dna blood mini kit  (Qiagen)


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    Name:
    QIAamp DNA Blood Mini Kit
    Description:
    For purification of up to 12 µg genomic mitochondrial or viral DNA from blood and related body fluids Kit contents Qiagen QIAamp DNA Blood Mini Kit 50 preps 1 to 200L Sample 50 to 200L Elution Volume Whole Blood Body Fluids Sample Spin Column Format Silica Technology Manual Processing 20 to 40 min Time Run 4 to 12g Yield For Purification of Up to 12g Genomic Mitochondrial or Viral DNA from Blood and Related Body Fluids Includes 50 QIAamp Mini Spin Columns Qiagen Protease Reagents Buffers 2mL Collection Tubes Benefits Rapid purification of high quality ready to use DNA No organic extraction or alcohol precipitation Consistent high yields Complete removal of contaminants and inhibitors for reliable results
    Catalog Number:
    51104
    Price:
    157
    Category:
    QIAamp DNA Blood Mini Kit
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    Structured Review

    Qiagen dna blood mini kit
    QIAamp DNA Blood Mini Kit
    For purification of up to 12 µg genomic mitochondrial or viral DNA from blood and related body fluids Kit contents Qiagen QIAamp DNA Blood Mini Kit 50 preps 1 to 200L Sample 50 to 200L Elution Volume Whole Blood Body Fluids Sample Spin Column Format Silica Technology Manual Processing 20 to 40 min Time Run 4 to 12g Yield For Purification of Up to 12g Genomic Mitochondrial or Viral DNA from Blood and Related Body Fluids Includes 50 QIAamp Mini Spin Columns Qiagen Protease Reagents Buffers 2mL Collection Tubes Benefits Rapid purification of high quality ready to use DNA No organic extraction or alcohol precipitation Consistent high yields Complete removal of contaminants and inhibitors for reliable results
    https://www.bioz.com/result/dna blood mini kit/product/Qiagen
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna blood mini kit - by Bioz Stars, 2021-03
    97/100 stars

    Images

    1) Product Images from "Inflammatory Genital Infections Mitigate a Severe Genetic Bottleneck in Heterosexual Transmission of Subtype A and C HIV-1"

    Article Title: Inflammatory Genital Infections Mitigate a Severe Genetic Bottleneck in Heterosexual Transmission of Subtype A and C HIV-1

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000274

    Transmission of multiple variants. Aligned nucleotide sequences for linked donors (green circles) and linked recipients (blue circles) were used to generate neighbor-joining trees for individual transmission pairs (A) RW57 and (B) ZM229. Horizontal branch lengths are drawn to scale with scale bar representing 1% divergence. Open circles represent sequences derived from uncultured PBMC DNA and closed circles represent sequences derived from plasma RNA. PBMC samples were unavailable for ZM229; therefore, only plasma sequences were derived for this transmission pair. Asterisks indicate branches with bootstrap values greater than 0.99.
    Figure Legend Snippet: Transmission of multiple variants. Aligned nucleotide sequences for linked donors (green circles) and linked recipients (blue circles) were used to generate neighbor-joining trees for individual transmission pairs (A) RW57 and (B) ZM229. Horizontal branch lengths are drawn to scale with scale bar representing 1% divergence. Open circles represent sequences derived from uncultured PBMC DNA and closed circles represent sequences derived from plasma RNA. PBMC samples were unavailable for ZM229; therefore, only plasma sequences were derived for this transmission pair. Asterisks indicate branches with bootstrap values greater than 0.99.

    Techniques Used: Transmission Assay, Derivative Assay

    2) Product Images from "Aberrant ZNF423 impedes B cell differentiation and is linked to adverse outcome of ETV6-RUNX1 negative B precursor acute lymphoblastic leukemia"

    Article Title: Aberrant ZNF423 impedes B cell differentiation and is linked to adverse outcome of ETV6-RUNX1 negative B precursor acute lymphoblastic leukemia

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20130497

    ZNF423 expression is regulated by DNA methylation. (A) Transactivation of ZNF423 isoform-specific promoters in dependence of central CGI. Reporter gene assay using ZNF423 promoters (α and β) with or without CGI after transfection in 293T cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing indicated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (B) Methylation map of CpGs in the central CGI at the ZNF423 locus. Genomic DNA from primary ALL ( n = 58), matched BM MNCs in complete continuous remission (MNC [CCR]; n = 58), H1, HES2 ESC lines, and normal hematopoietic cells at various stages of differentiation ( n = 42) from healthy donors were sequenced after bisulfite conversion. Each row represents one cytosine in a CG dinucleotide of the analyzed sequence. For cell lineage abbreviations, refer to Figure 1 . Significance is calculated by Student’s t test, comparing primary ALL to immunologically characterized control samples (in brackets; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Color code represents degree of methylation in percent. White boxes indicate no sequencing data available. (C) ZNF423 promoter activity in dependence of CPG mutational status. Disruption of CpGs was performed by site-directed mutagenesis as indicated by red marks. Combined deletion mutant (pCpGL-CGI-del_comb_α-prom) represents deletions at CGI positions 102, 183, 220, 250. Wild-type and mutated plasmids were treated with DNA-methylase M.SssI and transfected into 293T. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing M.SssI-treated wild-type and mutated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (D) DNA methylation pattern of central CGI. Diagram was created with BiQ Analyzer software from Max-Planck-Institute of Informatics. Drawing is to scale. White lollipop, unmethylated; black lollipop, methylated. Marked CpGs refer to heatmap in Fig. 4 B . (E and F) Expression of ZNF423 transcripts upon DNA demethylation and BMP2 stimulation. SEM cells (E) and CD34 + cord blood cells (F) were treated with 3 µM 5A2D for 24, 48, and 72 h. In SEM cells, additional BMP2 treatment was performed 6 h before lysis. Relative fold induction was measured by qPCR (2 −ΔΔCt ) using a ZNF423 isoform-specific primer design in SEM cells. mRNA levels were normalized to B2M and solvent control. Error bars represent SD out of three technical replicates. Significance is calculated using 2 −ΔCt values by Student’s t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant). Data were reproduced in three independent experiments.
    Figure Legend Snippet: ZNF423 expression is regulated by DNA methylation. (A) Transactivation of ZNF423 isoform-specific promoters in dependence of central CGI. Reporter gene assay using ZNF423 promoters (α and β) with or without CGI after transfection in 293T cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing indicated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (B) Methylation map of CpGs in the central CGI at the ZNF423 locus. Genomic DNA from primary ALL ( n = 58), matched BM MNCs in complete continuous remission (MNC [CCR]; n = 58), H1, HES2 ESC lines, and normal hematopoietic cells at various stages of differentiation ( n = 42) from healthy donors were sequenced after bisulfite conversion. Each row represents one cytosine in a CG dinucleotide of the analyzed sequence. For cell lineage abbreviations, refer to Figure 1 . Significance is calculated by Student’s t test, comparing primary ALL to immunologically characterized control samples (in brackets; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Color code represents degree of methylation in percent. White boxes indicate no sequencing data available. (C) ZNF423 promoter activity in dependence of CPG mutational status. Disruption of CpGs was performed by site-directed mutagenesis as indicated by red marks. Combined deletion mutant (pCpGL-CGI-del_comb_α-prom) represents deletions at CGI positions 102, 183, 220, 250. Wild-type and mutated plasmids were treated with DNA-methylase M.SssI and transfected into 293T. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing M.SssI-treated wild-type and mutated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (D) DNA methylation pattern of central CGI. Diagram was created with BiQ Analyzer software from Max-Planck-Institute of Informatics. Drawing is to scale. White lollipop, unmethylated; black lollipop, methylated. Marked CpGs refer to heatmap in Fig. 4 B . (E and F) Expression of ZNF423 transcripts upon DNA demethylation and BMP2 stimulation. SEM cells (E) and CD34 + cord blood cells (F) were treated with 3 µM 5A2D for 24, 48, and 72 h. In SEM cells, additional BMP2 treatment was performed 6 h before lysis. Relative fold induction was measured by qPCR (2 −ΔΔCt ) using a ZNF423 isoform-specific primer design in SEM cells. mRNA levels were normalized to B2M and solvent control. Error bars represent SD out of three technical replicates. Significance is calculated using 2 −ΔCt values by Student’s t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant). Data were reproduced in three independent experiments.

