistic PCR algorithm. Tests performed on Applied Biosystems 7500 Fast RT System on the IVD Cytomegalovirus PCR kit (GeneProof) according to the prescription. Elution buffers: (1) water, (2) AE elution buffer QIAamp DNA Mini Kit (Quiagen), (3) MBL5 NucleoMag Blood 200 uL (MACHEREY-NAGEL), (4) MagJET Whole Blood Genomic DNA Kit (Thermo Scientific). The gray line shows the expected distribution of results for Real-Time assay. ( B ) The graph shows the result of 24 runs of the synergistic assay, each on 16 partitions of the amplification mix. We conducted two series of 12 assays on the two elution buffers (1 and 4) that provided the largest difference in the result of the conventional qPCR analysis. The gray line shows the expected distribution of results from the synergistic assay used in the experiment, which should provide 60% precision of assessment. This distribution was also verified using 10,000 Monte-Carlo simulations.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification

    41) Product Images from "Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells"

    Article Title: Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2015.12.014

    Methyl Transfer from Methionine to DNA and RNA Is Impeded during Serine Starvation, yet Serine Does Not Contribute One-Carbon Units for Methylation under Methionine-Fed Conditions (A) A method was developed to directly track the incorporation of labeled one-carbons from extracellular nutrients into DNA and RNA. After starvation/drug treatment, cells were fed labeled nutrients for 3 hr. Following standard DNA and RNA isolation, nucleic acids were hydrolyzed and analyzed by LCMS, revealing the extent of methyl cytosine and methyl adenine labeling. (B and C) RKO cells were grown in the presence or absence of unlabeled methionine 0.1 mM (Met) and serine 0.8 mM (Ser) for 6 hr, after which the media were replaced with matched media containing either labeled methionine or labeled serine for 3 hr and analyzed as outlined in (A). The methionine-starved cells were supplemented with 0.8 mM homocysteine and 1 μM vitamin B 12 . The ratio of labeled to unlabeled methyl cytosine (mC+1/mC) in DNA and labeled to unlabeled methyl adenine (mA+1/mA) in RNA are shown. Data are averages of triplicate wells, and error bars are SD. Broken lines indicate the background labeling due to natural 13 C abundance, shown as control (Ctr.) blue bars. (D) HCT116 and RKO cells were grown with or without serine 0.8 mM (+/−Ser) for 6 hr, after which the media were replaced with matched media containing either unlabeled (Ctr.) or labeled methionine for 3 hr and analyzed as outlined in (A). Azacytidine 0.5 μM (AzaC) a DNA methyltransferase inhibitor was used as positive control. Data are averages of triplicate wells; error bars are SD. (E) HCT116 cells were treated as described in (D), either with an initial serine starvation (with unlabeled methionine) of 6 hr or 30 hr, followed by a 3 hr period with labeled methionine. Data are averages of triplicate wells; error bars are SD. See also Figure S1 .
    Figure Legend Snippet: Methyl Transfer from Methionine to DNA and RNA Is Impeded during Serine Starvation, yet Serine Does Not Contribute One-Carbon Units for Methylation under Methionine-Fed Conditions (A) A method was developed to directly track the incorporation of labeled one-carbons from extracellular nutrients into DNA and RNA. After starvation/drug treatment, cells were fed labeled nutrients for 3 hr. Following standard DNA and RNA isolation, nucleic acids were hydrolyzed and analyzed by LCMS, revealing the extent of methyl cytosine and methyl adenine labeling. (B and C) RKO cells were grown in the presence or absence of unlabeled methionine 0.1 mM (Met) and serine 0.8 mM (Ser) for 6 hr, after which the media were replaced with matched media containing either labeled methionine or labeled serine for 3 hr and analyzed as outlined in (A). The methionine-starved cells were supplemented with 0.8 mM homocysteine and 1 μM vitamin B 12 . The ratio of labeled to unlabeled methyl cytosine (mC+1/mC) in DNA and labeled to unlabeled methyl adenine (mA+1/mA) in RNA are shown. Data are averages of triplicate wells, and error bars are SD. Broken lines indicate the background labeling due to natural 13 C abundance, shown as control (Ctr.) blue bars. (D) HCT116 and RKO cells were grown with or without serine 0.8 mM (+/−Ser) for 6 hr, after which the media were replaced with matched media containing either unlabeled (Ctr.) or labeled methionine for 3 hr and analyzed as outlined in (A). Azacytidine 0.5 μM (AzaC) a DNA methyltransferase inhibitor was used as positive control. Data are averages of triplicate wells; error bars are SD. (E) HCT116 cells were treated as described in (D), either with an initial serine starvation (with unlabeled methionine) of 6 hr or 30 hr, followed by a 3 hr period with labeled methionine. Data are averages of triplicate wells; error bars are SD. See also Figure S1 .

    Techniques Used: Methylation, Labeling, Isolation, Liquid Chromatography with Mass Spectroscopy, Positive Control

    Under Methionine Starvation, Homocysteine Is Re-Methylated Using One-Carbon Units from Serine (A) RKO cells were grown with unlabeled methionine 0.1 mM and serine 0.8 mM (MS) or unlabeled serine plus homocysteine (SHc) for 21 hr, followed by matched medium containing 13 C 3 15 N 1 -labeled serine for 3 hr. DNA and RNA were isolated an analyzed by LCMS. Broken lines indicate the background labeling due to natural 13 C abundance, shown as control (Ctr.) blue bars. Data are averages of triplicate wells; error bars are SD. (B) RKO, HCT116, and SW480 cells were grown in medium with serine 0.8 mM (S), with or without methionine 0.1 mM (M), homocysteine 0.8 mM (Hc), and vitamin B 12 1uM (B12), and counted after 2 and 4 days. Data are averages of triplicate wells; error bars are SD.
    Figure Legend Snippet: Under Methionine Starvation, Homocysteine Is Re-Methylated Using One-Carbon Units from Serine (A) RKO cells were grown with unlabeled methionine 0.1 mM and serine 0.8 mM (MS) or unlabeled serine plus homocysteine (SHc) for 21 hr, followed by matched medium containing 13 C 3 15 N 1 -labeled serine for 3 hr. DNA and RNA were isolated an analyzed by LCMS. Broken lines indicate the background labeling due to natural 13 C abundance, shown as control (Ctr.) blue bars. Data are averages of triplicate wells; error bars are SD. (B) RKO, HCT116, and SW480 cells were grown in medium with serine 0.8 mM (S), with or without methionine 0.1 mM (M), homocysteine 0.8 mM (Hc), and vitamin B 12 1uM (B12), and counted after 2 and 4 days. Data are averages of triplicate wells; error bars are SD.

    Techniques Used: Methylation, Mass Spectrometry, Labeling, Isolation, Liquid Chromatography with Mass Spectroscopy

    42) Product Images from "Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function"

    Article Title: Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function

    Journal: Nature Communications

    doi: 10.1038/ncomms12723

    Catecholamine treatment triggers Perilipin 5 interaction with PGC-1α and SIRT1 that promotes PGC-1α deacetylation. ( a ) Immunoprecipitation (IP) of PGC-1α (left) and of Perilipin 5 (right) from nuclear extracts of C2C12 myotubes treated with vehicle or Iso/IBMX for 2 h. ( b ) IP of Perilipin 5 from BAT of wild-type mice treated with β3 adrenergic agonist CL 316,243 (CL) or vehicle. ( c , d ) IP of Perilipin 5 ( c ) or PGC-1α ( d ) from whole-cell lysate of brown adipocytes treated with vehicle or Iso/IBMX for 2 h. IgG control IP and siRNA duplexes for PGC-1α and/or Perilipin 5 were used as IP controls. ( e ) IP from whole-cell extract of C2C12 myoblasts transfected with myc-Perilipin 5 and flag-PGC-1α and treated with Iso/IBMX for 2 h. Non-transfected cells were used as IP control. ( f ) PLA reveals Perilipin 5 interaction with PGC-1α in C2C12 differentiating myoblasts treated with vehicle or Iso/IBMX for 2 h. Red dots indicate proximity of interaction partners within 20–40 nm (left) with quantification using Cell Profiler software (right). ( g ) IP of PGC-1α from whole-cell lysate of brown adypocytes treated with Iso/IBMX for 2 h. Cells were additionally treated with orlistat or vehicle and siATGL or siControl. IgG control and siPGC-1α were used as IP controls. ( h ) IP of PGC-1α from nuclear extracts of MLTC-1 cells transfected with siControl or siPerilipin 5. Non-immune rabbit IgG was used as the IP-negative control.
    Figure Legend Snippet: Catecholamine treatment triggers Perilipin 5 interaction with PGC-1α and SIRT1 that promotes PGC-1α deacetylation. ( a ) Immunoprecipitation (IP) of PGC-1α (left) and of Perilipin 5 (right) from nuclear extracts of C2C12 myotubes treated with vehicle or Iso/IBMX for 2 h. ( b ) IP of Perilipin 5 from BAT of wild-type mice treated with β3 adrenergic agonist CL 316,243 (CL) or vehicle. ( c , d ) IP of Perilipin 5 ( c ) or PGC-1α ( d ) from whole-cell lysate of brown adipocytes treated with vehicle or Iso/IBMX for 2 h. IgG control IP and siRNA duplexes for PGC-1α and/or Perilipin 5 were used as IP controls. ( e ) IP from whole-cell extract of C2C12 myoblasts transfected with myc-Perilipin 5 and flag-PGC-1α and treated with Iso/IBMX for 2 h. Non-transfected cells were used as IP control. ( f ) PLA reveals Perilipin 5 interaction with PGC-1α in C2C12 differentiating myoblasts treated with vehicle or Iso/IBMX for 2 h. Red dots indicate proximity of interaction partners within 20–40 nm (left) with quantification using Cell Profiler software (right). ( g ) IP of PGC-1α from whole-cell lysate of brown adypocytes treated with Iso/IBMX for 2 h. Cells were additionally treated with orlistat or vehicle and siATGL or siControl. IgG control and siPGC-1α were used as IP controls. ( h ) IP of PGC-1α from nuclear extracts of MLTC-1 cells transfected with siControl or siPerilipin 5. Non-immune rabbit IgG was used as the IP-negative control.

    Techniques Used: Pyrolysis Gas Chromatography, Immunoprecipitation, Mouse Assay, Transfection, Proximity Ligation Assay, Software, Negative Control

    Perilipin 5 promotes catecholamine-driven PGC-1α promoter activity in a PGC-1α /SIRT1-dependent manner. C2C12 myoblasts were co-transfected with the indicated plasmids (25–100 ng each, as labelled) ( a , b , d , f ) or the indicated plasmids and siRNA duplexes ( c , e ) together with the 2 kb PGC-1α promoter luciferase plasmid. Forty-eight hours after the transfection, cells were treated with vehicle or Iso/IBMX for 2 h and luciferase activity was measured. In all experiments Renilla luciferase vector was co-transfected to normalize for transfection efficiency. Values are expressed as mean±s.e.m. n =6 biological replicates for each data point with each biological replicate representing an independent transfection and treatment of C2C12 myoblasts. The data shown were replicated in two additional independent experiments. * P
    Figure Legend Snippet: Perilipin 5 promotes catecholamine-driven PGC-1α promoter activity in a PGC-1α /SIRT1-dependent manner. C2C12 myoblasts were co-transfected with the indicated plasmids (25–100 ng each, as labelled) ( a , b , d , f ) or the indicated plasmids and siRNA duplexes ( c , e ) together with the 2 kb PGC-1α promoter luciferase plasmid. Forty-eight hours after the transfection, cells were treated with vehicle or Iso/IBMX for 2 h and luciferase activity was measured. In all experiments Renilla luciferase vector was co-transfected to normalize for transfection efficiency. Values are expressed as mean±s.e.m. n =6 biological replicates for each data point with each biological replicate representing an independent transfection and treatment of C2C12 myoblasts. The data shown were replicated in two additional independent experiments. * P

    Techniques Used: Pyrolysis Gas Chromatography, Activity Assay, Transfection, Luciferase, Plasmid Preparation

    Perilipin 5 regulates catecholamine-stimulated SIRT1 deacetylation activity and mitochondrial biogenesis and respiration. ( a ) MLTC-1 cells were transfected with the indicated siRNA duplexes. After 72 h, cells were treated with Iso/IBMX or vehicle for 30 min and SIRT1 deacetylase activity was measured in the cell lysates (left). Western blotting for SIRT1, Perilipin 5 and GAPDH document siRNA specificity (bottom). Intracellular NAD + levels with Iso/IBMX or siPerilipin 5 treatments (right). ( b ) Immunoprecipitation of SIRT1 followed by WB for DBC1 and SIRT1 from MLTC-1 cells transfected with the indicated siRNA duplexes. Cells were treated with vehicle or Iso/IBMX for 30 min before immunoprecipitation. Relative quantification of immunoprecipitated DBC-1 normalized for immunoprecipitated SIRT1 is shown in the histogram. ( c ) PLA for SIRT1-DBC1 complexes (red dots) in MLTC-1 cells transfected with the indicated siRNA duplexes for 48 h followed by treatment with vehicle or Iso/IBMX for 30 min (left) with quantification using Cell Profiler (right). ( d ) mtDNA content analysed by qPCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX for 2 h. ( e ) OCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX. n =3 biological replicates/experimental condition with each biological replicate representing an independent transfection and treatment. Values are expressed as the mean±s.e.m. These data were replicated in a second independent experiment. * P
    Figure Legend Snippet: Perilipin 5 regulates catecholamine-stimulated SIRT1 deacetylation activity and mitochondrial biogenesis and respiration. ( a ) MLTC-1 cells were transfected with the indicated siRNA duplexes. After 72 h, cells were treated with Iso/IBMX or vehicle for 30 min and SIRT1 deacetylase activity was measured in the cell lysates (left). Western blotting for SIRT1, Perilipin 5 and GAPDH document siRNA specificity (bottom). Intracellular NAD + levels with Iso/IBMX or siPerilipin 5 treatments (right). ( b ) Immunoprecipitation of SIRT1 followed by WB for DBC1 and SIRT1 from MLTC-1 cells transfected with the indicated siRNA duplexes. Cells were treated with vehicle or Iso/IBMX for 30 min before immunoprecipitation. Relative quantification of immunoprecipitated DBC-1 normalized for immunoprecipitated SIRT1 is shown in the histogram. ( c ) PLA for SIRT1-DBC1 complexes (red dots) in MLTC-1 cells transfected with the indicated siRNA duplexes for 48 h followed by treatment with vehicle or Iso/IBMX for 30 min (left) with quantification using Cell Profiler (right). ( d ) mtDNA content analysed by qPCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX for 2 h. ( e ) OCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX. n =3 biological replicates/experimental condition with each biological replicate representing an independent transfection and treatment. Values are expressed as the mean±s.e.m. These data were replicated in a second independent experiment. * P