    Techniques Used: Expressing, DNA Methylation Assay, Reporter Gene Assay, Transfection, Luciferase, Activity Assay, Methylation, Sequencing, Mutagenesis, Software, Lysis, Real-time Polymerase Chain Reaction

    3) 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

    4) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    5) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    6) Product Images from "Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction"

    Article Title: Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2016.24

    HBV can be transmitted into NK cells through exosomes. ( a ) Identification of HBV DNA (rcDNA and cccDNA) and HBV RNA (HBx and HBs/p) in NK cells isolated from HD and CHB patients. ( b ) Transmission electron microscopy of ultrathin sections was used to observe freshly isolated NK cells from CHB patients with high virus loading. Cytoplasmic inclusion bodies with irregular electron density are shown on the left and middle panels (arrows and loop). Scale bar, 2 μm and 200 nm. ( c ) Confocal images of the co-localization of CFSE-labeled (green) primary NK cells from HD incubated with DiD-labeled exosomes (red) for 3 h. Scale bar, 20 μm. ( d ) The uptake of DiD-labeled exosomes by primary NK cells after 12 or 24 h incubation at 37 or 4 °C was determined by flow cytometry. ( e ) TGF-β (5 ng/ml) enhanced the ability of primary NK cells to take up DiD-labeled exosomes (red). ( f ) Analysis of HBV DNA (rcDNA and cccDNA) and RNA (HBx and HBs/p) in primary NK cells from HD infected with exosomes derived from CHB serum for 0–5 days. The results are representative of at least three independent experiments. The data are expressed as the mean±s.e.m. * P
    Figure Legend Snippet: HBV can be transmitted into NK cells through exosomes. ( a ) Identification of HBV DNA (rcDNA and cccDNA) and HBV RNA (HBx and HBs/p) in NK cells isolated from HD and CHB patients. ( b ) Transmission electron microscopy of ultrathin sections was used to observe freshly isolated NK cells from CHB patients with high virus loading. Cytoplasmic inclusion bodies with irregular electron density are shown on the left and middle panels (arrows and loop). Scale bar, 2 μm and 200 nm. ( c ) Confocal images of the co-localization of CFSE-labeled (green) primary NK cells from HD incubated with DiD-labeled exosomes (red) for 3 h. Scale bar, 20 μm. ( d ) The uptake of DiD-labeled exosomes by primary NK cells after 12 or 24 h incubation at 37 or 4 °C was determined by flow cytometry. ( e ) TGF-β (5 ng/ml) enhanced the ability of primary NK cells to take up DiD-labeled exosomes (red). ( f ) Analysis of HBV DNA (rcDNA and cccDNA) and RNA (HBx and HBs/p) in primary NK cells from HD infected with exosomes derived from CHB serum for 0–5 days. The results are representative of at least three independent experiments. The data are expressed as the mean±s.e.m. * P

    Techniques Used: Isolation, Transmission Assay, Electron Microscopy, Labeling, Incubation, Flow Cytometry, Cytometry, Infection, Derivative Assay

    Exosomes can transmit HBV to uninfected hepatoma cells. ( a ) The confocal image shows the co-localization of CFSE-labeled HLCZ01 cells (green) incubated with DiD-labeled exosomes (red) for 2 h. Scale bar, 20 μm. ( b ) Comparison of the ability to take up DiD-labeled exosomes (red) between live HLCZ01 cells and fixed HLCZ01 cells. Scale bar, 20 μm. ( c ) Immunocytochemical staining of HBsAg and HBcAg proteins in HLCZ01 cells infected with exosomes or HepG2.2.15 supernatant. Scale bar, 20 μm. ( d ) Quantitative PCR analysis of HBV DNA in HLCZ01 cells treated with HD/CHB exosomes, the supernatant of HepG2 cells or the supernatant of HepG2.2.15 cells at an multiplicity of infection of 50 per cell ( n =3). HepG2 and HepG2.2.15 cells were used as negative and positive controls, respectively. The results are representative of at least three independent experiments. The data are expressed as the mean±s.e.m. * P
    Figure Legend Snippet: Exosomes can transmit HBV to uninfected hepatoma cells. ( a ) The confocal image shows the co-localization of CFSE-labeled HLCZ01 cells (green) incubated with DiD-labeled exosomes (red) for 2 h. Scale bar, 20 μm. ( b ) Comparison of the ability to take up DiD-labeled exosomes (red) between live HLCZ01 cells and fixed HLCZ01 cells. Scale bar, 20 μm. ( c ) Immunocytochemical staining of HBsAg and HBcAg proteins in HLCZ01 cells infected with exosomes or HepG2.2.15 supernatant. Scale bar, 20 μm. ( d ) Quantitative PCR analysis of HBV DNA in HLCZ01 cells treated with HD/CHB exosomes, the supernatant of HepG2 cells or the supernatant of HepG2.2.15 cells at an multiplicity of infection of 50 per cell ( n =3). HepG2 and HepG2.2.15 cells were used as negative and positive controls, respectively. The results are representative of at least three independent experiments. The data are expressed as the mean±s.e.m. * P

    Techniques Used: Labeling, Incubation, Staining, Infection, Real-time Polymerase Chain Reaction

    Exosomes derived from CHB patients contained viral components. ( a ) Electron microscope image of purified exosomes from CHB serum. Scale bar, 100 nm. ( b ) CD81 protein was used to identify the exosomes isolated from CHB serum using flow cytometry. The shaded histograms represent the isotype control. ( c ) HBV DNA (rcDNA and cccDNA) and RNA (HBx and HBs/p) in exosomes isolated from CHB serum were detected using PCR and RT-PCR, respectively. ( d ) Western blot analysis of HBsAg and CD63 in exosomes from HD and CHB patient serum (10 μg/well). (Because of the limited volume of each sample, six fresh serum samples from CHB patients or healthy donors were harvested and mixed for exosome isolation). One representative of at least three independent experiments is shown. CHB, chronic hepatitis B; HBV, hepatitis B virus; DNA, deoxyribonucleic acid; rcDNA, relaxed circular DNA; cccDNA, closed-circular DNA; RNA, ribonucleic acid; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; HD, healthy donors.
    Figure Legend Snippet: Exosomes derived from CHB patients contained viral components. ( a ) Electron microscope image of purified exosomes from CHB serum. Scale bar, 100 nm. ( b ) CD81 protein was used to identify the exosomes isolated from CHB serum using flow cytometry. The shaded histograms represent the isotype control. ( c ) HBV DNA (rcDNA and cccDNA) and RNA (HBx and HBs/p) in exosomes isolated from CHB serum were detected using PCR and RT-PCR, respectively. ( d ) Western blot analysis of HBsAg and CD63 in exosomes from HD and CHB patient serum (10 μg/well). (Because of the limited volume of each sample, six fresh serum samples from CHB patients or healthy donors were harvested and mixed for exosome isolation). One representative of at least three independent experiments is shown. CHB, chronic hepatitis B; HBV, hepatitis B virus; DNA, deoxyribonucleic acid; rcDNA, relaxed circular DNA; cccDNA, closed-circular DNA; RNA, ribonucleic acid; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; HD, healthy donors.

    Techniques Used: Derivative Assay, Microscopy, Purification, Isolation, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    7) Product Images from "JAK2V617F Allele Burden Measurement in Peripheral Blood of Iranian Patients with Myeloproliferative Neoplasms and Effect of Hydroxyurea on JAK2V617F Allele Burden"

    Article Title: JAK2V617F Allele Burden Measurement in Peripheral Blood of Iranian Patients with Myeloproliferative Neoplasms and Effect of Hydroxyurea on JAK2V617F Allele Burden

    Journal: International Journal of Hematology-Oncology and Stem Cell Research

    doi:

    Agarose gel analysis for the detection of JAK2V617F mutation in genomic DNA by ARMS- PCR. The 463- bp fragment was amplified as a control band for all PCR products. wild-type specific primers produce a fragment of 229- bp and mutant specific primers generate a fragment of 279-bp. Lane 2, 3 and 4 samples from patients with mutation; lane 5 sample from patients without mutation; lane 6 is a healthy person, lane 7 is negative PCR control. Lane 1 is 100-base pair (bp).
    Figure Legend Snippet: Agarose gel analysis for the detection of JAK2V617F mutation in genomic DNA by ARMS- PCR. The 463- bp fragment was amplified as a control band for all PCR products. wild-type specific primers produce a fragment of 229- bp and mutant specific primers generate a fragment of 279-bp. Lane 2, 3 and 4 samples from patients with mutation; lane 5 sample from patients without mutation; lane 6 is a healthy person, lane 7 is negative PCR control. Lane 1 is 100-base pair (bp).

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

    8) Product Images from "Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood"

    Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

    Journal: Journal of Biomolecular Techniques : JBT

    doi: 10.7171/jbt.18-2902-001

    PCR of different HLA targets using gDNA prepared using the PES membrane or QIAamp kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore ( A ) or QIAamp DNA Blood Mini Kit from Qiagen ( B ). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter . PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).
    Figure Legend Snippet: PCR of different HLA targets using gDNA prepared using the PES membrane or QIAamp kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore ( A ) or QIAamp DNA Blood Mini Kit from Qiagen ( B ). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter . PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).

    Techniques Used: Polymerase Chain Reaction, Amplification, Isolation, Agarose Gel Electrophoresis, Marker

    Agarose gel electrophoresis of gDNA samples prepared using different PES membranes or the QIAamp commercial kit. A ) Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B ) Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; and lane 3: Sterlitech). C ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb). Representative of a minimum of 4 independent experiments.
    Figure Legend Snippet: Agarose gel electrophoresis of gDNA samples prepared using different PES membranes or the QIAamp commercial kit. A ) Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B ) Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; and lane 3: Sterlitech). C ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb). Representative of a minimum of 4 independent experiments.

    Techniques Used: Agarose Gel Electrophoresis, Marker

    Agarose gel electrophoresis of gDNA samples digested with either Hin dIII or Eco RI. DNAs digested with Hin dIII ( A – C ); Eco R1 digested gDNAs ( D – F ). A , D ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B , E ) Buffy coat from 2 different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; lane 3: Sterlitech). C , F ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either the 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb).
    Figure Legend Snippet: Agarose gel electrophoresis of gDNA samples digested with either Hin dIII or Eco RI. DNAs digested with Hin dIII ( A – C ); Eco R1 digested gDNAs ( D – F ). A , D ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B , E ) Buffy coat from 2 different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; lane 3: Sterlitech). C , F ) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either the 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb).

    Techniques Used: Agarose Gel Electrophoresis, Marker

    9) Product Images from "Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood"

    Article Title: Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood

    Journal: Journal of Applied Microbiology

    doi: 10.1111/jam.12769

    Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.
    Figure Legend Snippet: Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.

    Techniques Used: Real-time Polymerase Chain Reaction, Lysis

    10) Product Images from "Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood"

    Article Title: Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood

    Journal: Journal of Applied Microbiology

    doi: 10.1111/jam.12769

    Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.
    Figure Legend Snippet: Real-time PCR detection of Salmonella DNA. Two millilitres of whole blood was lysed using erythrocyte lysis buffer and spiked with the shown CFU of Salmonella Typhi CVD 909. DNA was extracted using a QIAamp Mini blood kit and S. Typhi was detected by qPCR.

    Techniques Used: Real-time Polymerase Chain Reaction, Lysis

    11) Product Images from "Lack of Detection of Xenotropic Murine Leukemia Virus-Related Virus in HIV-1 Lymphoma Patients"

    Article Title: Lack of Detection of Xenotropic Murine Leukemia Virus-Related Virus in HIV-1 Lymphoma Patients

    Journal: Advances in Virology

    doi: 10.1155/2011/797820

    Real-time PCR analysis of HIV-1 lymphoma positive patients for XMRV. XMRV gag qPCR failed to detect XMRV from DNA isolated from PBMCs for the 26 HIV-1 lymphoma patients (green lines). Inset shows single-copy sensitivity of the assay.
    Figure Legend Snippet: Real-time PCR analysis of HIV-1 lymphoma positive patients for XMRV. XMRV gag qPCR failed to detect XMRV from DNA isolated from PBMCs for the 26 HIV-1 lymphoma patients (green lines). Inset shows single-copy sensitivity of the assay.

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation

    12) Product Images from "A novel function for spumaretrovirus integrase: an early requirement for integrase-mediated cleavage of 2 LTR circles"

    Article Title: A novel function for spumaretrovirus integrase: an early requirement for integrase-mediated cleavage of 2 LTR circles

    Journal: Retrovirology

    doi: 10.1186/1742-4690-2-31

    Decreased viral DNA production by IN-defective viruses is concomitant with an abnormal accumulation of LTR-LTR junctions . Quantification of viral DNA synthesis was carried out by real-time PCR amplification of total DNA extracts from U373-MG infected cells (equal virion levels as measured by reverse transcriptase activity), collected 3, 6, 10, and 24 hours post-infection. An m.o.i. of 1 for the WT infection as determined by the FAB assay was used. Data are presented for 10 6 cells as measured by quantification of the nuclear β-globin gene and standard deviations representing variations between two quantifications of the same sample are given. To ensure that only freshly synthesized DNA, and not contaminating DNA contained in the viral particles input, was analyzed, all infections were performed in parallel control experiments under AZT treatment that inhibits viral neosynthesis. Representative kinetics from 4 independent experiments is presented. (A) Total viral DNA was detected using primers allowing amplification of the region of the PFV cDNA at the 5' end of the gag gene [40]. (B) Viral DNA with 2-LTR junctions was measured using primers that cross the junction between the two LTRs as previously described [40]. (C) The abundance of 2-LTR molecules is expressed as the percentage of 2-LTR copies relative to the total viral DNA ( gag ) at each infection time-point.
    Figure Legend Snippet: Decreased viral DNA production by IN-defective viruses is concomitant with an abnormal accumulation of LTR-LTR junctions . Quantification of viral DNA synthesis was carried out by real-time PCR amplification of total DNA extracts from U373-MG infected cells (equal virion levels as measured by reverse transcriptase activity), collected 3, 6, 10, and 24 hours post-infection. An m.o.i. of 1 for the WT infection as determined by the FAB assay was used. Data are presented for 10 6 cells as measured by quantification of the nuclear β-globin gene and standard deviations representing variations between two quantifications of the same sample are given. To ensure that only freshly synthesized DNA, and not contaminating DNA contained in the viral particles input, was analyzed, all infections were performed in parallel control experiments under AZT treatment that inhibits viral neosynthesis. Representative kinetics from 4 independent experiments is presented. (A) Total viral DNA was detected using primers allowing amplification of the region of the PFV cDNA at the 5' end of the gag gene [40]. (B) Viral DNA with 2-LTR junctions was measured using primers that cross the junction between the two LTRs as previously described [40]. (C) The abundance of 2-LTR molecules is expressed as the percentage of 2-LTR copies relative to the total viral DNA ( gag ) at each infection time-point.