    Techniques Used: Activity Assay, Transfection, Histone Deacetylase Assay, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Real-time Polymerase Chain Reaction

    43) Product Images from "A Pilot Study into the Association between Oral Health Status and Human Papillomavirus—16 Infection"

    Article Title: A Pilot Study into the Association between Oral Health Status and Human Papillomavirus—16 Infection

    Journal: Diagnostics

    doi: 10.3390/diagnostics7010011

    The detection of human papillomavirus (HPV)-16 DNA in patient oral rinse samples from UQ Dental School Clinic by PCR. Representative gel image showing the detection of HPV-16 DNA in patient oral rinse samples using HPV-16 NB2 primers (~140 bp) with beta-globin used as an internal control (~250 bp). Lane 1 represents DNA Ladder; Lane 2 represents non-template control; Lanes 3–5 (134, 144 and 145) represent the absence of HPV-16 DNA in patient samples; Lanes 6–8 (236, 1338 and 1368) represent the presence of HPV-16 DNA in patient samples and Lane 9 (93VU147T) and 10 (Caski) represent HPV-16 positive cancer cell lines.
    Figure Legend Snippet: The detection of human papillomavirus (HPV)-16 DNA in patient oral rinse samples from UQ Dental School Clinic by PCR. Representative gel image showing the detection of HPV-16 DNA in patient oral rinse samples using HPV-16 NB2 primers (~140 bp) with beta-globin used as an internal control (~250 bp). Lane 1 represents DNA Ladder; Lane 2 represents non-template control; Lanes 3–5 (134, 144 and 145) represent the absence of HPV-16 DNA in patient samples; Lanes 6–8 (236, 1338 and 1368) represent the presence of HPV-16 DNA in patient samples and Lane 9 (93VU147T) and 10 (Caski) represent HPV-16 positive cancer cell lines.

    Techniques Used: Polymerase Chain Reaction

    44) Product Images from "The transcription factor XBP1s restores hippocampal synaptic plasticity and memory by control of the Kalirin-7 pathway in Alzheimer model"

    Article Title: The transcription factor XBP1s restores hippocampal synaptic plasticity and memory by control of the Kalirin-7 pathway in Alzheimer model

    Journal: Molecular Psychiatry

    doi: 10.1038/mp.2016.152

    Kalirin-7 levels are reduced in the hippocampus of patients with Alzheimer’s disease. ( a – d ) Representative immunohistochemical images depicting cytoplasmic Kalirin-7 (Kal7) in neurons of hippocampal dentate gyrus (DG) ( a , b ) and CA1 region ( c , d ) from patients with Braak and Braak (BB) stages V–VI ( n =12) compared with healthy BB0 control brains (CON, n =5). Scale bar: 100 μm (upper images), 20 μm (lower images). PCs, pyramidal cells; GCs, granule cells. ( e , f ) Quantitative assessment of Kal7 levels in a – d . Mann–Whitney test: Kal7 levels DG: U =−3.5529, P
    Figure Legend Snippet: Kalirin-7 levels are reduced in the hippocampus of patients with Alzheimer’s disease. ( a – d ) Representative immunohistochemical images depicting cytoplasmic Kalirin-7 (Kal7) in neurons of hippocampal dentate gyrus (DG) ( a , b ) and CA1 region ( c , d ) from patients with Braak and Braak (BB) stages V–VI ( n =12) compared with healthy BB0 control brains (CON, n =5). Scale bar: 100 μm (upper images), 20 μm (lower images). PCs, pyramidal cells; GCs, granule cells. ( e , f ) Quantitative assessment of Kal7 levels in a – d . Mann–Whitney test: Kal7 levels DG: U =−3.5529, P

    Techniques Used: Immunohistochemistry, MANN-WHITNEY

    Reducing Kalirin-7 levels induces synaptic and cognitive deficits in naive mice. ( a ) Left, bilateral stereotaxic injection of lentiviral vectors into the hippocampus of naive mice. Right, timeline of Morris water maze (MWM) test and subsequent biochemical analysis of mice. ( b ) qRT-PCR analysis of EphB2 and Kal7 transcripts in the whole hippocampus ( n =5–6 mice per group). Kruskal–Wallis test: EphB2: H =14.4855, P
    Figure Legend Snippet: Reducing Kalirin-7 levels induces synaptic and cognitive deficits in naive mice. ( a ) Left, bilateral stereotaxic injection of lentiviral vectors into the hippocampus of naive mice. Right, timeline of Morris water maze (MWM) test and subsequent biochemical analysis of mice. ( b ) qRT-PCR analysis of EphB2 and Kal7 transcripts in the whole hippocampus ( n =5–6 mice per group). Kruskal–Wallis test: EphB2: H =14.4855, P

    Techniques Used: Mouse Assay, Injection, Quantitative RT-PCR

    45) Product Images from "Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer"

    Article Title: Epigenetic Suppression of the T-box Subfamily 2 (TBX2) in Human Non-Small Cell Lung Cancer

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20051159

    Suppressed expression of TBX2 subfamily in non-small cell lung cancer (NSCLC) cell lines relative to normal pulmonary alveolar epithelial cells. Messenger RNA expression levels of all four members of TBX2 subfamily in NSCLC cell lines compared to normal pulmonary alveolar epithelial cells (NAC) were analyzed in triplicates by qRT-PCR. Analysis was performed on NSCLC cell lines grown in serum-containing medium (10% FBS; left panels) as well as on cells maintained in media with reduced serum (1% FBS; right panels). Relative expression data in each of the NSCLC cell lines and for each of the TBX genes were computed using the 2 −ΔΔ C t calculation method by normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and are presented as mean ± SEM (* p
    Figure Legend Snippet: Suppressed expression of TBX2 subfamily in non-small cell lung cancer (NSCLC) cell lines relative to normal pulmonary alveolar epithelial cells. Messenger RNA expression levels of all four members of TBX2 subfamily in NSCLC cell lines compared to normal pulmonary alveolar epithelial cells (NAC) were analyzed in triplicates by qRT-PCR. Analysis was performed on NSCLC cell lines grown in serum-containing medium (10% FBS; left panels) as well as on cells maintained in media with reduced serum (1% FBS; right panels). Relative expression data in each of the NSCLC cell lines and for each of the TBX genes were computed using the 2 −ΔΔ C t calculation method by normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and are presented as mean ± SEM (* p

    Techniques Used: Expressing, RNA Expression, Quantitative RT-PCR

    Members of the TBX2 subfamily are hypermethylated in NSCLC cell lines. Methylation levels of members of the TBX2 subfamily were examined in eight NSCLC cell lines and normal alveolar epithelial cells by quantitative methylation-specific PCR using the Epitect Methyl II PCR assay from QIAGEN according to the manufacturer’s instructions (see Section 4 ). Percentages of un-methylated (UM) and methylated (M) TBX2 subfamily alleles are depicted and represent an average of two experiments (* p
    Figure Legend Snippet: Members of the TBX2 subfamily are hypermethylated in NSCLC cell lines. Methylation levels of members of the TBX2 subfamily were examined in eight NSCLC cell lines and normal alveolar epithelial cells by quantitative methylation-specific PCR using the Epitect Methyl II PCR assay from QIAGEN according to the manufacturer’s instructions (see Section 4 ). Percentages of un-methylated (UM) and methylated (M) TBX2 subfamily alleles are depicted and represent an average of two experiments (* p

    Techniques Used: Methylation, Polymerase Chain Reaction

    Augmented expression of the TBX2 subfamily in NSCLC cell lines treated with the DNA methyltransferase 5-azacytidine. ( A ) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to assess cellular proliferation as described in Section 4 . Cells were seeded in 96-well plates, at varying seeding densities following optimization, and treated with differing doses of Aza or dimethyl sulfoxide (DMSO) as control for 24, 48 and 72 h. Aza treatment was prepared and replenished every day as per previous reports [ 14 ]. Cell numbers (% of control) were determined by MTT assays and represent percentages of absorbance readings from Aza-treated cells (at each dose) versus DMSO-treated cells. Data depicted represent means ± standard deviations. ( B ) The same NSCLC cell lines were seeded in 96-well plates and treated according to IC50s (1 µM) for 24 h and 72 h. Assessment of growth inhibition was performed using the trypan blue dye exclusion method as detailed in Section 4 . Growth inhibition was calculated according to the following formula: 100 − (treated/non-treated) × 100). Data depicted comprise the means ± SEM of two independent experiments (* p
    Figure Legend Snippet: Augmented expression of the TBX2 subfamily in NSCLC cell lines treated with the DNA methyltransferase 5-azacytidine. ( A ) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to assess cellular proliferation as described in Section 4 . Cells were seeded in 96-well plates, at varying seeding densities following optimization, and treated with differing doses of Aza or dimethyl sulfoxide (DMSO) as control for 24, 48 and 72 h. Aza treatment was prepared and replenished every day as per previous reports [ 14 ]. Cell numbers (% of control) were determined by MTT assays and represent percentages of absorbance readings from Aza-treated cells (at each dose) versus DMSO-treated cells. Data depicted represent means ± standard deviations. ( B ) The same NSCLC cell lines were seeded in 96-well plates and treated according to IC50s (1 µM) for 24 h and 72 h. Assessment of growth inhibition was performed using the trypan blue dye exclusion method as detailed in Section 4 . Growth inhibition was calculated according to the following formula: 100 − (treated/non-treated) × 100). Data depicted comprise the means ± SEM of two independent experiments (* p

    Techniques Used: Expressing, MTT Assay, Inhibition

    46) Product Images from "Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors"

    Article Title: Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors

    Journal: Epigenetics

    doi: 10.1080/15592294.2015.1048953

    Treatment with 5-Aza does not enhance radiation-induced DNA damage and cell death. ( A ) SCC-61 and rSCC-61 cells were incubated with 2 µM of 5-Aza or AraC overnight and then irradiated with a single dose of 2, 4, or 8 Gy. Cell viability
    Figure Legend Snippet: Treatment with 5-Aza does not enhance radiation-induced DNA damage and cell death. ( A ) SCC-61 and rSCC-61 cells were incubated with 2 µM of 5-Aza or AraC overnight and then irradiated with a single dose of 2, 4, or 8 Gy. Cell viability

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Irradiation

    Targeted analysis of CCND2 regulation by promoter methylation. SCC-61 and rSCC-61 cells were treated with 0–5 µM 5-Aza or 0–1 µM AraC for 4 days. After this, the cells were either lysed for Western blot
    Figure Legend Snippet: Targeted analysis of CCND2 regulation by promoter methylation. SCC-61 and rSCC-61 cells were treated with 0–5 µM 5-Aza or 0–1 µM AraC for 4 days. After this, the cells were either lysed for Western blot

    Techniques Used: Methylation, Western Blot

    Treatment of rSCC-61 cells with 5-Aza decreases PTEN levels and upregulates downstream AKT signaling. SCC-61 and rSCC-61 cells were treated with 0–5 µM 5-Aza or 0–1 µM AraC for 4 days. Cells were then lysed
    Figure Legend Snippet: Treatment of rSCC-61 cells with 5-Aza decreases PTEN levels and upregulates downstream AKT signaling. SCC-61 and rSCC-61 cells were treated with 0–5 µM 5-Aza or 0–1 µM AraC for 4 days. Cells were then lysed

    Techniques Used:

    47) Product Images from "AZFc deletions do not affect the function of human spermatogonia in vitro"

    Article Title: AZFc deletions do not affect the function of human spermatogonia in vitro

    Journal: Molecular Human Reproduction

    doi: 10.1093/molehr/gav022

    Confirmation of the absence of the complete AZFc region in testis tissues of all AZFc -deleted patients. The complete absence or presence of the AZFc region in testicular genomic DNA of all patients and controls was performed in a multiplex PCR by testing
    Figure Legend Snippet: Confirmation of the absence of the complete AZFc region in testis tissues of all AZFc -deleted patients. The complete absence or presence of the AZFc region in testicular genomic DNA of all patients and controls was performed in a multiplex PCR by testing

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction

    48) Product Images from "Comparative haploid genetic screens reveal divergent pathways in the biogenesis and trafficking of glycophosphatidylinositol-anchored proteins"

    Article Title: Comparative haploid genetic screens reveal divergent pathways in the biogenesis and trafficking of glycophosphatidylinositol-anchored proteins

    Journal: Cell reports

    doi: 10.1016/j.celrep.2015.05.026

    Haploid genetic screen of the PrP pathway (A) Diagram showing the haploid genetic screen of the PrP pathway using the mutant HAP1 cell library. ( B ) Mutant HAP1 cells were labeled with anti-PrP antibodies and APC-conjugated anti-mouse antibodies. FACS was used to collect cells with the lowest fluorescence signal. After the third round of sorting, cells were divided into two populations – dim and dark – according to their fluorescence intensity. The dark population contained cells with log fluorescence intensity less than 10, while the dim population contained cells with log fluorescence intensity between 10 and 500. Genomic DNA samples from the dark and dim populations were combined in equal amounts and retroviral gene-trap insertions were mapped by deep sequencing.
    Figure Legend Snippet: Haploid genetic screen of the PrP pathway (A) Diagram showing the haploid genetic screen of the PrP pathway using the mutant HAP1 cell library. ( B ) Mutant HAP1 cells were labeled with anti-PrP antibodies and APC-conjugated anti-mouse antibodies. FACS was used to collect cells with the lowest fluorescence signal. After the third round of sorting, cells were divided into two populations – dim and dark – according to their fluorescence intensity. The dark population contained cells with log fluorescence intensity less than 10, while the dim population contained cells with log fluorescence intensity between 10 and 500. Genomic DNA samples from the dark and dim populations were combined in equal amounts and retroviral gene-trap insertions were mapped by deep sequencing.