    Techniques Used: DNA Synthesis, Real-time Polymerase Chain Reaction, Amplification, Infection, Activity Assay, Synthesized

    Integration of IN-defective viruses . (A) A quantitative assay based on a real-time RACE-PCR reaction was designed, amplifying Alu-LTR junctions between the cell genome and integrated proviruses twenty-four hours post-infection. PCR amplifications of existing Alu-PFV-1 LTR junctions were subjected to a second quantitative round of real time PCR with PFV-1 LTR-specific primers. Fluorogenic hybridization probes were used to quantify the amplification products. Infected cells with known copy numbers of integrated proviruses were used as quantification standards. The assay is highly sensitive since it allows detecting 25 proviruses copies in 50,000 human cells. Control reactions are detailed in the Material and methods section. (B) Detection of integrated viral DNA following infection of IN-mutated viruses. Quantitation of viral DNA accumulated in PFV-1 infected cells was carried out by real-time PCR of total DNA extracts from U373-MG infected cells (m.o.i. of 1) collected at the completion of the first viral replication cycle, 24 hours post-infection. Total viral DNA ( gag quantifications) and integrated proviruses were quantified in duplicate using real-time PCRs. Data obtained in one representative infection from four independent experiments are expressed as integrated DNA copies per million cells (logarithmic scale) as determined by a human β-globin quantification in cell extracts (
    Figure Legend Snippet: Integration of IN-defective viruses . (A) A quantitative assay based on a real-time RACE-PCR reaction was designed, amplifying Alu-LTR junctions between the cell genome and integrated proviruses twenty-four hours post-infection. PCR amplifications of existing Alu-PFV-1 LTR junctions were subjected to a second quantitative round of real time PCR with PFV-1 LTR-specific primers. Fluorogenic hybridization probes were used to quantify the amplification products. Infected cells with known copy numbers of integrated proviruses were used as quantification standards. The assay is highly sensitive since it allows detecting 25 proviruses copies in 50,000 human cells. Control reactions are detailed in the Material and methods section. (B) Detection of integrated viral DNA following infection of IN-mutated viruses. Quantitation of viral DNA accumulated in PFV-1 infected cells was carried out by real-time PCR of total DNA extracts from U373-MG infected cells (m.o.i. of 1) collected at the completion of the first viral replication cycle, 24 hours post-infection. Total viral DNA ( gag quantifications) and integrated proviruses were quantified in duplicate using real-time PCRs. Data obtained in one representative infection from four independent experiments are expressed as integrated DNA copies per million cells (logarithmic scale) as determined by a human β-globin quantification in cell extracts ("Integrated provirus" panel). Total DNA copies per million cells (logarithmic scale) present in the same extracts are presented in the lower panel. Standard deviations representing variations between two quantifications of the same sample are given. (C) Integration efficiency in PFV-1 infected cells. Integration efficiency was determined by normalizing the number of integrated proviruses (mean of duplicates) with the total number of viral DNA molecules (mean of duplicates) present in the same extract. Raw LightCycler data from four independent experiments are presented in the upper table. Mean of integration efficiencies from these four experiments are figured in the lower histogram.

    Techniques Used: Polymerase Chain Reaction, Infection, Real-time Polymerase Chain Reaction, Hybridization, Amplification, Quantitation Assay

    13) Product Images from "PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids"

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.8.3.499-502.2001

    Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.
    Figure Legend Snippet: Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.

    Techniques Used: Concentration Assay, Polymerase Chain Reaction

    Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).
    Figure Legend Snippet: Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).

    Techniques Used: Polymerase Chain Reaction, DNA Extraction

    Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.
    Figure Legend Snippet: Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.

    Techniques Used: DNA Extraction, Polymerase Chain Reaction

    Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.
    Figure Legend Snippet: Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry, Marker, Positive Control, Negative Control, DNA Extraction

    14) Product Images from "PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids"

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.8.3.499-502.2001

    Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.
    Figure Legend Snippet: Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.

    Techniques Used: Concentration Assay, Polymerase Chain Reaction

    Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).
    Figure Legend Snippet: Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).

    Techniques Used: Polymerase Chain Reaction, DNA Extraction

    Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.
    Figure Legend Snippet: Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.

    Techniques Used: DNA Extraction, Polymerase Chain Reaction

    Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.
    Figure Legend Snippet: Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry, Marker, Positive Control, Negative Control, DNA Extraction

    15) Product Images from "Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome"

    Article Title: Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome

    Journal: International Journal of Medical Sciences

    doi:

    Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.
    Figure Legend Snippet: Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.

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

    16) Product Images from "Assessing the Performance of Dried-Blood-Spot DNA Extraction Methods in Next Generation Sequencing"

    Article Title: Assessing the Performance of Dried-Blood-Spot DNA Extraction Methods in Next Generation Sequencing

    Journal: International journal of neonatal screening

    doi: 10.3390/ijns6020036

    Sequence coverage (average) per CFTR target region for the MiSeqDx cystic fibrosis clinical sequencing assay for each DNA extraction method. Each bar represents the average sequence coverage of a single target across nineteen samples (each containing at least one pathogenic variant in the CFTR gene). The sequence coverage for each DNA extraction method was determined and graphed from highest to lowest using the Qiagen QIAamp® micro DNA extraction method as the standard. Target regions are color coded according to the exon or intron region sequenced.
    Figure Legend Snippet: Sequence coverage (average) per CFTR target region for the MiSeqDx cystic fibrosis clinical sequencing assay for each DNA extraction method. Each bar represents the average sequence coverage of a single target across nineteen samples (each containing at least one pathogenic variant in the CFTR gene). The sequence coverage for each DNA extraction method was determined and graphed from highest to lowest using the Qiagen QIAamp® micro DNA extraction method as the standard. Target regions are color coded according to the exon or intron region sequenced.

    Techniques Used: Sequencing, DNA Extraction, Variant Assay

    17) Product Images from "Detection of pathogenic microorganisms from bloodstream infection specimens using TaqMan array card technology"

    Article Title: Detection of pathogenic microorganisms from bloodstream infection specimens using TaqMan array card technology

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31200-3

    TAC optimization. ( A ) 50% Tween-20/Triton X-100 and 0.1% saponin treatment on the yield of pathogen cells from whole blood specimens. 10 3 CFU S. aureus was mixed in 1 mL blood from healthy donors and then lysed by Tween-20/Triton X-100 or saponin. The colony numbers were determined by a plate count. ( B , C ). TNA extraction kit performance on blood culture specimens ( B ) and mock whole blood specimens ( C ). Kit 1 is the BiOstic bacteremia DNA isolation kit; Kit 2 is the QIAamp DNA Blood Mini Kit; Kit 3 is the QIAamp UCP Pathogen Mini Kit; Kit 4 is the TIANamp Blood DNA Kit; Kit 5 is the QIAamp cador Pathogen Mini Kit. M represents the benzyl alcohol-guanidine hydrochloride method. For blood culture specimens, three blood culture samples positive for S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used. For whole blood specimens, three whole blood mock specimens spiked with 10 1 CFU/mL of S. aureus , E. coli , and C. albicans were used. ( D ) The effect of TNA combined with reverse transcription on amplifying efficiency. 10 1 and 10 2 CFU S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used to make mock specimens. The cycle threshold (Ct) was compared with RT or without RT. ( E ) The effect of 0.1% blue dextran 2000 on the TAC assay. 10 6 CFU S. aureus (G + ), 10 7 CFU P. aeruginosa (G − ) and 10 4 CFU C. albicans (fungi) were used to make mock specimens. B+, TAC assay with blue dextran 2000; B−, TAC assay without blue dextran 2000.
    Figure Legend Snippet: TAC optimization. ( A ) 50% Tween-20/Triton X-100 and 0.1% saponin treatment on the yield of pathogen cells from whole blood specimens. 10 3 CFU S. aureus was mixed in 1 mL blood from healthy donors and then lysed by Tween-20/Triton X-100 or saponin. The colony numbers were determined by a plate count. ( B , C ). TNA extraction kit performance on blood culture specimens ( B ) and mock whole blood specimens ( C ). Kit 1 is the BiOstic bacteremia DNA isolation kit; Kit 2 is the QIAamp DNA Blood Mini Kit; Kit 3 is the QIAamp UCP Pathogen Mini Kit; Kit 4 is the TIANamp Blood DNA Kit; Kit 5 is the QIAamp cador Pathogen Mini Kit. M represents the benzyl alcohol-guanidine hydrochloride method. For blood culture specimens, three blood culture samples positive for S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used. For whole blood specimens, three whole blood mock specimens spiked with 10 1 CFU/mL of S. aureus , E. coli , and C. albicans were used. ( D ) The effect of TNA combined with reverse transcription on amplifying efficiency. 10 1 and 10 2 CFU S. aureus (G + ), E. coli (G − ), or C. albicans (fungi) were used to make mock specimens. The cycle threshold (Ct) was compared with RT or without RT. ( E ) The effect of 0.1% blue dextran 2000 on the TAC assay. 10 6 CFU S. aureus (G + ), 10 7 CFU P. aeruginosa (G − ) and 10 4 CFU C. albicans (fungi) were used to make mock specimens. B+, TAC assay with blue dextran 2000; B−, TAC assay without blue dextran 2000.

    Techniques Used: DNA Extraction

    18) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    19) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    20) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    21) Product Images from "Extracellular vesicles produced in B cells deliver tumor suppressor miR-335 to breast cancer cells disrupting oncogenic programming in vitro and in vivo"

    Article Title: Extracellular vesicles produced in B cells deliver tumor suppressor miR-335 to breast cancer cells disrupting oncogenic programming in vitro and in vivo

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35968-2

    Experimental model and cartoon of dual miR-335 plasmid. ( A ) Schematic of experimental model involving the transfection of murine J558L B cells with pCMVmir.335, the production of induced extracellular vesicles (iEVs), and subsequent treatment on LM2 cells. ( B ) Schematic diagram of tandem hsa-mir335 stem (pre-miR) loops with an intervening spacer sequence. ( C ) Differential abundance of miR-335 in iEVs from J558L cells transfected with pCMVmir.335 containing a single or dual pre-miR-335 sequence, respectively. 10 6 J558L cells were transfected with 1 µg of plasmid DNA, and the supernatant was collected 48 hrs later. iEVs were isolated by precipitation, counted and analyzed (10 6 ) by RT-qPCR amplification using RT-specific primers for miR-335 and SnoRNA202 as a control. RQ (Relative Quantity). Results refer to the mean ± SD of a representative experiment out of three independent experiments. ( D ) Negative staining electron micrographs of iEVs-335. Magnification: Inset (6800x), Main frame 9300x.
    Figure Legend Snippet: Experimental model and cartoon of dual miR-335 plasmid. ( A ) Schematic of experimental model involving the transfection of murine J558L B cells with pCMVmir.335, the production of induced extracellular vesicles (iEVs), and subsequent treatment on LM2 cells. ( B ) Schematic diagram of tandem hsa-mir335 stem (pre-miR) loops with an intervening spacer sequence. ( C ) Differential abundance of miR-335 in iEVs from J558L cells transfected with pCMVmir.335 containing a single or dual pre-miR-335 sequence, respectively. 10 6 J558L cells were transfected with 1 µg of plasmid DNA, and the supernatant was collected 48 hrs later. iEVs were isolated by precipitation, counted and analyzed (10 6 ) by RT-qPCR amplification using RT-specific primers for miR-335 and SnoRNA202 as a control. RQ (Relative Quantity). Results refer to the mean ± SD of a representative experiment out of three independent experiments. ( D ) Negative staining electron micrographs of iEVs-335. Magnification: Inset (6800x), Main frame 9300x.

    Techniques Used: Plasmid Preparation, Transfection, Sequencing, Isolation, Quantitative RT-PCR, Amplification, Negative Staining

    22) Product Images from "PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids"

    Article Title: PCR-Based Method for Isolation and Detection of Chlamydia pneumoniae DNA in Cerebrospinal Fluids

    Journal: Clinical and Diagnostic Laboratory Immunology

    doi: 10.1128/CDLI.8.3.499-502.2001

    Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.
    Figure Legend Snippet: Optimization of annealing temperature and Mg 2+ concentration for PCR with primers for omp1 . The annealing temperature was optimized in a Mastercycler gradient (Eppendorf). The Mg concentration for PCR specific for omp1 was optimized with a PCR optimization kit (Invitrogen). The target DNA for PCR was extracted from C. pneumoniae with the QIAmp DNA Mini Kit.

    Techniques Used: Concentration Assay, Polymerase Chain Reaction

    Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).
    Figure Legend Snippet: Detection sensitivity of PCR for omp1 versus the 16S rRNA gene. Two hundred microliters of the mock CSF containing a specific number of C. pneumoniae organisms (as indicated), was used for DNA extraction with the QIAmp DNA Mini Kit. A 2-μl portion of the 50-μl volume of DNA extracts was subjected to PCR. The optimized PCRs for omp1 and 16S rRNA gene were conducted (see Materials and Methods).

    Techniques Used: Polymerase Chain Reaction, DNA Extraction

    Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.
    Figure Legend Snippet: Comparison of extraction efficacy of C. pneumoniae DNA from mock CSFs using two DNA extraction kits. The mock CSFs (200 μl) spiked with serially diluted bacteria were extracted either with the QIAmp DNA Mini Kit with a bacterial DNA extraction protocol or with the QIAmp DNA Blood Mini Kit. Two microliters of the extracted DNA (50 μl) was subjected to PCR with primers for omp1 . Results are representative of three experiments.

    Techniques Used: DNA Extraction, Polymerase Chain Reaction

    Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.
    Figure Legend Snippet: Detection of C. pneumoniae DNA by PCR in CSFs obtained from patients with MS. Two hundred microliters of CSFs obtained from MS patients was processed for extraction of DNA utilizing the QIAmp DNA Mini Kit, and 2 μl of the resulting 50-μl DNA solution was subjected to PCR with primers for either omp1 or the 16S rRNA gene. Data are representative of three PCR experiments. Boxed numbers indicate CSFs obtained from the same patient at different time points. Each number is the sample number. M, molecular marker; PC, positive control for PCR; NC, negative control for PCR; IC, negative control for DNA extraction.