    Techniques Used: Mutagenesis, Labeling, FACS, Fluorescence, Sequencing

    49) Product Images from "Development of Sensitive Detection of Cryptosporidium and Giardia from Surface Water in Iran"

    Article Title: Development of Sensitive Detection of Cryptosporidium and Giardia from Surface Water in Iran

    Journal: Iranian Journal of Parasitology

    doi:

    1.5% agarose gel electrophoresis of PCR product. Lane 1: Giardia positive control by Phenol-chloroform extraction; Lanes 2 and 3: DNA extraction by Phenol-chloroform of surface water samples; Lanes 4 and 5: DNA extraction by QIAamp DNA mini kit of surface water samples
    Figure Legend Snippet: 1.5% agarose gel electrophoresis of PCR product. Lane 1: Giardia positive control by Phenol-chloroform extraction; Lanes 2 and 3: DNA extraction by Phenol-chloroform of surface water samples; Lanes 4 and 5: DNA extraction by QIAamp DNA mini kit of surface water samples

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Positive Control, DNA Extraction

    50) Product Images from "Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases"

    Article Title: Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases

    Journal: Parasite

    doi: 10.1051/parasite/2013051

    Influence of proteinase K digestion (56 °C overnight) on DNA extraction. Graph A shows the Ct values obtained by quantifying THP1 cell DNA derived from direct extraction with the NucliSENS easyMAG system and extraction performed on the same quantity of cells following overnight (ON) digestion with Proteinase K. Graph B shows Leishmania quantification after extraction with the NucliSENS easyMAG system both with and without PK and quantification after extraction using a QIAamp DNA Mini kit after ON digestion with PK.
    Figure Legend Snippet: Influence of proteinase K digestion (56 °C overnight) on DNA extraction. Graph A shows the Ct values obtained by quantifying THP1 cell DNA derived from direct extraction with the NucliSENS easyMAG system and extraction performed on the same quantity of cells following overnight (ON) digestion with Proteinase K. Graph B shows Leishmania quantification after extraction with the NucliSENS easyMAG system both with and without PK and quantification after extraction using a QIAamp DNA Mini kit after ON digestion with PK.

    Techniques Used: DNA Extraction, Derivative Assay

    51) Product Images from "Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR"

    Article Title: Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169846

    Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns. Genomic and plasmid DNA were isolated from E . coli DH5α and E . coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P
    Figure Legend Snippet: Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns. Genomic and plasmid DNA were isolated from E . coli DH5α and E . coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P

    Techniques Used: Plasmid Preparation, Isolation, Spectrophotometry, Lysis, Standard Deviation

    Quantification of pBR322 plasmid copy number by digital droplet PCR. E . coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A ( Table 1A ). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs , respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs . Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.
    Figure Legend Snippet: Quantification of pBR322 plasmid copy number by digital droplet PCR. E . coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A ( Table 1A ). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs , respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs . Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Isolation, Amplification

    52) Product Images from "Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols"

    Article Title: Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

    Journal: Journal of Applied Oral Science

    doi: 10.1590/1678-77572016-0046

    DNA quantity, purity, integrity and whether each sample contained amplifiable human DNA obtained from fresh saliva and saliva frozen at different time periods using five different extraction protocols. Protocol 1 (1) used the Oragene™ kit; protocol 2 (2) used the QIAamp® DNA Mini kit; protocol 3 (3) used ammonium acetate, protocol 4 (4) used the InstaGene™ Matrix kit; protocol 5 (5) used the InstaGene™ kit with proteinase K and 1% SDS. Quantity in terms of concentration (ng/µL) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges. (A) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (B) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (C) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (D) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Purity was assessed using spectrophotometry and expressed as a percentage of positive samples, absorbance ratio of A260/280 between 1.6 and 2.0 (black fill) or negative, outside the 1.6 to 2.0 range (white fill); (E) Samples from fresh saliva using each of the 5 DNA extraction protocols; (F) Samples from saliva frozen for 3 months using each of the 5 DNA extraction protocols; (G) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (H) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Integrity of DNA, visualized as a percentage of unfragmented (black fill), fragmented (white fill) or undetected (gray fill) DNA, as assessed using electrophoretic analysis; (I) Samples from fresh saliva using each of the 5 DNA extraction protocols; (J) Samples from saliva frozen for 3 months using each of the 5 DNA extraction protocols; (K) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (L) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Conventional PCR using primers specific for human DNA was used to amplify exon 3 of the interferon regulatory factor 6 (IRF6) gene in all samples at all time points investigated, and then visualized on an agarose gel; samples were either positive (black fill) or negative (white fill) for the presence of amplified human DNA; (M) Human DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (N) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (O) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (P) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Absolute quantity (µg) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges; (Q) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (R) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (S) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (T) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. For each test at each time point, all protocols were tested overall (p-value
    Figure Legend Snippet: DNA quantity, purity, integrity and whether each sample contained amplifiable human DNA obtained from fresh saliva and saliva frozen at different time periods using five different extraction protocols. Protocol 1 (1) used the Oragene™ kit; protocol 2 (2) used the QIAamp® DNA Mini kit; protocol 3 (3) used ammonium acetate, protocol 4 (4) used the InstaGene™ Matrix kit; protocol 5 (5) used the InstaGene™ kit with proteinase K and 1% SDS. Quantity in terms of concentration (ng/µL) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges. (A) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (B) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (C) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (D) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Purity was assessed using spectrophotometry and expressed as a percentage of positive samples, absorbance ratio of A260/280 between 1.6 and 2.0 (black fill) or negative, outside the 1.6 to 2.0 range (white fill); (E) Samples from fresh saliva using each of the 5 DNA extraction protocols; (F) Samples from saliva frozen for 3 months using each of the 5 DNA extraction protocols; (G) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (H) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Integrity of DNA, visualized as a percentage of unfragmented (black fill), fragmented (white fill) or undetected (gray fill) DNA, as assessed using electrophoretic analysis; (I) Samples from fresh saliva using each of the 5 DNA extraction protocols; (J) Samples from saliva frozen for 3 months using each of the 5 DNA extraction protocols; (K) Samples from saliva frozen for 6 months using each of the 5 DNA extraction protocols; (L) Samples from saliva frozen for 12 months using each of the 5 DNA extraction protocols. Conventional PCR using primers specific for human DNA was used to amplify exon 3 of the interferon regulatory factor 6 (IRF6) gene in all samples at all time points investigated, and then visualized on an agarose gel; samples were either positive (black fill) or negative (white fill) for the presence of amplified human DNA; (M) Human DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (N) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (O) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (P) Human DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. Absolute quantity (µg) was assessed using spectrophotometry and reported using medians and their respective interquartile ranges; (Q) DNA obtained by each of the 5 DNA extraction protocols from fresh saliva; (R) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 3 months; (S) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 6 months; (T) DNA obtained by each of the 5 DNA extraction protocols from saliva frozen for 12 months. For each test at each time point, all protocols were tested overall (p-value

    Techniques Used: Concentration Assay, Spectrophotometry, DNA Extraction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Significance Assay

    An example of a gel from 5 different extraction protocols when DNA was extracted from fresh saliva or saliva stored for 3, 6 or 12 months investigating whether samples were fragmented. DNA samples from 2 individuals (A, B) were electrophoresed using a 0.8% agarose gel in Tris-acetate (200 mM) with EDTA (50 mM) buffer. Lanes 1 and 25 contain the 100 bp molecular weight standard (M); lanes 2 and 26 contain the positive control (+), 115 ng of human DNA; lanes 3 and 27 contain the negative control (-), 8 µL of ddH2O; lanes 24 and 48 were left blank; all other lanes contain 8 µL of extracted DNA from either volunteer A or B. Protocol 1 (P1) used the Oragene™ kit; protocol 2 (P2) used the QIAamp® DNA Mini kit; protocol 3 (P3) used ammonium acetate, protocol 4 (P4) used the InstaGene™ Matrix kit; protocol 5 (P5) used the InstaGene™ kit with proteinase K and 1% SDS
    Figure Legend Snippet: An example of a gel from 5 different extraction protocols when DNA was extracted from fresh saliva or saliva stored for 3, 6 or 12 months investigating whether samples were fragmented. DNA samples from 2 individuals (A, B) were electrophoresed using a 0.8% agarose gel in Tris-acetate (200 mM) with EDTA (50 mM) buffer. Lanes 1 and 25 contain the 100 bp molecular weight standard (M); lanes 2 and 26 contain the positive control (+), 115 ng of human DNA; lanes 3 and 27 contain the negative control (-), 8 µL of ddH2O; lanes 24 and 48 were left blank; all other lanes contain 8 µL of extracted DNA from either volunteer A or B. Protocol 1 (P1) used the Oragene™ kit; protocol 2 (P2) used the QIAamp® DNA Mini kit; protocol 3 (P3) used ammonium acetate, protocol 4 (P4) used the InstaGene™ Matrix kit; protocol 5 (P5) used the InstaGene™ kit with proteinase K and 1% SDS

    Techniques Used: Agarose Gel Electrophoresis, Molecular Weight, Positive Control, Negative Control

    53) Product Images from "Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR"

    Article Title: Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    doi: 10.1007/s10096-011-1191-4

    DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles
    Figure Legend Snippet: DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles

    Techniques Used: DNA Extraction

    Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water
    Figure Legend Snippet: Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water

    Techniques Used: Inhibition, DNA Extraction

    DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit
    Figure Legend Snippet: DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit

    Techniques Used: DNA Extraction

    54) Product Images from "Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice"

    Article Title: Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33930-w

    Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P
    Figure Legend Snippet: Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P

    Techniques Used: Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Mouse Assay

    55) Product Images from "Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice"

    Article Title: Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33930-w

    Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P
    Figure Legend Snippet: Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P

    Techniques Used: Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Mouse Assay

    56) Product Images from "An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer"

    Article Title: An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer

    Journal: eLife

    doi: 10.7554/eLife.37184

    CD44 status serves as a marker for the mesenchymal and stem-like cell state in human mammary epithelial cells. ( A ) HMLER cells were stained with CD44-PE-CY7 antibody. CD44 high and low cell populations were sorted by flow cytometry. The gating for CD44 high and low cells is shown. ( B ) qPCR analysis of the expression of mesenchymal ( ZEB1 , FN1 and VIM ) and epithelial ( CDH1 ) markers in CD44 high and low HMLER cells. n = 4. *, p
    Figure Legend Snippet: CD44 status serves as a marker for the mesenchymal and stem-like cell state in human mammary epithelial cells. ( A ) HMLER cells were stained with CD44-PE-CY7 antibody. CD44 high and low cell populations were sorted by flow cytometry. The gating for CD44 high and low cells is shown. ( B ) qPCR analysis of the expression of mesenchymal ( ZEB1 , FN1 and VIM ) and epithelial ( CDH1 ) markers in CD44 high and low HMLER cells. n = 4. *, p

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

    QKI and RBFOX1 promote an intermediate mesenchymal cell state. ( A ) Cell morphologies of the CD44-high cells induced by QKI, RBFOX1 and SNAI1. (Upper) HMLER cells expressing QKI, RBFOX1 or SNAI1 were subjected to cell sorting for CD44 high populations by FACS. (Lower) CD44-high cells induced by QKI, RBFOX1 or SNAI1 were stained for actin structures by Phalloidin. Representative pictures were shown for each cell lines. ( B ) Levels of CD44 as measured by flow cytometry in multiple cell lines including MCF7 and ZR75-1 with ectopic expression of EGFP, RBFOX1 or QKI. ( C ) The impact of QKI and RBFOX1 overexpression in cell proliferation. 10,000 HME cells expressing EGFP, QKI, RBFOX1 and SNAI1 were plated in six-well plates and cell numbers were counted after 6 days of propagation. (Left) Relative cell number quantification. (Right) Immunoblot showing the expression of the indicated V5-tagged ORFs. n = 5; *p
    Figure Legend Snippet: QKI and RBFOX1 promote an intermediate mesenchymal cell state. ( A ) Cell morphologies of the CD44-high cells induced by QKI, RBFOX1 and SNAI1. (Upper) HMLER cells expressing QKI, RBFOX1 or SNAI1 were subjected to cell sorting for CD44 high populations by FACS. (Lower) CD44-high cells induced by QKI, RBFOX1 or SNAI1 were stained for actin structures by Phalloidin. Representative pictures were shown for each cell lines. ( B ) Levels of CD44 as measured by flow cytometry in multiple cell lines including MCF7 and ZR75-1 with ectopic expression of EGFP, RBFOX1 or QKI. ( C ) The impact of QKI and RBFOX1 overexpression in cell proliferation. 10,000 HME cells expressing EGFP, QKI, RBFOX1 and SNAI1 were plated in six-well plates and cell numbers were counted after 6 days of propagation. (Left) Relative cell number quantification. (Right) Immunoblot showing the expression of the indicated V5-tagged ORFs. n = 5; *p

    Techniques Used: Expressing, FACS, Staining, Flow Cytometry, Cytometry, Over Expression

    57) Product Images from "A Molecular Epidemiology Survey of Respiratory Adenoviruses Circulating in Children Residing in Southern Palestine"

    Article Title: A Molecular Epidemiology Survey of Respiratory Adenoviruses Circulating in Children Residing in Southern Palestine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042732

    Circulating HAdV types in southern Palestine. HAdV types circulating in Southern Palestine from 2005 to 2010 were detected using nested PCR and DNA sequencing assay of the HAdV hexon gene HVR1–6 followed by sequencing and sequence analysis. The 44 sequences were assigned the GenBank accession numbers [JQ796022–JQ796065]. The percentage of each serotype was calculated relatively to the number of total sequenced samples (44 samples).
    Figure Legend Snippet: Circulating HAdV types in southern Palestine. HAdV types circulating in Southern Palestine from 2005 to 2010 were detected using nested PCR and DNA sequencing assay of the HAdV hexon gene HVR1–6 followed by sequencing and sequence analysis. The 44 sequences were assigned the GenBank accession numbers [JQ796022–JQ796065]. The percentage of each serotype was calculated relatively to the number of total sequenced samples (44 samples).