    Techniques Used: Polymerase Chain Reaction, Mass Spectrometry, Marker, Positive Control, Negative Control, DNA Extraction

    23) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    24) Product Images from "White blood cell and cell-free DNA analyses for detection of residual disease in gastric cancer"

    Article Title: White blood cell and cell-free DNA analyses for detection of residual disease in gastric cancer

    Journal: Nature Communications

    doi: 10.1038/s41467-020-14310-3

    Analysis of cfDNA in patients with resectable gastric cancer. Study schematic ( a ). Patients with confirmed stage IB–IVA gastric adenocarcinoma eligible for perioperative treatment with systemic chemotherapy were randomized upfront to receive three cycles of preoperative chemotherapy, followed by three cycles of postoperative chemotherapy, or to receive the same preoperative regimen, followed by postoperative radiotherapy combined with chemotherapy. A blood draw was collected for each patient at the time of study enrollment (baseline), after three cycles of preoperative chemotherapy (preoperative timepoint), and after surgery (postoperative timepoint). Blood samples were initially processed to allow extraction of cfDNA from plasma and genomic DNA (gDNA) from white blood cells (WBC). Both cfDNA and WBC gDNA libraries were captured with custom RNA oligo pools encompassing 80,930 bases across 58 cancer driver genes. Capture libraries were sequenced at high coverage ( > 30,000×), followed by sequence alignment, error correction, and variant calling. Mutations detected in WBCs were identified in cfDNA and removed, allowing the identification of tumor-specific mutations in cfDNA. Clinicopathological characteristics, type of surgical treatment, pathological stage, and type of postoperative treatment for the subset of patients were analyzed in this translational study ( n = 50) ( b ). MSI, microsatellite instability; MSS, microsatellite stability; EBV, Epstein–Barr virus; ECC, epirubicin, cisplatin, capecitabine; CC, cisplatin, capecitabine.
    Figure Legend Snippet: Analysis of cfDNA in patients with resectable gastric cancer. Study schematic ( a ). Patients with confirmed stage IB–IVA gastric adenocarcinoma eligible for perioperative treatment with systemic chemotherapy were randomized upfront to receive three cycles of preoperative chemotherapy, followed by three cycles of postoperative chemotherapy, or to receive the same preoperative regimen, followed by postoperative radiotherapy combined with chemotherapy. A blood draw was collected for each patient at the time of study enrollment (baseline), after three cycles of preoperative chemotherapy (preoperative timepoint), and after surgery (postoperative timepoint). Blood samples were initially processed to allow extraction of cfDNA from plasma and genomic DNA (gDNA) from white blood cells (WBC). Both cfDNA and WBC gDNA libraries were captured with custom RNA oligo pools encompassing 80,930 bases across 58 cancer driver genes. Capture libraries were sequenced at high coverage ( > 30,000×), followed by sequence alignment, error correction, and variant calling. Mutations detected in WBCs were identified in cfDNA and removed, allowing the identification of tumor-specific mutations in cfDNA. Clinicopathological characteristics, type of surgical treatment, pathological stage, and type of postoperative treatment for the subset of patients were analyzed in this translational study ( n = 50) ( b ). MSI, microsatellite instability; MSS, microsatellite stability; EBV, Epstein–Barr virus; ECC, epirubicin, cisplatin, capecitabine; CC, cisplatin, capecitabine.

    Techniques Used: Sequencing, Variant Assay

    25) Product Images from "Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance"

    Article Title: Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136728

    Effect of rtL269I substitution on the resistance to entecavir (ETV). (A) HBV 1.2mer DNA of all the mutants was transfected into Huh7 cells, which were treated with ETV for 3 days. The intracellular HBV DNA level was determined by Southern blot analysis. A representative result was displayed. (B) The relative replication levels of each HBV mutant (no drug vs ETV treatment) was calculated based on the results displayed in Figs 2C and 4A. (C) The relative replication ability of the HBV mutants treated with ETV were determined by Southern blot analysis, and quantified by Phosphorimager (*, P
    Figure Legend Snippet: Effect of rtL269I substitution on the resistance to entecavir (ETV). (A) HBV 1.2mer DNA of all the mutants was transfected into Huh7 cells, which were treated with ETV for 3 days. The intracellular HBV DNA level was determined by Southern blot analysis. A representative result was displayed. (B) The relative replication levels of each HBV mutant (no drug vs ETV treatment) was calculated based on the results displayed in Figs 2C and 4A. (C) The relative replication ability of the HBV mutants treated with ETV were determined by Southern blot analysis, and quantified by Phosphorimager (*, P

    Techniques Used: Transfection, Southern Blot, Mutagenesis

    Molecular modeling of rtL269I of HBV polymerase. (A) The three dimensional structure of the modeled HBV polymerase-DNA-TTP complex and (B) the expanded structure around rtL269. N-3 denotes the third nucleotide in the primer strand, starting from the 3′-end, just above the substrate-binding site. The domains of fingers, palm and thumb are shown in green, gray and pink colors, respectively. Also, the primer and template strand are shown in yellow and blue, respectively. TTP and active site residues (Asp83, Asp205 and Asp206) are colored by atom-types. The active site is noted as a dotted box. (C) When Leucine is mutated to isoleucine, residue 269 experiences steric clash with the neighboring rtR289 (block arrow), which would result in a conformational change in the primer binding residue, rtW284, and consequently, in the primer nucleotide, N-3 (dotted arrow).
    Figure Legend Snippet: Molecular modeling of rtL269I of HBV polymerase. (A) The three dimensional structure of the modeled HBV polymerase-DNA-TTP complex and (B) the expanded structure around rtL269. N-3 denotes the third nucleotide in the primer strand, starting from the 3′-end, just above the substrate-binding site. The domains of fingers, palm and thumb are shown in green, gray and pink colors, respectively. Also, the primer and template strand are shown in yellow and blue, respectively. TTP and active site residues (Asp83, Asp205 and Asp206) are colored by atom-types. The active site is noted as a dotted box. (C) When Leucine is mutated to isoleucine, residue 269 experiences steric clash with the neighboring rtR289 (block arrow), which would result in a conformational change in the primer binding residue, rtW284, and consequently, in the primer nucleotide, N-3 (dotted arrow).

    Techniques Used: Binding Assay, Blocking Assay

    Characterization of multi-drug resistant HBV mutant 50–2 isolated from a chronic HBV patient. (A) Schematic representation of the mutations in RT and overlapping surface gene of clone 50–2. The amino acid changes in polymerase and corresponding surface gene are indicated by arrows. (B) Comparison of intracellular and extracellular secreted HBV DNA levels between WT virus and clone 50–2. The HBV 1.2mer construct plasmids (2 μg) were transfected into Huh7 hepatoma cells and harvested at 72h post-transfection. The linearized 3.2kb HBV genome was loaded in lane 1 as a marker. (C) (D) In vitro susceptibility WT HBV (C) and clone 50–2 (D) to lamivudine (LMV), clevudine (CLV), entecavir (ETV), adefovir (ADV), and tenofovir (TDF). Cells were treated for 3 days with each drug, and the replication level was compared with that of the WT (without drug treatment). The relative HBV replication level was quantified using Phosphorimager. The standard deviation of three independent experiments was measured (***, P
    Figure Legend Snippet: Characterization of multi-drug resistant HBV mutant 50–2 isolated from a chronic HBV patient. (A) Schematic representation of the mutations in RT and overlapping surface gene of clone 50–2. The amino acid changes in polymerase and corresponding surface gene are indicated by arrows. (B) Comparison of intracellular and extracellular secreted HBV DNA levels between WT virus and clone 50–2. The HBV 1.2mer construct plasmids (2 μg) were transfected into Huh7 hepatoma cells and harvested at 72h post-transfection. The linearized 3.2kb HBV genome was loaded in lane 1 as a marker. (C) (D) In vitro susceptibility WT HBV (C) and clone 50–2 (D) to lamivudine (LMV), clevudine (CLV), entecavir (ETV), adefovir (ADV), and tenofovir (TDF). Cells were treated for 3 days with each drug, and the replication level was compared with that of the WT (without drug treatment). The relative HBV replication level was quantified using Phosphorimager. The standard deviation of three independent experiments was measured (***, P

    Techniques Used: Mutagenesis, Isolation, Construct, Transfection, Marker, In Vitro, Standard Deviation

    Effect of rtL269I and other substitutions on resistance to lamivudine (LMV). (A) HBV DNA constructs were transfected into Huh7 cells, which were treated with LMV for 3 days. The intracellular HBV DNA was prepared for Southern blot analysis. A representative result has been shown. (B) The relative replication levels of each HBV mutant (no drug vs LMV treatment) were calculated based on the results of Figs 2C and 3A. (C) The relative replication ability of the HBV mutants treated with LMV were determined by Southern blotting and quantified by Phosphorimager (*, P
    Figure Legend Snippet: Effect of rtL269I and other substitutions on resistance to lamivudine (LMV). (A) HBV DNA constructs were transfected into Huh7 cells, which were treated with LMV for 3 days. The intracellular HBV DNA was prepared for Southern blot analysis. A representative result has been shown. (B) The relative replication levels of each HBV mutant (no drug vs LMV treatment) were calculated based on the results of Figs 2C and 3A. (C) The relative replication ability of the HBV mutants treated with LMV were determined by Southern blotting and quantified by Phosphorimager (*, P

    Techniques Used: Construct, Transfection, Southern Blot, Mutagenesis

    rtL269I substitution enhances the replication of both WT and drug-resistant hepatitis B virus (HBV). (A) Schematic diagram of each HBV mutant construct used in this study. (B–C) Effect of mutations at positions 204, 173, 129, 337, and 269 on HBV DNA replication. Huh7 cells cultured in six-well plates were transfected with HBV plasmids. HBV DNA levels were analyzed by Southern blotting. HBeAg in culture supernatant was determined by ELISA. (D) Phosphor-imager analysis of the relative replication capacities of the HBV mutants. The standard deviation from three independent experiments was calculated (**, P
    Figure Legend Snippet: rtL269I substitution enhances the replication of both WT and drug-resistant hepatitis B virus (HBV). (A) Schematic diagram of each HBV mutant construct used in this study. (B–C) Effect of mutations at positions 204, 173, 129, 337, and 269 on HBV DNA replication. Huh7 cells cultured in six-well plates were transfected with HBV plasmids. HBV DNA levels were analyzed by Southern blotting. HBeAg in culture supernatant was determined by ELISA. (D) Phosphor-imager analysis of the relative replication capacities of the HBV mutants. The standard deviation from three independent experiments was calculated (**, P

    Techniques Used: Mutagenesis, Construct, Cell Culture, Transfection, Southern Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

    26) Product Images from "Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification"

    Article Title: Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification

    Journal: Analytical and Bioanalytical Chemistry

    doi: 10.1007/s00216-014-7835-3

    Assessment of cell-free DNA (cfDNA) yield using four extraction methods. The mean yield ± one standard deviation from replicate extractions using plasma pool A (i) (with or without ADH ) performed with the QIAamp circulating nucleic acid ( CNA ), NucleoSpin Plasma XS ( NS ) and FitAmp plasma/serum DNA isolation ( FA ) kits ( n = 10) and the QIAamp DNA blood mini ( DBM ) kit ( n = 9) is displayed relative to the mean yield of the DBM kit. The yield of cfDNA was quantified by quantitative PCR (qPCR) assays to TERT and ALUJ
    Figure Legend Snippet: Assessment of cell-free DNA (cfDNA) yield using four extraction methods. The mean yield ± one standard deviation from replicate extractions using plasma pool A (i) (with or without ADH ) performed with the QIAamp circulating nucleic acid ( CNA ), NucleoSpin Plasma XS ( NS ) and FitAmp plasma/serum DNA isolation ( FA ) kits ( n = 10) and the QIAamp DNA blood mini ( DBM ) kit ( n = 9) is displayed relative to the mean yield of the DBM kit. The yield of cfDNA was quantified by quantitative PCR (qPCR) assays to TERT and ALUJ

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

    27) Product Images from "Aging-associated distinctive DNA methylation changes of LINE-1 retrotransposons in pure cell-free DNA from human blood"

    Article Title: Aging-associated distinctive DNA methylation changes of LINE-1 retrotransposons in pure cell-free DNA from human blood

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-79126-z

    Cellular and cfDNA isolation using various commercially available kits. Lane 1: genomic DNA by QiAamp DNA Blood Mini Kit (kit A ). Lane 2/3: cfDNA by QiAamp DNA Blood Mini Kit (kit A). Lane 4/5: cfDNA by PME free-circulating DNA Extraction Kit (kit B ). Lane 6/7: cfDNA by Quick-cfDNA Serum Plasma Kit (kit C ). Lane 8/9: cfDNA by QIAamp MinElute ccfDNA Kit (kit D ).
    Figure Legend Snippet: Cellular and cfDNA isolation using various commercially available kits. Lane 1: genomic DNA by QiAamp DNA Blood Mini Kit (kit A ). Lane 2/3: cfDNA by QiAamp DNA Blood Mini Kit (kit A). Lane 4/5: cfDNA by PME free-circulating DNA Extraction Kit (kit B ). Lane 6/7: cfDNA by Quick-cfDNA Serum Plasma Kit (kit C ). Lane 8/9: cfDNA by QIAamp MinElute ccfDNA Kit (kit D ).

    Techniques Used: Isolation, DNA Extraction

    28) Product Images from "Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting"

    Article Title: Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

    Journal: The Journal of molecular diagnostics : JMD

    doi: 10.2353/jmoldx.2007.060136

    Derivative melting plots of MTHFR samples extracted using different methods. Samples were extracted in parallel using the MagNA Pure LC system (thick black line), QIAamp DNA blood mini kit (thin black line), and the Puregene DNA blood kit (dotted black
    Figure Legend Snippet: Derivative melting plots of MTHFR samples extracted using different methods. Samples were extracted in parallel using the MagNA Pure LC system (thick black line), QIAamp DNA blood mini kit (thin black line), and the Puregene DNA blood kit (dotted black

    Techniques Used:

    29) Product Images from "Epigenetic silencing of miR-342-3p in B cell lymphoma and its impact on autophagy"

    Article Title: Epigenetic silencing of miR-342-3p in B cell lymphoma and its impact on autophagy

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-020-00926-1

    Methylation of EVL/MIR342 in healthy normal controls and NHL cell lines. a Direct sequencing of M-MSP products from enzymatically methylated positive control DNA showed complete bisulfite conversion and MSP specificity. b , c Results of both M- and U-MSP showed absence of EVL/MIR342 methylation in healthy normal controls, including normal peripheral blood buffy coats (P1-P10) ( b ) and normal tonsil tissues (T1–T11) ( c ). d In lymphoma cell lines, M- and U-MSP showed that EVL/MIR342 was completely methylated (MM) in SU-DHL-16, partially methylated (MU) in JEKO-1, GRANTA-519, SU-DHL-6, and KARPAS-299, and completely unmethylated (UU) in MINO, REC-1, SP-53, SU-DHL-1, and SUP-T1
    Figure Legend Snippet: Methylation of EVL/MIR342 in healthy normal controls and NHL cell lines. a Direct sequencing of M-MSP products from enzymatically methylated positive control DNA showed complete bisulfite conversion and MSP specificity. b , c Results of both M- and U-MSP showed absence of EVL/MIR342 methylation in healthy normal controls, including normal peripheral blood buffy coats (P1-P10) ( b ) and normal tonsil tissues (T1–T11) ( c ). d In lymphoma cell lines, M- and U-MSP showed that EVL/MIR342 was completely methylated (MM) in SU-DHL-16, partially methylated (MU) in JEKO-1, GRANTA-519, SU-DHL-6, and KARPAS-299, and completely unmethylated (UU) in MINO, REC-1, SP-53, SU-DHL-1, and SUP-T1