    Techniques Used: Nested PCR, DNA Sequencing, Sequencing

    58) Product Images from "Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function"

    Article Title: Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function

    Journal: Nature Communications

    doi: 10.1038/ncomms12723

    Catecholamine treatment triggers Perilipin 5 interaction with PGC-1α and SIRT1 that promotes PGC-1α deacetylation. ( a ) Immunoprecipitation (IP) of PGC-1α (left) and of Perilipin 5 (right) from nuclear extracts of C2C12 myotubes treated with vehicle or Iso/IBMX for 2 h. ( b ) IP of Perilipin 5 from BAT of wild-type mice treated with β3 adrenergic agonist CL 316,243 (CL) or vehicle. ( c , d ) IP of Perilipin 5 ( c ) or PGC-1α ( d ) from whole-cell lysate of brown adipocytes treated with vehicle or Iso/IBMX for 2 h. IgG control IP and siRNA duplexes for PGC-1α and/or Perilipin 5 were used as IP controls. ( e ) IP from whole-cell extract of C2C12 myoblasts transfected with myc-Perilipin 5 and flag-PGC-1α and treated with Iso/IBMX for 2 h. Non-transfected cells were used as IP control. ( f ) PLA reveals Perilipin 5 interaction with PGC-1α in C2C12 differentiating myoblasts treated with vehicle or Iso/IBMX for 2 h. Red dots indicate proximity of interaction partners within 20–40 nm (left) with quantification using Cell Profiler software (right). ( g ) IP of PGC-1α from whole-cell lysate of brown adypocytes treated with Iso/IBMX for 2 h. Cells were additionally treated with orlistat or vehicle and siATGL or siControl. IgG control and siPGC-1α were used as IP controls. ( h ) IP of PGC-1α from nuclear extracts of MLTC-1 cells transfected with siControl or siPerilipin 5. Non-immune rabbit IgG was used as the IP-negative control.
    Figure Legend Snippet: Catecholamine treatment triggers Perilipin 5 interaction with PGC-1α and SIRT1 that promotes PGC-1α deacetylation. ( a ) Immunoprecipitation (IP) of PGC-1α (left) and of Perilipin 5 (right) from nuclear extracts of C2C12 myotubes treated with vehicle or Iso/IBMX for 2 h. ( b ) IP of Perilipin 5 from BAT of wild-type mice treated with β3 adrenergic agonist CL 316,243 (CL) or vehicle. ( c , d ) IP of Perilipin 5 ( c ) or PGC-1α ( d ) from whole-cell lysate of brown adipocytes treated with vehicle or Iso/IBMX for 2 h. IgG control IP and siRNA duplexes for PGC-1α and/or Perilipin 5 were used as IP controls. ( e ) IP from whole-cell extract of C2C12 myoblasts transfected with myc-Perilipin 5 and flag-PGC-1α and treated with Iso/IBMX for 2 h. Non-transfected cells were used as IP control. ( f ) PLA reveals Perilipin 5 interaction with PGC-1α in C2C12 differentiating myoblasts treated with vehicle or Iso/IBMX for 2 h. Red dots indicate proximity of interaction partners within 20–40 nm (left) with quantification using Cell Profiler software (right). ( g ) IP of PGC-1α from whole-cell lysate of brown adypocytes treated with Iso/IBMX for 2 h. Cells were additionally treated with orlistat or vehicle and siATGL or siControl. IgG control and siPGC-1α were used as IP controls. ( h ) IP of PGC-1α from nuclear extracts of MLTC-1 cells transfected with siControl or siPerilipin 5. Non-immune rabbit IgG was used as the IP-negative control.

    Techniques Used: Pyrolysis Gas Chromatography, Immunoprecipitation, Mouse Assay, Transfection, Proximity Ligation Assay, Software, Negative Control

    Perilipin 5 regulates catecholamine-stimulated SIRT1 deacetylation activity and mitochondrial biogenesis and respiration. ( a ) MLTC-1 cells were transfected with the indicated siRNA duplexes. After 72 h, cells were treated with Iso/IBMX or vehicle for 30 min and SIRT1 deacetylase activity was measured in the cell lysates (left). Western blotting for SIRT1, Perilipin 5 and GAPDH document siRNA specificity (bottom). Intracellular NAD + levels with Iso/IBMX or siPerilipin 5 treatments (right). ( b ) Immunoprecipitation of SIRT1 followed by WB for DBC1 and SIRT1 from MLTC-1 cells transfected with the indicated siRNA duplexes. Cells were treated with vehicle or Iso/IBMX for 30 min before immunoprecipitation. Relative quantification of immunoprecipitated DBC-1 normalized for immunoprecipitated SIRT1 is shown in the histogram. ( c ) PLA for SIRT1-DBC1 complexes (red dots) in MLTC-1 cells transfected with the indicated siRNA duplexes for 48 h followed by treatment with vehicle or Iso/IBMX for 30 min (left) with quantification using Cell Profiler (right). ( d ) mtDNA content analysed by qPCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX for 2 h. ( e ) OCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX. n =3 biological replicates/experimental condition with each biological replicate representing an independent transfection and treatment. Values are expressed as the mean±s.e.m. These data were replicated in a second independent experiment. * P
    Figure Legend Snippet: Perilipin 5 regulates catecholamine-stimulated SIRT1 deacetylation activity and mitochondrial biogenesis and respiration. ( a ) MLTC-1 cells were transfected with the indicated siRNA duplexes. After 72 h, cells were treated with Iso/IBMX or vehicle for 30 min and SIRT1 deacetylase activity was measured in the cell lysates (left). Western blotting for SIRT1, Perilipin 5 and GAPDH document siRNA specificity (bottom). Intracellular NAD + levels with Iso/IBMX or siPerilipin 5 treatments (right). ( b ) Immunoprecipitation of SIRT1 followed by WB for DBC1 and SIRT1 from MLTC-1 cells transfected with the indicated siRNA duplexes. Cells were treated with vehicle or Iso/IBMX for 30 min before immunoprecipitation. Relative quantification of immunoprecipitated DBC-1 normalized for immunoprecipitated SIRT1 is shown in the histogram. ( c ) PLA for SIRT1-DBC1 complexes (red dots) in MLTC-1 cells transfected with the indicated siRNA duplexes for 48 h followed by treatment with vehicle or Iso/IBMX for 30 min (left) with quantification using Cell Profiler (right). ( d ) mtDNA content analysed by qPCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX for 2 h. ( e ) OCR in brown adipocytes transfected with siControl or siPerilipin 5 followed by treatment with vehicle or Iso/IBMX. n =3 biological replicates/experimental condition with each biological replicate representing an independent transfection and treatment. Values are expressed as the mean±s.e.m. These data were replicated in a second independent experiment. * P

    Techniques Used: Activity Assay, Transfection, Histone Deacetylase Assay, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Real-time Polymerase Chain Reaction

    59) Product Images from "Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid"

    Article Title: Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22416-4

    Sample collection, DNA isolation and shotgun metagenomic sequencing. ( A ) (I.) Sample collection: Vitreous body (intraocular body fluid) was collected through vitrectomy from 14 patients with endophthalmitis following cataract surgery (n = 7) and intravitreal injection (n = 7). As control, vitreous was collected from 7 patients without postoperative endophthalmitis during macula hole surgery. Six aliquots (3 sample pairs) were obtained from balanced salt solution (BSS) that is infused into the eye during vitrectomy. Three aliquots were collected from separate BSS bottles (BSS-B) and the second set of aliquots was collected from the vitrectomy surgical system (BSS-S) after it had passed through the vitrectomy infusion line, respectively. The samples were examined using (II.) Cultivation-based analyses and (III.) DNA isolation (2 methods) Metagenomic shotgun sequencing, including the examination of DNA extraction (blank) controls. A total of 62 samples were sequenced using Illumina MiSeq sequencing technology. ( B ) More details to steps (II.) and (III.): (II.) Cultivation-based analyses: Aliquots of the vitreous body fluid and balanced salt solution samples were subjected to cultivation-based analyses separately at the hospital and research laboratories. Obtained isolates were analyzed using mass spectrometry and whole genome sequencing. (III.) DNA isolation Metagenomic shotgun sequencing: Samples were extracted using two DNA isolation procedures: QIAamp DNA Mini Kit (QIA) and QIAamp UCP Pathogen Mini kit (UCP). A DNA extraction (blank) control was included at each round of DNA isolation, i.e. one DNA extraction control for 12–14 samples in total per extraction round (more vitreous samples were extracted than analyzed in this study). To verify the presence of the main microorganisms detected in the metagenomics analysis, the shotgun metagenomics reads were mapped to the genome assemblies of the isolates obtained from the vitreous samples. Not displayed here is the mapping of metagenomic shotgun reads to microbial reference genomes in the database (Provided in Fig. 4 ). As an additional verification, PCR analyses were carried out to detect the presence of the most abundant microorganisms in the vitreous samples using organism-specific primer sets.
    Figure Legend Snippet: Sample collection, DNA isolation and shotgun metagenomic sequencing. ( A ) (I.) Sample collection: Vitreous body (intraocular body fluid) was collected through vitrectomy from 14 patients with endophthalmitis following cataract surgery (n = 7) and intravitreal injection (n = 7). As control, vitreous was collected from 7 patients without postoperative endophthalmitis during macula hole surgery. Six aliquots (3 sample pairs) were obtained from balanced salt solution (BSS) that is infused into the eye during vitrectomy. Three aliquots were collected from separate BSS bottles (BSS-B) and the second set of aliquots was collected from the vitrectomy surgical system (BSS-S) after it had passed through the vitrectomy infusion line, respectively. The samples were examined using (II.) Cultivation-based analyses and (III.) DNA isolation (2 methods) Metagenomic shotgun sequencing, including the examination of DNA extraction (blank) controls. A total of 62 samples were sequenced using Illumina MiSeq sequencing technology. ( B ) More details to steps (II.) and (III.): (II.) Cultivation-based analyses: Aliquots of the vitreous body fluid and balanced salt solution samples were subjected to cultivation-based analyses separately at the hospital and research laboratories. Obtained isolates were analyzed using mass spectrometry and whole genome sequencing. (III.) DNA isolation Metagenomic shotgun sequencing: Samples were extracted using two DNA isolation procedures: QIAamp DNA Mini Kit (QIA) and QIAamp UCP Pathogen Mini kit (UCP). A DNA extraction (blank) control was included at each round of DNA isolation, i.e. one DNA extraction control for 12–14 samples in total per extraction round (more vitreous samples were extracted than analyzed in this study). To verify the presence of the main microorganisms detected in the metagenomics analysis, the shotgun metagenomics reads were mapped to the genome assemblies of the isolates obtained from the vitreous samples. Not displayed here is the mapping of metagenomic shotgun reads to microbial reference genomes in the database (Provided in Fig. 4 ). As an additional verification, PCR analyses were carried out to detect the presence of the most abundant microorganisms in the vitreous samples using organism-specific primer sets.

    Techniques Used: DNA Extraction, Sequencing, Injection, Shotgun Sequencing, Mass Spectrometry, Polymerase Chain Reaction

    Summary of cultivation-based, metagenomics and whole genome sequence analyses. Bacterial isolates were obtained at the hospital laboratory (1 st cultivation) from vitreous from endophthalmitis patients following cataract surgery (C1-7) and intravitreal injection (I1-7) and the taxonomic affiliation of the isolates were determined by MALDI-TOF mass spectrometry (MS). Vitreous was analyzed through metagenomics at the research laboratory using two DNA isolation methods (QIAamp DNA Mini Kit, QIA; QIAamp UCP Pathogen Mini kit, UCP) and the taxonomic affiliation of reads was determined. The detected amount of human DNA sequences in percent (%) is provided in the first column of the Metagenomics tab. In the horizontal bar charts, the taxonomic identity and relative fraction of microbial reads for the most abundant identified organisms based on the Kraken + Bracken analysis is indicated for both DNA isolation methods. The read counts for the most abundant organism according to the Kraken + Bracken (all reads) and BLASTN (forward read) analyses are indicated to the right. The read counts for the most abundant organisms per sample as determined by Kraken, Bracken and BLASTn analyses are available through figshare at https://figshare.com/s/5feabfad1d8c495bf7a3 . Bacterial isolates for some samples were obtained in a second round of cultivation at the research laboratory (2 nd cultivation) and one representative per colony morphotype per vitreous sample was subjected to MS and whole genome sequencing (WGS). The taxonomic affiliation of isolates was determined through classification of assembled genomes using a k-mer based approach and genomic MLST, and antibiotic resistance genes were identified using ResFinder. Furthermore, metagenomic assemblies were generated from the shotgun metagenomic reads and analyzed with regards to taxonomic affiliation and selected functional characteristics (Supplementary Table S6 ). A video summary is available from figshare at https://figshare.com/s/38fe043f6a8ef1710444 .
    Figure Legend Snippet: Summary of cultivation-based, metagenomics and whole genome sequence analyses. Bacterial isolates were obtained at the hospital laboratory (1 st cultivation) from vitreous from endophthalmitis patients following cataract surgery (C1-7) and intravitreal injection (I1-7) and the taxonomic affiliation of the isolates were determined by MALDI-TOF mass spectrometry (MS). Vitreous was analyzed through metagenomics at the research laboratory using two DNA isolation methods (QIAamp DNA Mini Kit, QIA; QIAamp UCP Pathogen Mini kit, UCP) and the taxonomic affiliation of reads was determined. The detected amount of human DNA sequences in percent (%) is provided in the first column of the Metagenomics tab. In the horizontal bar charts, the taxonomic identity and relative fraction of microbial reads for the most abundant identified organisms based on the Kraken + Bracken analysis is indicated for both DNA isolation methods. The read counts for the most abundant organism according to the Kraken + Bracken (all reads) and BLASTN (forward read) analyses are indicated to the right. The read counts for the most abundant organisms per sample as determined by Kraken, Bracken and BLASTn analyses are available through figshare at https://figshare.com/s/5feabfad1d8c495bf7a3 . Bacterial isolates for some samples were obtained in a second round of cultivation at the research laboratory (2 nd cultivation) and one representative per colony morphotype per vitreous sample was subjected to MS and whole genome sequencing (WGS). The taxonomic affiliation of isolates was determined through classification of assembled genomes using a k-mer based approach and genomic MLST, and antibiotic resistance genes were identified using ResFinder. Furthermore, metagenomic assemblies were generated from the shotgun metagenomic reads and analyzed with regards to taxonomic affiliation and selected functional characteristics (Supplementary Table S6 ). A video summary is available from figshare at https://figshare.com/s/38fe043f6a8ef1710444 .

    Techniques Used: Sequencing, Injection, Mass Spectrometry, DNA Extraction, Generated, Functional Assay

    60) Product Images from "p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *"

    Article Title: p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.656116

    Mapping of a CncC binding site in the ref(2)P promoter. A , CncC, but not CncA or CncB, transactivates the ref(2)P promoter. S2R+ cells were co-transfected with an empty vector (pActin5C-3xFLAG) or the indicated Cnc constructs (75 ng each) together with
    Figure Legend Snippet: Mapping of a CncC binding site in the ref(2)P promoter. A , CncC, but not CncA or CncB, transactivates the ref(2)P promoter. S2R+ cells were co-transfected with an empty vector (pActin5C-3xFLAG) or the indicated Cnc constructs (75 ng each) together with

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

    61) Product Images from "The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer"

    Article Title: The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0137-x

    UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in ( e and g ) (* P
    Figure Legend Snippet: UBC13 controls DNMT1 stability via ubiquitination and DNMT1 participates in UBC13 regulation of the paclitaxel sensitivity DNMT1 ubiquitination in a A2780 and b SKOV3 cells with UBC13-overexpression or c A2780 and d SKOV3 with UBC13-knockdown without or with HA-ubiquitin. Cells were treated with MG-132 (20 μM, 8 h) prior to preparation of lysates and then subjected to IP followed by western blot with the indicated antibodies. e Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days, and treated with paclitaxel at the indicated concentrations. f Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown, which were transfected in advance with UBC13-specific shRNA and selected with G418 (400 μg/mL) for 14 days. g Cell viability assays in A2780 and SKOV3 cells with DNMT1-knockdown, which were treated with paclitaxel at the indicated concentrations. h Western blotting of UBC13, DNMT1, CHFR, and Aurora A in A2780 and SKOV3 cells with DNMT1-knockdown. Results are shown as means ± SEM for at least three separate experiments in ( e and g ) (* P

    Techniques Used: Over Expression, Western Blot, Transfection, shRNA

    62) Product Images from "Epidemiological and molecular investigation of resurgent cutaneous leishmaniasis in Sudan"

    Article Title: Epidemiological and molecular investigation of resurgent cutaneous leishmaniasis in Sudan

    Journal: International Journal of Infectious Diseases

    doi: 10.1016/j.ijid.2019.08.018

    PCR-RFLP identification of Leishmania species from cutaneous leishmaniasis lesions. (A) DNA extracted from ATL buffer, CL05 and CL06 produced the RFLP pattern characteristic of Leishmania major , with band sizes as described in the text. (B) DNA extracted from culture of the isolate from CL08 (MHOM/SD/2017/ELOBIED) also gave the pattern typical of L. major ; PCR-RFLP of L. tropica was included as control. The L. major identification was verified by DNA sequencing (see text).
    Figure Legend Snippet: PCR-RFLP identification of Leishmania species from cutaneous leishmaniasis lesions. (A) DNA extracted from ATL buffer, CL05 and CL06 produced the RFLP pattern characteristic of Leishmania major , with band sizes as described in the text. (B) DNA extracted from culture of the isolate from CL08 (MHOM/SD/2017/ELOBIED) also gave the pattern typical of L. major ; PCR-RFLP of L. tropica was included as control. The L. major identification was verified by DNA sequencing (see text).

    Techniques Used: Polymerase Chain Reaction, Produced, DNA Sequencing

    63) Product Images from "Microbial Signatures and Innate Immune Gene Expression in Lamina Propria Phagocytes of Inflammatory Bowel Disease Patients"

    Article Title: Microbial Signatures and Innate Immune Gene Expression in Lamina Propria Phagocytes of Inflammatory Bowel Disease Patients

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2019.10.013

    Immune gene expression in CD11b+ cells relates to the inflammation state of the disease. ( A ) Principal component analysis (PCA) based on log 2 transformed gene expression counts obtained from inflamed and noninflamed lamina propria phagocytes of UC and CD patients (n = 6–14 samples per group). Numbers in parentheses indicate the percentage variation explained by the axis. ( B ) Venn diagram showing the number of differentially expressed genes that were shared between inflamed and noninflamed samples obtained from CD and UC patients. ( C and D ) Volcano plots showing genes differentially expressed between inflamed and noninflamed samples from CD and UC patients, respectively. Colored symbols in the volcano plots represent genes that were significantly more abundant (log 2 fold change greater than 1 and P adjusted ≤ .1) in the respective groups: CD inflamed (grey), CD noninflamed (green), UC inflamed (blue), UC noninflamed (pink). The P values were obtained by t test followed by adjustment for false-discovery rate. I, inflamed; NI, non-inflamed.
    Figure Legend Snippet: Immune gene expression in CD11b+ cells relates to the inflammation state of the disease. ( A ) Principal component analysis (PCA) based on log 2 transformed gene expression counts obtained from inflamed and noninflamed lamina propria phagocytes of UC and CD patients (n = 6–14 samples per group). Numbers in parentheses indicate the percentage variation explained by the axis. ( B ) Venn diagram showing the number of differentially expressed genes that were shared between inflamed and noninflamed samples obtained from CD and UC patients. ( C and D ) Volcano plots showing genes differentially expressed between inflamed and noninflamed samples from CD and UC patients, respectively. Colored symbols in the volcano plots represent genes that were significantly more abundant (log 2 fold change greater than 1 and P adjusted ≤ .1) in the respective groups: CD inflamed (grey), CD noninflamed (green), UC inflamed (blue), UC noninflamed (pink). The P values were obtained by t test followed by adjustment for false-discovery rate. I, inflamed; NI, non-inflamed.

    Techniques Used: Expressing, Transformation Assay

    Flow cytometric characterization of lamina propria CD11b+ cells isolated from biopsy specimens obtained from IBD patients. ( A ) The CD11b+ population was examined by determining the expression of CD33 (Siglec-3) (monocytes, macrophages, granulocytes, dendritic cells, and mast cells) and the activation marker CD206 (mannose receptor) (macrophages and dendritic cells). Cells also were assessed for expression of CD14 (part of the lipopolysaccharide-receptor complex) and HLA-DR (antigen presentation) in the 2 subsets. ( B ) Number (means ± 95% CI) of CD11b+ cells isolated from inflamed/uninflamed patient biopsy specimens. FSC-A, forward scatter area; SSC-A, side scatter area.
    Figure Legend Snippet: Flow cytometric characterization of lamina propria CD11b+ cells isolated from biopsy specimens obtained from IBD patients. ( A ) The CD11b+ population was examined by determining the expression of CD33 (Siglec-3) (monocytes, macrophages, granulocytes, dendritic cells, and mast cells) and the activation marker CD206 (mannose receptor) (macrophages and dendritic cells). Cells also were assessed for expression of CD14 (part of the lipopolysaccharide-receptor complex) and HLA-DR (antigen presentation) in the 2 subsets. ( B ) Number (means ± 95% CI) of CD11b+ cells isolated from inflamed/uninflamed patient biopsy specimens. FSC-A, forward scatter area; SSC-A, side scatter area.

    Techniques Used: Isolation, Expressing, Activation Assay, Marker

    64) Product Images from "Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases"

    Article Title: Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases

    Journal: Parasite

    doi: 10.1051/parasite/2013051

    Influence of proteinase K digestion (56 °C overnight) on DNA extraction. Graph A shows the Ct values obtained by quantifying THP1 cell DNA derived from direct extraction with the NucliSENS easyMAG system and extraction performed on the same quantity of cells following overnight (ON) digestion with Proteinase K. Graph B shows Leishmania quantification after extraction with the NucliSENS easyMAG system both with and without PK and quantification after extraction using a QIAamp DNA Mini kit after ON digestion with PK.
    Figure Legend Snippet: Influence of proteinase K digestion (56 °C overnight) on DNA extraction. Graph A shows the Ct values obtained by quantifying THP1 cell DNA derived from direct extraction with the NucliSENS easyMAG system and extraction performed on the same quantity of cells following overnight (ON) digestion with Proteinase K. Graph B shows Leishmania quantification after extraction with the NucliSENS easyMAG system both with and without PK and quantification after extraction using a QIAamp DNA Mini kit after ON digestion with PK.

    Techniques Used: DNA Extraction, Derivative Assay

    65) Product Images from "Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases"

    Article Title: Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases

    Journal: Parasite

    doi: 10.1051/parasite/2013051

    Influence of proteinase K digestion (56 °C overnight) on DNA extraction. Graph A shows the Ct values obtained by quantifying THP1 cell DNA derived from direct extraction with the NucliSENS easyMAG system and extraction performed on the same quantity of cells following overnight (ON) digestion with Proteinase K. Graph B shows Leishmania quantification after extraction with the NucliSENS easyMAG system both with and without PK and quantification after extraction using a QIAamp DNA Mini kit after ON digestion with PK.
    Figure Legend Snippet: Influence of proteinase K digestion (56 °C overnight) on DNA extraction. Graph A shows the Ct values obtained by quantifying THP1 cell DNA derived from direct extraction with the NucliSENS easyMAG system and extraction performed on the same quantity of cells following overnight (ON) digestion with Proteinase K. Graph B shows Leishmania quantification after extraction with the NucliSENS easyMAG system both with and without PK and quantification after extraction using a QIAamp DNA Mini kit after ON digestion with PK.

    Techniques Used: DNA Extraction, Derivative Assay

    66) Product Images from "Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases"

    Article Title: Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases

    Journal: Parasite

    doi: 10.1051/parasite/2013051

    Influence of proteinase K digestion (56 °C overnight) on DNA extraction. Graph A shows the Ct values obtained by quantifying THP1 cell DNA derived from direct extraction with the NucliSENS easyMAG system and extraction performed on the same quantity of cells following overnight (ON) digestion with Proteinase K. Graph B shows Leishmania quantification after extraction with the NucliSENS easyMAG system both with and without PK and quantification after extraction using a QIAamp DNA Mini kit after ON digestion with PK.
    Figure Legend Snippet: Influence of proteinase K digestion (56 °C overnight) on DNA extraction. Graph A shows the Ct values obtained by quantifying THP1 cell DNA derived from direct extraction with the NucliSENS easyMAG system and extraction performed on the same quantity of cells following overnight (ON) digestion with Proteinase K. Graph B shows Leishmania quantification after extraction with the NucliSENS easyMAG system both with and without PK and quantification after extraction using a QIAamp DNA Mini kit after ON digestion with PK.

    Techniques Used: DNA Extraction, Derivative Assay

    67) Product Images from "Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR"

    Article Title: Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169846

    Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns. Genomic and plasmid DNA were isolated from E . coli DH5α and E . coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P
    Figure Legend Snippet: Efficiency of genomic and plasmid DNA recovery with the QIAamp DNA mini kit columns. Genomic and plasmid DNA were isolated from E . coli DH5α and E . coli DH5α [pBR322] with the use of Genomic and Plasmid DNA mini kits, respectively (A A Biotechnology). DNA concentrations were measured by NanoDrop 1000 UV-VIS spectrophotometer (Thermo Scientific). Genomic DNA in the following amounts: 2110 ng, 1855 ng and 3922 ng, was coupled with 524 ng, 1100 ng and 1684 ng of plasmid DNA, respectively. Then, 100 μl of the lysis buffer (Qiagen) was added separately to genomic and plasmid DNA and the nucleic acids isolation was performed according to the QIAamp DNA mini kit manufacturer’s manual. The level of isolated DNA is indicated as a percentage relative to the unprocessed sample. The diagram represents three independent experiments. Error bars represent standard deviation (n = 3); (* P

    Techniques Used: Plasmid Preparation, Isolation, Spectrophotometry, Lysis, Standard Deviation

    Quantification of pBR322 plasmid copy number by digital droplet PCR. E . coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A ( Table 1A ). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs , respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs . Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.
    Figure Legend Snippet: Quantification of pBR322 plasmid copy number by digital droplet PCR. E . coli DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the bla and dxs ddPCR amplification with the use of primer set A ( Table 1A ). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of bla and dxs , respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of bla by the copy number of dxs . Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Isolation, Amplification

    68) Product Images from "Development and Validation of a New Reliable Method for the Diagnosis of Avian Botulism"

    Article Title: Development and Validation of a New Reliable Method for the Diagnosis of Avian Botulism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169640

    Sample analysis workflow for suspected avian botulism cultures. Parameters evaluated during the study: test sample type (whole organ means that the whole liver or up to 25g of the liver was analyzed), enrichment culturing conditions, DNA extraction methods. The CFX96 thermocycler (Bio-Rad, Marne-la-Coquette, France) was used for Real-Time PCR. Analyses performed by ANSES are indicated in black, and performed by LABOCEA are in grey. Kit 1: QIAamp ® DNA Mini kit (Qiagen, Courtaboeuf, France), Kit2: InstaGene Matrix (Bio-Rad, Marne-la-Coquette, France), Kit3: Mericon Bacteria+ (Qiagen, Courtaboeuf, France). CII, CIII, DII, DIII, E are the primers and probe used to perform real-time PCR for the detection of type C, D, C/D, D/C and E BoNT genes [ 4 , 14 ]. L: Liver; L1 to L5: Liver N° 1 to Liver N° 5. M1 to M4: Method 1 to method 4.
    Figure Legend Snippet: Sample analysis workflow for suspected avian botulism cultures. Parameters evaluated during the study: test sample type (whole organ means that the whole liver or up to 25g of the liver was analyzed), enrichment culturing conditions, DNA extraction methods. The CFX96 thermocycler (Bio-Rad, Marne-la-Coquette, France) was used for Real-Time PCR. Analyses performed by ANSES are indicated in black, and performed by LABOCEA are in grey. Kit 1: QIAamp ® DNA Mini kit (Qiagen, Courtaboeuf, France), Kit2: InstaGene Matrix (Bio-Rad, Marne-la-Coquette, France), Kit3: Mericon Bacteria+ (Qiagen, Courtaboeuf, France). CII, CIII, DII, DIII, E are the primers and probe used to perform real-time PCR for the detection of type C, D, C/D, D/C and E BoNT genes [ 4 , 14 ]. L: Liver; L1 to L5: Liver N° 1 to Liver N° 5. M1 to M4: Method 1 to method 4.

    Techniques Used: DNA Extraction, Real-time Polymerase Chain Reaction

    69) Product Images from "Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)"

    Article Title: Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120997

    (A) The Lower limit detection of LAMP and PCR results in spiked water samples ( Table 2A ) was determined by making 10-fold dilutions from N . fowleri ranging from 10 4– 1 cells/250 ml of water, processed and extracted by QIAamp DNA minikit. Analytical sensitivity showed identical results in the LAMP (A1-A6) and PCR assays (B7-B12). (B) Electrophoresis results of the LAMP products from A1-A6 (B1-B6) and PCR products (110 bp) (B7-B12). M, 100 bp DNA Ladder; Neg, negative control.
    Figure Legend Snippet: (A) The Lower limit detection of LAMP and PCR results in spiked water samples ( Table 2A ) was determined by making 10-fold dilutions from N . fowleri ranging from 10 4– 1 cells/250 ml of water, processed and extracted by QIAamp DNA minikit. Analytical sensitivity showed identical results in the LAMP (A1-A6) and PCR assays (B7-B12). (B) Electrophoresis results of the LAMP products from A1-A6 (B1-B6) and PCR products (110 bp) (B7-B12). M, 100 bp DNA Ladder; Neg, negative control.

    Techniques Used: Polymerase Chain Reaction, Electrophoresis, Negative Control

    70) Product Images from "Reappraisal of the Therapeutic Role of Celecoxib in Cholangiocarcinoma"

    Article Title: Reappraisal of the Therapeutic Role of Celecoxib in Cholangiocarcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069928

    COX-2 expression in rat CCA and apoptosis detection in treated rat CCA. (A) Rat CCA tissue revealed positive cytoplasmic immunoreactivity for COX-2 (×400). (B) Apoptotic DNA fragmentation of rat CCA tissue after treatment with celecoxib (160 mg/kg body weight) for 5 weeks visualised by the DNA laddering assay. No apoptotic DNA fragmentation was observed in the control group treated with DMSO buffer for 5 weeks. (C, D, E) TUNNEL assay for determination of rat CCA apoptosis. The positive cells for TUNNEL assay with brown stains are shown in the positive control (TACS-Nuclease treated tissues ) figure (D). No apoptosis was detected in the control group (C). (E) Apoptosis cells were detected in rat CCA tissues treated with celecoxib (160 mg/kg body weight).
    Figure Legend Snippet: COX-2 expression in rat CCA and apoptosis detection in treated rat CCA. (A) Rat CCA tissue revealed positive cytoplasmic immunoreactivity for COX-2 (×400). (B) Apoptotic DNA fragmentation of rat CCA tissue after treatment with celecoxib (160 mg/kg body weight) for 5 weeks visualised by the DNA laddering assay. No apoptotic DNA fragmentation was observed in the control group treated with DMSO buffer for 5 weeks. (C, D, E) TUNNEL assay for determination of rat CCA apoptosis. The positive cells for TUNNEL assay with brown stains are shown in the positive control (TACS-Nuclease treated tissues ) figure (D). No apoptosis was detected in the control group (C). (E) Apoptosis cells were detected in rat CCA tissues treated with celecoxib (160 mg/kg body weight).

    Techniques Used: Expressing, DNA Laddering, Positive Control

    Celecoxib induced apoptosis in CGCCA cells. CGCCA cells were treated with the indicated concentrations of celecoxib for 2 days. (A) TUNNEL assay was used to identify apoptotic cells under different concentrations of celecoxib. Cell morphology and the nuclei were revealed by Harris modified hematoxylin solution counter-staining. The arrows indicated cells positive (+) for TUNNEL assay. The examples of positive (+) and negative (–) cells for TUNNEL assay were shown in the right lower corner panel (B) The apoptotic index of CGCCA cells treated with different concentrations of celecoxib. Each value is a mean ± SD of 3 to 5 determinations. *p
    Figure Legend Snippet: Celecoxib induced apoptosis in CGCCA cells. CGCCA cells were treated with the indicated concentrations of celecoxib for 2 days. (A) TUNNEL assay was used to identify apoptotic cells under different concentrations of celecoxib. Cell morphology and the nuclei were revealed by Harris modified hematoxylin solution counter-staining. The arrows indicated cells positive (+) for TUNNEL assay. The examples of positive (+) and negative (–) cells for TUNNEL assay were shown in the right lower corner panel (B) The apoptotic index of CGCCA cells treated with different concentrations of celecoxib. Each value is a mean ± SD of 3 to 5 determinations. *p

    Techniques Used: Modification, Staining

    71) Product Images from "CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome"

    Article Title: CRISPR-Cas9-Based Knockout of the Prion Protein and Its Effect on the Proteome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114594

    Strategy for generation of mouse PrP knockout clones based on CRISPR/Cas9-system.
    Figure Legend Snippet: Strategy for generation of mouse PrP knockout clones based on CRISPR/Cas9-system.

    Techniques Used: Knock-Out, Clone Assay, CRISPR

    Generation of Prnp knockout clones in three different mouse cell lines. ( A ) Identification of clones that exhibit loss of PrP expression by Western blot analyses of cellular lysates. ( B ) Crude characterization of Prnp gene editing by genomic PCR analyses. Note the appearance of additional slower or faster migrating PCR products in clones that exhibit loss of PrP expression. C, control lane derived from genomic PCR analyses of cells not subjected to CRISPR/Cas9 gene editing. The asterisk identifies the NMuMG clone that was employed for the global proteome comparison described below. ( C ) Detailed insertion/deletion (indel) analysis of one Prnp -deficient NMuMG cell clone by DNA-sequencing of genomic PCR products. Consistent with the observed loss of detectable PrP by Western blot analysis, the analysis established the deletion of 2 and 4 nucleotides within the two Prnp alleles present in this NMuMG cell clone.
    Figure Legend Snippet: Generation of Prnp knockout clones in three different mouse cell lines. ( A ) Identification of clones that exhibit loss of PrP expression by Western blot analyses of cellular lysates. ( B ) Crude characterization of Prnp gene editing by genomic PCR analyses. Note the appearance of additional slower or faster migrating PCR products in clones that exhibit loss of PrP expression. C, control lane derived from genomic PCR analyses of cells not subjected to CRISPR/Cas9 gene editing. The asterisk identifies the NMuMG clone that was employed for the global proteome comparison described below. ( C ) Detailed insertion/deletion (indel) analysis of one Prnp -deficient NMuMG cell clone by DNA-sequencing of genomic PCR products. Consistent with the observed loss of detectable PrP by Western blot analysis, the analysis established the deletion of 2 and 4 nucleotides within the two Prnp alleles present in this NMuMG cell clone.

    Techniques Used: Knock-Out, Clone Assay, Expressing, Western Blot, Polymerase Chain Reaction, Derivative Assay, CRISPR, DNA Sequencing

    PrP deficiency generated by CRISPR/Cas9-mediated gene knockout or stable shRNA-mediated knockdown manifests in highly reproducible changes to the expression of more than hundred proteins in NMuMG cell model. (A) Chart depicting number of peptide-spectrum matches versus false discovery rate (FDR). 33,132 and 33,969 peptide-to-spectrum matches (PSMs) passed the set FDR threshold of 0.5 in PrP knockout and knockdown datasets, respectively. (B) Venn diagram depicting overlap in proteins (peptides) whose expression was altered in clones made deficient for PrP expression by the two aforementioned methods. (C) Several gene ontology (GO) classifiers were significantly overrepresented when their occurrence was compared amongst the 120 proteins whose abundance levels were consistently altered by PrP knockout (or knockdown). The list of all 3254 mouse NMuMG protein groups detected in this study served as the reference data set for these analyses. Colour code: dark brown, proteins whose abundance levels were changed in both PrP kd and ko data sets; grey, subset of proteins within reference data set assigned to a given ‘GO’ classifier.
    Figure Legend Snippet: PrP deficiency generated by CRISPR/Cas9-mediated gene knockout or stable shRNA-mediated knockdown manifests in highly reproducible changes to the expression of more than hundred proteins in NMuMG cell model. (A) Chart depicting number of peptide-spectrum matches versus false discovery rate (FDR). 33,132 and 33,969 peptide-to-spectrum matches (PSMs) passed the set FDR threshold of 0.5 in PrP knockout and knockdown datasets, respectively. (B) Venn diagram depicting overlap in proteins (peptides) whose expression was altered in clones made deficient for PrP expression by the two aforementioned methods. (C) Several gene ontology (GO) classifiers were significantly overrepresented when their occurrence was compared amongst the 120 proteins whose abundance levels were consistently altered by PrP knockout (or knockdown). The list of all 3254 mouse NMuMG protein groups detected in this study served as the reference data set for these analyses. Colour code: dark brown, proteins whose abundance levels were changed in both PrP kd and ko data sets; grey, subset of proteins within reference data set assigned to a given ‘GO’ classifier.

    Techniques Used: Generated, CRISPR, Gene Knockout, shRNA, Expressing, Knock-Out, Clone Assay

    72) Product Images from "HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy"

    Article Title: HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2016.1219825

    Spontaneous memory T cell responses are detectable in a leukemia patient. The presence of memory T cell responses in leukemia and lymphoma patients was analyzed using 12-d recall IFNγ ELISPOT assays. (A) In a single (out of 22 tested) MYD88 L265P+ NHL patients (CLL-05-R) IFNγ secretion was observed after stimulation with the MYD88 L265P -derived peptides P5 B*15/B * 40 (HQKRPIPI) and P1 C*03 (RPIPIKYKAM). (B) Representative example of a MYD88 L265P+ patient (CLL-03-R) where no IFNγ secretion was observed after stimulation with the MYD88 L265P -derived peptides P5 B*15 (HQKRPIPI) and P1 C*03 (RPIPIKYKAM). An EBV epitope mix containing the frequently recognized peptides BRLF1 109–117 YVLDHLIVV (HLA-A*02) and EBNA3 247–255 RPPIFIRRL (HLA-B*07) served as positive control. Benign-tissue derived peptide DDX5 YLLPAIVHI (HLA-A*02) served as negative control. The dotted line indicates the 3-fold number of spot forming unit of the negative control. Error bars indicate ± SEM of two independent replicates. Abbreviations: SFU, spot forming unit; neg., negative; pos., positive; SEM, standard error of the mean.
    Figure Legend Snippet: Spontaneous memory T cell responses are detectable in a leukemia patient. The presence of memory T cell responses in leukemia and lymphoma patients was analyzed using 12-d recall IFNγ ELISPOT assays. (A) In a single (out of 22 tested) MYD88 L265P+ NHL patients (CLL-05-R) IFNγ secretion was observed after stimulation with the MYD88 L265P -derived peptides P5 B*15/B * 40 (HQKRPIPI) and P1 C*03 (RPIPIKYKAM). (B) Representative example of a MYD88 L265P+ patient (CLL-03-R) where no IFNγ secretion was observed after stimulation with the MYD88 L265P -derived peptides P5 B*15 (HQKRPIPI) and P1 C*03 (RPIPIKYKAM). An EBV epitope mix containing the frequently recognized peptides BRLF1 109–117 YVLDHLIVV (HLA-A*02) and EBNA3 247–255 RPPIFIRRL (HLA-B*07) served as positive control. Benign-tissue derived peptide DDX5 YLLPAIVHI (HLA-A*02) served as negative control. The dotted line indicates the 3-fold number of spot forming unit of the negative control. Error bars indicate ± SEM of two independent replicates. Abbreviations: SFU, spot forming unit; neg., negative; pos., positive; SEM, standard error of the mean.

    Techniques Used: Enzyme-linked Immunospot, Derivative Assay, Positive Control, Negative Control

    Functionality and specificity of MYD88 L265P -specific T cells. Functionality and specificity of MYD88 L265P -specific CD8 + T cells were analyzed by (A) IFNγ ELISPOT assay or (B, C) intracellular cytokine staining. Both assays showed increased production of IFNγ or TNFα after stimulation with the mutation-derived peptide (P1 B*07 ) in comparison with the corresponding WT peptide (P1 WT ). Representative examples of two different donors are shown. The frequency of P1 B*07 -specific CD8 + T cell populations was 2.69% (A) and 0.40% (B and C), respectively, as detected by tetramer staining (not shown). Error bars indicate ± SEM of two independent replicates. Abbreviations: SFU, spot forming unit; neg., negative; pos., positive; SEM, standard error of the mean.
    Figure Legend Snippet: Functionality and specificity of MYD88 L265P -specific T cells. Functionality and specificity of MYD88 L265P -specific CD8 + T cells were analyzed by (A) IFNγ ELISPOT assay or (B, C) intracellular cytokine staining. Both assays showed increased production of IFNγ or TNFα after stimulation with the mutation-derived peptide (P1 B*07 ) in comparison with the corresponding WT peptide (P1 WT ). Representative examples of two different donors are shown. The frequency of P1 B*07 -specific CD8 + T cell populations was 2.69% (A) and 0.40% (B and C), respectively, as detected by tetramer staining (not shown). Error bars indicate ± SEM of two independent replicates. Abbreviations: SFU, spot forming unit; neg., negative; pos., positive; SEM, standard error of the mean.

    Techniques Used: Enzyme-linked Immunospot, Staining, Mutagenesis, Derivative Assay

    Prediction of MYD88 L265P -derived HLA class I ligands. 50 MYD88 L265P -derived 8- to 12-mer peptides were scored by the online prediction tools SYFPEITHI and NetMHC 3.4, as well as an extended in-house database. Peptides with NetMHC 3.4 IC 50 ≤ 500 nM were defined as binders (region left of the dotted line). SYFPEITHI scores are displayed as percent of the maximum score for the respective HLA allotype. The threshold for binders is defined as ≥ 50% of the maximum score (above the dotted line). For some HLA:peptide combinations scoring was possible with only one of the prediction tools due to limited availability of predictors. The figure illustrates the predicted ligands for HLA-A and -B allotypes. Fold-change ratios in binding scores of mutated peptides compared with the corresponding WT peptides are indicated by the size of the respective dot: large dots indicate an at least 2-fold better score, mutated ligands illustrated by small dots exhibit no increased binding score in comparison to their corresponding WT ligand. Out of 50 unique peptide sequences, 23 were scored as potential HLA class I ligands. Four ligands were concordantly designated as ligands of the same HLA allotype by both algorithms with three of them having an at least 2-fold higher score as their corresponding WT peptide. The black dots indicate the peptides which were tested in aAPC-based in vitro primings in HBDs or CLL patients. Abbreviations: max., maximal.
    Figure Legend Snippet: Prediction of MYD88 L265P -derived HLA class I ligands. 50 MYD88 L265P -derived 8- to 12-mer peptides were scored by the online prediction tools SYFPEITHI and NetMHC 3.4, as well as an extended in-house database. Peptides with NetMHC 3.4 IC 50 ≤ 500 nM were defined as binders (region left of the dotted line). SYFPEITHI scores are displayed as percent of the maximum score for the respective HLA allotype. The threshold for binders is defined as ≥ 50% of the maximum score (above the dotted line). For some HLA:peptide combinations scoring was possible with only one of the prediction tools due to limited availability of predictors. The figure illustrates the predicted ligands for HLA-A and -B allotypes. Fold-change ratios in binding scores of mutated peptides compared with the corresponding WT peptides are indicated by the size of the respective dot: large dots indicate an at least 2-fold better score, mutated ligands illustrated by small dots exhibit no increased binding score in comparison to their corresponding WT ligand. Out of 50 unique peptide sequences, 23 were scored as potential HLA class I ligands. Four ligands were concordantly designated as ligands of the same HLA allotype by both algorithms with three of them having an at least 2-fold higher score as their corresponding WT peptide. The black dots indicate the peptides which were tested in aAPC-based in vitro primings in HBDs or CLL patients. Abbreviations: max., maximal.

    Techniques Used: Derivative Assay, Binding Assay, In Vitro

    Efficient in vitro generation of P4 B*15 - and P1 B*07 -specific CD8 + T cells from naive T cells of CLL patients and HBDs. Representative tetramer stainings of CD8 + T cells after three cycles of aAPC-based in vitro priming using CD8 + T cells derived from HLA-matched HBDs primed with (A) the HLA-B*15-restricted peptide HQKRPIPIKY (P4 B*15 ) and (B) the HLA-B*07-restricted peptide RPIPIKYKAM (P1 B*07 ) as well as from HLA-matched MYD88 WT CLL patient (CLL-05) primed with (C) the HLA-B*07-restricted peptide RPIPIKYKAM (P1 B*07 ): 1st column: tetramer staining of CD8 + T cells primed with the MYD88 L265P -derived peptide; 2nd column: control staining with HLA-matched tetramer containing a non-relevant control peptide on CD8 + T cells derived from the same population as T cells depicted in the 1st column; 3rd column: ex vivo tetramer staining of CD8 + T cells. In vitro primings with HBD-derived PBMCs were performed in six (P1 B*07 ) and three (P4 B*15 ) independent replicates, respectively. For the in vitro priming with PBMCs of CLL patients two independent replicates were conducted. Abbreviations: neg., negative.
    Figure Legend Snippet: Efficient in vitro generation of P4 B*15 - and P1 B*07 -specific CD8 + T cells from naive T cells of CLL patients and HBDs. Representative tetramer stainings of CD8 + T cells after three cycles of aAPC-based in vitro priming using CD8 + T cells derived from HLA-matched HBDs primed with (A) the HLA-B*15-restricted peptide HQKRPIPIKY (P4 B*15 ) and (B) the HLA-B*07-restricted peptide RPIPIKYKAM (P1 B*07 ) as well as from HLA-matched MYD88 WT CLL patient (CLL-05) primed with (C) the HLA-B*07-restricted peptide RPIPIKYKAM (P1 B*07 ): 1st column: tetramer staining of CD8 + T cells primed with the MYD88 L265P -derived peptide; 2nd column: control staining with HLA-matched tetramer containing a non-relevant control peptide on CD8 + T cells derived from the same population as T cells depicted in the 1st column; 3rd column: ex vivo tetramer staining of CD8 + T cells. In vitro primings with HBD-derived PBMCs were performed in six (P1 B*07 ) and three (P4 B*15 ) independent replicates, respectively. For the in vitro priming with PBMCs of CLL patients two independent replicates were conducted. Abbreviations: neg., negative.

    Techniques Used: In Vitro, Derivative Assay, Staining, Ex Vivo

    MYD88 L265P -selective cytotoxicity of P4 B*15 -specific effector cells. The MYD88 L265P -specific cytotoxicity was analyzed in a VITAL cytotoxicity assay with CD8 + effector cells of in vitro primed cells of HBDs. (A, B) Tetramer staining of polyclonal effector cells one day before the VITAL assay determined the number of P4 B*15 -specific effector cells in the (A) population of successfully P4 B*15 -primed CD8 + T cells and in the (B) population of control cells primed with a HLA-matched non-relevant peptide. These control cells were used as unspecific effectors for the determination of the unspecific lysis of target cells. (C) At an effector to target ratio of 1:1 P4 B*15 -specific effectors (▴) exerted 17.9% (±1.2%) MYD88 L265P -specific and significant higher lysis of P4 B*15 -loaded autologous target cells in comparison to P4 WT -loaded cells. P4 B*15 -unspecific effectors (×) only caused 2.6% (±1.2%) unspecific lysis of the same targets. Results are shown for three independent replicates. Error bars indicate ± SEM. Abbreviations: SEM, standard error of the mean; n.s., not significant; * p > 0.05; ** p > 0.01; *** p > 0.001.
    Figure Legend Snippet: MYD88 L265P -selective cytotoxicity of P4 B*15 -specific effector cells. The MYD88 L265P -specific cytotoxicity was analyzed in a VITAL cytotoxicity assay with CD8 + effector cells of in vitro primed cells of HBDs. (A, B) Tetramer staining of polyclonal effector cells one day before the VITAL assay determined the number of P4 B*15 -specific effector cells in the (A) population of successfully P4 B*15 -primed CD8 + T cells and in the (B) population of control cells primed with a HLA-matched non-relevant peptide. These control cells were used as unspecific effectors for the determination of the unspecific lysis of target cells. (C) At an effector to target ratio of 1:1 P4 B*15 -specific effectors (▴) exerted 17.9% (±1.2%) MYD88 L265P -specific and significant higher lysis of P4 B*15 -loaded autologous target cells in comparison to P4 WT -loaded cells. P4 B*15 -unspecific effectors (×) only caused 2.6% (±1.2%) unspecific lysis of the same targets. Results are shown for three independent replicates. Error bars indicate ± SEM. Abbreviations: SEM, standard error of the mean; n.s., not significant; * p > 0.05; ** p > 0.01; *** p > 0.001.

    Techniques Used: Cytotoxicity Assay, In Vitro, Staining, Lysis

    73) Product Images from "HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy"

    Article Title: HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2016.1219825

    Efficient in vitro generation of P4 B*15 - and P1 B*07 -specific CD8 + T cells from naive T cells of CLL patients and HBDs. Representative tetramer stainings of CD8 + T cells after three cycles of aAPC-based in vitro priming using CD8 + T cells derived from HLA-matched HBDs primed with (A) the HLA-B*15-restricted peptide HQKRPIPIKY (P4 B*15 ) and (B) the HLA-B*07-restricted peptide RPIPIKYKAM (P1 B*07 ) as well as from HLA-matched MYD88 WT CLL patient (CLL-05) primed with (C) the HLA-B*07-restricted peptide RPIPIKYKAM (P1 B*07 ): 1st column: tetramer staining of CD8 + T cells primed with the MYD88 L265P -derived peptide; 2nd column: control staining with HLA-matched tetramer containing a non-relevant control peptide on CD8 + T cells derived from the same population as T cells depicted in the 1st column; 3rd column: ex vivo tetramer staining of CD8 + T cells. In vitro primings with HBD-derived PBMCs were performed in six (P1 B*07 ) and three (P4 B*15 ) independent replicates, respectively. For the in vitro priming with PBMCs of CLL patients two independent replicates were conducted. Abbreviations: neg., negative.
    Figure Legend Snippet: Efficient in vitro generation of P4 B*15 - and P1 B*07 -specific CD8 + T cells from naive T cells of CLL patients and HBDs. Representative tetramer stainings of CD8 + T cells after three cycles of aAPC-based in vitro priming using CD8 + T cells derived from HLA-matched HBDs primed with (A) the HLA-B*15-restricted peptide HQKRPIPIKY (P4 B*15 ) and (B) the HLA-B*07-restricted peptide RPIPIKYKAM (P1 B*07 ) as well as from HLA-matched MYD88 WT CLL patient (CLL-05) primed with (C) the HLA-B*07-restricted peptide RPIPIKYKAM (P1 B*07 ): 1st column: tetramer staining of CD8 + T cells primed with the MYD88 L265P -derived peptide; 2nd column: control staining with HLA-matched tetramer containing a non-relevant control peptide on CD8 + T cells derived from the same population as T cells depicted in the 1st column; 3rd column: ex vivo tetramer staining of CD8 + T cells. In vitro primings with HBD-derived PBMCs were performed in six (P1 B*07 ) and three (P4 B*15 ) independent replicates, respectively. For the in vitro priming with PBMCs of CLL patients two independent replicates were conducted. Abbreviations: neg., negative.

    Techniques Used: In Vitro, Derivative Assay, Staining, Ex Vivo

    74) Product Images from "The transcription factor XBP1s restores hippocampal synaptic plasticity and memory by control of the Kalirin-7 pathway in Alzheimer model"

    Article Title: The transcription factor XBP1s restores hippocampal synaptic plasticity and memory by control of the Kalirin-7 pathway in Alzheimer model

    Journal: Molecular Psychiatry

    doi: 10.1038/mp.2016.152

    Reducing Kalirin-7 levels induces synaptic and cognitive deficits in naive mice. ( a ) Left, bilateral stereotaxic injection of lentiviral vectors into the hippocampus of naive mice. Right, timeline of Morris water maze (MWM) test and subsequent biochemical analysis of mice. ( b ) qRT-PCR analysis of EphB2 and Kal7 transcripts in the whole hippocampus ( n =5–6 mice per group). Kruskal–Wallis test: EphB2: H =14.4855, P
    Figure Legend Snippet: Reducing Kalirin-7 levels induces synaptic and cognitive deficits in naive mice. ( a ) Left, bilateral stereotaxic injection of lentiviral vectors into the hippocampus of naive mice. Right, timeline of Morris water maze (MWM) test and subsequent biochemical analysis of mice. ( b ) qRT-PCR analysis of EphB2 and Kal7 transcripts in the whole hippocampus ( n =5–6 mice per group). Kruskal–Wallis test: EphB2: H =14.4855, P

    Techniques Used: Mouse Assay, Injection, Quantitative RT-PCR

    Depletion of Kalirin-7, but not EphB2, abolishes XBP1s-associated rescue of synaptic plasticity and memory in 3xTg-AD mice. ( a ) Left, stereotaxic injection of lentiviral vectors into the hippocampus of 3xTg-AD (AD) and nontransgenic (NTG) control mice. Right, timeline of Morris water maze (MWM) test and subsequent biochemical analysis of mice. ( b ) Immunofluorescence images depicting GFP-positive cells with DAPI and XBP1s stainings in CA1 region. Scale bar, 20 μm. ( c , d ) Spine density in CA1 region (number of slice per mouse/number of mice: 5/7 per group). Kruskal–Wallis test: H =74.2712, P
    Figure Legend Snippet: Depletion of Kalirin-7, but not EphB2, abolishes XBP1s-associated rescue of synaptic plasticity and memory in 3xTg-AD mice. ( a ) Left, stereotaxic injection of lentiviral vectors into the hippocampus of 3xTg-AD (AD) and nontransgenic (NTG) control mice. Right, timeline of Morris water maze (MWM) test and subsequent biochemical analysis of mice. ( b ) Immunofluorescence images depicting GFP-positive cells with DAPI and XBP1s stainings in CA1 region. Scale bar, 20 μm. ( c , d ) Spine density in CA1 region (number of slice per mouse/number of mice: 5/7 per group). Kruskal–Wallis test: H =74.2712, P

    Techniques Used: Mouse Assay, Injection, Immunofluorescence

    75) Product Images from "Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts"

    Article Title: Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts

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

    doi: 10.1073/pnas.1806305115

    Ectopically expressed PDGFRα renders ARPE-19 and THP-1 cells susceptible to trimer-only virus entry. Cell-surface staining of PDGFRα on ARPE-19 ( A ) or THP-1 ( B ) cells following no treatment (control), infection with a lentivirus lacking an insert (vector), or infection with a lentivirus expressing PDGFRα. ( Top ) Cells were subjected to flow cytometry using the indicated antibodies. ( Bottom ) Cells were infected with AD169-fibro, Merlin-fibro (pAL1111), or TB40/E-fibro at a multiplicity of 3 FFU per cell, and IE1 expression was measured at 24 hpi (ARPE-19) or 18 hpi (THP-1) to assay the percentage of infected cells.
    Figure Legend Snippet: Ectopically expressed PDGFRα renders ARPE-19 and THP-1 cells susceptible to trimer-only virus entry. Cell-surface staining of PDGFRα on ARPE-19 ( A ) or THP-1 ( B ) cells following no treatment (control), infection with a lentivirus lacking an insert (vector), or infection with a lentivirus expressing PDGFRα. ( Top ) Cells were subjected to flow cytometry using the indicated antibodies. ( Bottom ) Cells were infected with AD169-fibro, Merlin-fibro (pAL1111), or TB40/E-fibro at a multiplicity of 3 FFU per cell, and IE1 expression was measured at 24 hpi (ARPE-19) or 18 hpi (THP-1) to assay the percentage of infected cells.

    Techniques Used: Staining, Infection, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry

    HFFs expressing Cas9 and gRNAs targeting PDGFRα survived trimer-only HCMV infection in a genome-wide CRISPR screen. Statistical analysis identified PDGFRα as the most significant hit from the screens of AD169 ( A ) and Merlin ( B ), using the MAGeCK algorithm.
    Figure Legend Snippet: HFFs expressing Cas9 and gRNAs targeting PDGFRα survived trimer-only HCMV infection in a genome-wide CRISPR screen. Statistical analysis identified PDGFRα as the most significant hit from the screens of AD169 ( A ) and Merlin ( B ), using the MAGeCK algorithm.

    Techniques Used: Expressing, Infection, Genome Wide, CRISPR

    PDGFRα is important for cell-to-cell spread of trimer-only virus, but not for spread of virus containing the pentameric complex. ( A ) AD169-GFP, Merlin-GFP (pAL1158), or Merlin-GFP (pAL1160) BAC DNA was electroporated into CN-9 and PDGFRα-KO cells. The culture medium was supplemented with 6% CytoGam or PBS control and medium was changed daily. At 16 d after electroporation, immunofluorescence analysis of IE1-positive cells was performed to assay the spread of virus. White arrows point to the single cells producing IE1 from HCMV BACs. ( B ) Supernatants from CN-9 and 1-10 cells of the PBS group in A between day 8 and day 12 after electroporation were added to HFF cells to assay the presence of progeny viruses. Images (GFP or bright field) were recorded at 17 dpi. ( C ) CN-9 and 1-10 cells were electroporated with AD169-GFP BAC DNA, and GFP-positive cells were sorted at 6 d after electroporation. The same number (140) of GFP-positive CN-9 or 1-10 cells was added to cultures of HFF or 1-10 cells. GFP expression was recorded by fluorescent microscopy after 14 d of coculture. White arrows point to the single cells producing GFP from HCMV BACs.
    Figure Legend Snippet: PDGFRα is important for cell-to-cell spread of trimer-only virus, but not for spread of virus containing the pentameric complex. ( A ) AD169-GFP, Merlin-GFP (pAL1158), or Merlin-GFP (pAL1160) BAC DNA was electroporated into CN-9 and PDGFRα-KO cells. The culture medium was supplemented with 6% CytoGam or PBS control and medium was changed daily. At 16 d after electroporation, immunofluorescence analysis of IE1-positive cells was performed to assay the spread of virus. White arrows point to the single cells producing IE1 from HCMV BACs. ( B ) Supernatants from CN-9 and 1-10 cells of the PBS group in A between day 8 and day 12 after electroporation were added to HFF cells to assay the presence of progeny viruses. Images (GFP or bright field) were recorded at 17 dpi. ( C ) CN-9 and 1-10 cells were electroporated with AD169-GFP BAC DNA, and GFP-positive cells were sorted at 6 d after electroporation. The same number (140) of GFP-positive CN-9 or 1-10 cells was added to cultures of HFF or 1-10 cells. GFP expression was recorded by fluorescent microscopy after 14 d of coculture. White arrows point to the single cells producing GFP from HCMV BACs.

    Techniques Used: BAC Assay, Electroporation, Immunofluorescence, Expressing, Microscopy

    IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. ( A ) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. ( B and C ) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. ( D ) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.
    Figure Legend Snippet: IgG-like domain 3 of PDGFRα is required for trimer-only HCMV entry to fibroblasts. ( A ) PDGFRα-KO cells (clone 1-10) were transduced with lentiviruses expressing V5-tagged, full-length, or deleted variants of PDGFRα. Transduced cells were infected with Merlin-GFP (pAL1158) at a multiplicity of 3 FFU per cell, and IE1-positive cells were quantified at 24 hpi by flow cytometry. ( B and C ) Expression of V5-tagged full-length and deleted PDGFRα variants was confirmed by Western blot. ( D ) HFF or PDGFRα-KO cells (clone 1-10) expressing full-length or C-terminus-deleted PDGFRα in C was infected by AD169, TB40/E, or Merlin (pAL1111) at multiplicity of infection of 3 FFU per cell. Viral genome replication in infected cells was measured by qPCR at 4 dpi. D, IgG-like domain; SP, signal peptide; TM, transmembrane domain.

    Techniques Used: Transduction, Expressing, Infection, Flow Cytometry, Cytometry, Western Blot, Real-time Polymerase Chain Reaction

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    Standard Deviation:

    Article Title: Comparison of Commercial Extraction Systems and PCR Assays for Quantification of Epstein-Barr Virus DNA Load in Whole Blood
    Article Snippet: The comparison of the MagNA and QIAcube automated extraction platforms showed an excellent correlation of the EBV load results in 200 whole-blood specimens ( R = 0.958; P < 0.0001), with fewer than 8% of VL differing by more than 0.5 log10 , and with a low standard deviation of the differences (0.31). .. The QIAcube is not totally automated but offers the advantage of being more versatile, since almost all the manual extraction kits available from Qiagen can be adapted for the QIAcube with comparable results.

    Expressing:

    Article Title: An Algorithm for Generating Small RNAs Capable of Epigenetically Modulating Transcriptional Gene Silencing and Activation in Human Cells
    Article Snippet: RNA isolation and gene expression profiling. .. The transiently transfected cells were collected at predetermined time points and RNA was extracted (Qiacube Qiagen; Qiagen).

    Modification:

    Article Title: Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.)
    Article Snippet: .. DNA extraction and quantification optimization Genomic DNA from 500 mg dry seeds (~45 seeds) was extracted by the following six methods to determine which one was optimal and most reliable for PCR: (1) DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA, USA), (2) TRIzol reagent method (Life Technologies, Carlsbad, CA, USA), (3) QIAcube kit (Qiagen Inc., Valencia, CA, USA), (4) Promega Maxwell 16 kit (Promega, Madison, WI, USA), (5) CTAB method [ ], and (6) modified CTAB method. ..

    Sequencing:

    Article Title: Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells
    Article Snippet: The results obtained with the short sequence are used to monitor the copy number of the mitochondrial genome and, more importantly, to normalize the data obtained with the large (7–15 kb) fragment. .. As noted above if DNA is extracted using an automated system employing the QIAcube (QIAGEN, catalog number 9001292) with the QIAamp DNA mini kit for human samples (QIAGEN, catalog number 51304), linearization of the mtDNA is necessary to get an accurate level of mtDNA.

    Western Blot:

    Article Title: Automated Nucleic Acid Extraction Systems for Detecting Cytomegalovirus and Epstein-Barr Virus Using Real-Time PCR: A Comparison Study Between the QIAsymphony RGQ and QIAcube Systems
    Article Snippet: One hundred and fifty-three whole blood (WB) samples were tested for CMV detection, and 117 WB samples were tested for EBV detection. .. Viral nucleic acid was extracted in parallel by using QIAsymphony RGQ and QIAcube (Qiagen GmbH, Germany), and real-time PCR assays for CMV and EBV were performed with a Rotor-Gene Q real-time PCR cycler (Qiagen).

    Article Title: An Algorithm for Generating Small RNAs Capable of Epigenetically Modulating Transcriptional Gene Silencing and Activation in Human Cells
    Article Snippet: Western blot analysis was carried out on CFPAC cells 72 hours following transfection with either the as-AK4 or control U6M2 plasmids. .. The transiently transfected cells were collected at predetermined time points and RNA was extracted (Qiacube Qiagen; Qiagen).

    Article Title: Automated Nucleic Acid Extraction Systems for Detecting Cytomegalovirus and Epstein-Barr Virus Using Real-Time PCR: A Comparison Study Between the QIAsymphony RGQ and QIAcube Systems
    Article Snippet: .. In this study, we evaluated two automated nucleic acid extraction systems used for detecting CMV and EBV DNA from WB: the QIAsymphony system and the QIAcube system (Qiagen GmbH, Hilden, Germany). .. The QIAcube system consists of the QIAcube and Rotor-Gene Q real-time PCR cycler (RGQ, Qiagen).

    Inhibition:

    Article Title: Comparison of Commercial Extraction Systems and PCR Assays for Quantification of Epstein-Barr Virus DNA Load in Whole Blood
    Article Snippet: No PCR inhibition precluded the measurement of EBV load, since none of the samples inhibited the internal positive control in any of the PCR assays. .. The QIAcube is not totally automated but offers the advantage of being more versatile, since almost all the manual extraction kits available from Qiagen can be adapted for the QIAcube with comparable results.

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    Qiagen qiaamp dna mini kit
    <t>DNA</t> extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and <t>QIAamp®</t> DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles
    Qiaamp Dna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    Article Title: Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    doi: 10.1007/s10096-011-1191-4

    Figure Lengend Snippet: DNA extraction efficiencies of the three methods. Percentage of DNA extraction efficiency ( n = 12) of FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit for ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. The horizontal bars show the median and quartiles

    Article Snippet: The efficiency of the Reischl et al. protocol was significantly lower with 9.0% as compared to the QIAamp® DNA Mini Kit (P = 0.001) and FTA® Elute paper (P = 0.001).

    Techniques: DNA Extraction

    Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    Article Title: Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    doi: 10.1007/s10096-011-1191-4

    Figure Lengend Snippet: Inhibition of the E. coli uidA qrtPCR assay. The mean levels of DNA were determined with qrtPCR by adding a template of 1 ng of the standard together with either water or an extraction control of one of the three following DNA extraction methods to triplicate reactions ( n = 3): FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The dashed line represents part of the standard curve. (Ο) FTA® Elute, (□) QIAamp® DNA Mini Kit, (◊) Reischl, (Δ) Water

    Article Snippet: The efficiency of the Reischl et al. protocol was significantly lower with 9.0% as compared to the QIAamp® DNA Mini Kit (P = 0.001) and FTA® Elute paper (P = 0.001).

    Techniques: Inhibition, DNA Extraction

    DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit

    Journal: European Journal of Clinical Microbiology & Infectious Diseases

    Article Title: Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR

    doi: 10.1007/s10096-011-1191-4

    Figure Lengend Snippet: DNA levels extracted by three methods with varying amounts of cells. Median DNA levels as determined by qrtPCR resulting from quadruplicate extractions ( n = 4) of different amounts of cells of ( a ) E. coli ATCC 11775 T and ( b ) S. aureus subsp. aureus ATCC 25923. Extractions were performed by three different DNA extraction methods: FTA® Elute, the Reischl et al. method and QIAamp® DNA Mini Kit. The error bars indicate minimum and maximum values. Extrapolated quantification limits of the methods are represented by dotted lines: - - -, FTA® Elute; ─ ─ ─, Reischl et al. and QIAamp® DNA Mini Kit

    Article Snippet: The efficiency of the Reischl et al. protocol was significantly lower with 9.0% as compared to the QIAamp® DNA Mini Kit (P = 0.001) and FTA® Elute paper (P = 0.001).

    Techniques: DNA Extraction

    Amplified pfmdr1 gene products after nested PCR. Standard DNA extraction was carried out using the QIAamp DNA mini blood kit (Qiagen, Hilden, Germany). DNA extraction by microwave irradiation was performed using a microwave oven (MDA, model number: MW17M70G-AU, 230 V, 50 HZ, operated at 800 W). 1 μl of condensed droplets after microwave treatment were utilized for the PCR procedures. First lane: DNA ladder; NC: Negative Control; PC1 and PC2: Standard extraction from archived blood sample and pfmdr1 amplicons at expected sizes; PC3 and PC4: Standard extraction from 3D7 P. falciparum parasites in culture and pfmdr1 amplicons at expected sizes; ME1 and ME2: Microwave based extraction from archived blood sample and pfmdr1 amplicons at expected sizes; ME3 and ME4: Microwave based extraction in 3D7 culture parasites and pfmdr1 amplicons at expected sizes; ME5: Microwave based DNA extraction from fresh blood sample and pfmdr1 amplicons at expected sizes.

    Journal: Malaria Journal

    Article Title: A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification

    doi: 10.1186/1475-2875-13-454

    Figure Lengend Snippet: Amplified pfmdr1 gene products after nested PCR. Standard DNA extraction was carried out using the QIAamp DNA mini blood kit (Qiagen, Hilden, Germany). DNA extraction by microwave irradiation was performed using a microwave oven (MDA, model number: MW17M70G-AU, 230 V, 50 HZ, operated at 800 W). 1 μl of condensed droplets after microwave treatment were utilized for the PCR procedures. First lane: DNA ladder; NC: Negative Control; PC1 and PC2: Standard extraction from archived blood sample and pfmdr1 amplicons at expected sizes; PC3 and PC4: Standard extraction from 3D7 P. falciparum parasites in culture and pfmdr1 amplicons at expected sizes; ME1 and ME2: Microwave based extraction from archived blood sample and pfmdr1 amplicons at expected sizes; ME3 and ME4: Microwave based extraction in 3D7 culture parasites and pfmdr1 amplicons at expected sizes; ME5: Microwave based DNA extraction from fresh blood sample and pfmdr1 amplicons at expected sizes.

    Article Snippet: DNA extraction: microwave irradiation First, DNA was extracted from whole blood samples as well as from the cultured parasites of the dilution series using the conventional QIAamp DNA mini blood kit based extraction procedure (Qiagen, Hilden, Germany) following the manufacturer’s instructions.

    Techniques: Amplification, Nested PCR, DNA Extraction, Irradiation, Multiple Displacement Amplification, Polymerase Chain Reaction, Negative Control

    DNA extraction method impacts leukocyte telomere length (T/S ratio). Bars are mean telomere length and error bars are standard deviations. DNA was extracted using the Purelink, QiaAmp and Lahiri and Nurnberger methods, respectively. ** P = 0.003.

    Journal: BMC Research Notes

    Article Title: Leukocyte telomere length variation due to DNA extraction method

    doi: 10.1186/1756-0500-7-877

    Figure Lengend Snippet: DNA extraction method impacts leukocyte telomere length (T/S ratio). Bars are mean telomere length and error bars are standard deviations. DNA was extracted using the Purelink, QiaAmp and Lahiri and Nurnberger methods, respectively. ** P = 0.003.

    Article Snippet: The Lahiri-extracted telomeres, however, were significantly shorter than those extracted using the QiaAmp DNA Mini Kit (mean T/S ratio: 2.71, range: 2.32 – 3.02; P = 0.003).

    Techniques: DNA Extraction

    5 different extraction methods for three sizes, (a) Figure: 110 bp size, (b) 256 and (c) 512bp of amplified β-globin gene primers. 1. Phenol-chloroform method, 2. QIAamp DNA FFPE Tissue Kit method, 3. Proteinase K and xylol method, 4. Heat alkaline method, 5. Heat alkaline plus mineral oil method and 6 is Negative control

    Journal: Nigerian Medical Journal : Journal of the Nigeria Medical Association

    Article Title: The effect of deoxyribonucleic acid extraction methods from lymphoid tissue on the purity, content, and amplifying ability

    doi: 10.4103/0300-1652.188321

    Figure Lengend Snippet: 5 different extraction methods for three sizes, (a) Figure: 110 bp size, (b) 256 and (c) 512bp of amplified β-globin gene primers. 1. Phenol-chloroform method, 2. QIAamp DNA FFPE Tissue Kit method, 3. Proteinase K and xylol method, 4. Heat alkaline method, 5. Heat alkaline plus mineral oil method and 6 is Negative control

    Article Snippet: They showed that the method, using phenol-chloroform and the QIAamp DNA Mini Kit (Qiagen, Germany), was the most efficient DNA extraction and purification method and that the DNA quantity extracted from liver is statistically more important than that extracted from heart.

    Techniques: Amplification, Formalin-fixed Paraffin-Embedded, Negative Control