    Techniques Used: Methylation, Sequencing, Positive Control

    30) Product Images from "Gene expression profiling of hematologic malignant cell lines resistant to oncolytic virus treatment"

    Article Title: Gene expression profiling of hematologic malignant cell lines resistant to oncolytic virus treatment

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13598

    Physical viral particle count by qPCR assay The number of physical DNA copies targeting E9L gene of vaccinia virus is plotted against time after viral infection on IM-9 and Ramos cell lines. The total DNA was isolated from infected cell harvest using QIAamp DNA blood mini kit. Data is shown on average of duplicate runs. JX-594:Pexa-Vec, WR: western reserve virus.
    Figure Legend Snippet: Physical viral particle count by qPCR assay The number of physical DNA copies targeting E9L gene of vaccinia virus is plotted against time after viral infection on IM-9 and Ramos cell lines. The total DNA was isolated from infected cell harvest using QIAamp DNA blood mini kit. Data is shown on average of duplicate runs. JX-594:Pexa-Vec, WR: western reserve virus.

    Techniques Used: Real-time Polymerase Chain Reaction, Infection, Isolation, Western Blot

    31) Product Images from "Follicular lymphoma-like B cells in healthy individuals: a novel intermediate step in early lymphomagenesis"

    Article Title: Follicular lymphoma-like B cells in healthy individuals: a novel intermediate step in early lymphomagenesis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20061292

    t(14;18) + B cells in the peripheral blood of HI are not naive B cells and constitute an expanding population of FL-like clones. (A) Human PBMCs from 10 consecutive blood samples were collected from anonymous healthy donors, and IgD + /CD27 − naive, IgD + /CD27 + memory, and IgD − /CD27 + switched memory B cell populations were isolated by cell sorting. Results from a representative cell-sorting experiment are shown. (B) Genomic DNA from the three purified subsets was tested for the presence of t(14;18) using the short-range BCL2/J H PCR assay (mbr20A/21A and J H coB/cointB nested primers; see Fig. 1 A ). A two-step “fluctuation” nested PCR assay was used, consisting of at least five parallel replicates performed for each sample (lanes 1–5). Results from sample #20 using 100 ng per replicate is depicted. (C) The contribution of naive (CD27 − , white circles) and memory (CD27 + , black triangles) B cells to the total t(14;18) + frequency (CD19 + , gray squares) in the healthy donors is shown. The translocation frequency in each subset was plotted for each donor ( Table II ). The 10 samples are ordered according to increasing CD19 + total B cell translocation frequencies (gray squares). (D) PCR fragments from CD27 + memory B cells were cloned and sequenced, and clonality was identified on the basis of the BCL2/J H junction, which is unique for a given translocation. The frequency of each independent BCL2/J H clone was calculated, and the contribution of each clone (vertical bars, each color represents a given clone in a given sample) was superimposed on the t(14;18) frequency from the CD27 + subset (black triangles). All positive amplicons were sequenced except one in sample #12, three in #15, and two in #24 (percentage on top of histograms corresponds to the number of sequenced amplicons on the total number of positive replicates; Table II ). For #99, which required DNA dilution for the calculation of the frequency, 100% corresponds to the sequencing of 10 out of 10 clones obtained from amplifications performed on diluted DNA (1:5; Table II ) and undiluted DNA (9:9; not depicted). (E) BCL2-Sγ (top) and BCL2/Sμ LR-PCR (bottom) were performed in the 3 IgD + /CD27 − naive, IgD + /CD27 + memory, and IgD − /CD27 + switched memory B cell subsets for four HI (#15, #20, #21, and #24). Results from sample #24 are shown. β-globin/TCR-β controls were performed as control for long-range amplifications (unpublished data). Most positive amplicons were confirmed by cloning/sequencing and alignment to germline BCL2 , Sμ, and Sγ loci (not depicted). Samples presenting multiple size bands revealed oligo/polyclonality. (F) The overall distribution of switched (gray histograms) versus unswitched (white histograms) translocated alleles in the three B cell subsets from samples #15, #20, #21, and #24 is represented. The ratio of positive replicates is indicated at the top of the bars.
    Figure Legend Snippet: t(14;18) + B cells in the peripheral blood of HI are not naive B cells and constitute an expanding population of FL-like clones. (A) Human PBMCs from 10 consecutive blood samples were collected from anonymous healthy donors, and IgD + /CD27 − naive, IgD + /CD27 + memory, and IgD − /CD27 + switched memory B cell populations were isolated by cell sorting. Results from a representative cell-sorting experiment are shown. (B) Genomic DNA from the three purified subsets was tested for the presence of t(14;18) using the short-range BCL2/J H PCR assay (mbr20A/21A and J H coB/cointB nested primers; see Fig. 1 A ). A two-step “fluctuation” nested PCR assay was used, consisting of at least five parallel replicates performed for each sample (lanes 1–5). Results from sample #20 using 100 ng per replicate is depicted. (C) The contribution of naive (CD27 − , white circles) and memory (CD27 + , black triangles) B cells to the total t(14;18) + frequency (CD19 + , gray squares) in the healthy donors is shown. The translocation frequency in each subset was plotted for each donor ( Table II ). The 10 samples are ordered according to increasing CD19 + total B cell translocation frequencies (gray squares). (D) PCR fragments from CD27 + memory B cells were cloned and sequenced, and clonality was identified on the basis of the BCL2/J H junction, which is unique for a given translocation. The frequency of each independent BCL2/J H clone was calculated, and the contribution of each clone (vertical bars, each color represents a given clone in a given sample) was superimposed on the t(14;18) frequency from the CD27 + subset (black triangles). All positive amplicons were sequenced except one in sample #12, three in #15, and two in #24 (percentage on top of histograms corresponds to the number of sequenced amplicons on the total number of positive replicates; Table II ). For #99, which required DNA dilution for the calculation of the frequency, 100% corresponds to the sequencing of 10 out of 10 clones obtained from amplifications performed on diluted DNA (1:5; Table II ) and undiluted DNA (9:9; not depicted). (E) BCL2-Sγ (top) and BCL2/Sμ LR-PCR (bottom) were performed in the 3 IgD + /CD27 − naive, IgD + /CD27 + memory, and IgD − /CD27 + switched memory B cell subsets for four HI (#15, #20, #21, and #24). Results from sample #24 are shown. β-globin/TCR-β controls were performed as control for long-range amplifications (unpublished data). Most positive amplicons were confirmed by cloning/sequencing and alignment to germline BCL2 , Sμ, and Sγ loci (not depicted). Samples presenting multiple size bands revealed oligo/polyclonality. (F) The overall distribution of switched (gray histograms) versus unswitched (white histograms) translocated alleles in the three B cell subsets from samples #15, #20, #21, and #24 is represented. The ratio of positive replicates is indicated at the top of the bars.

    Techniques Used: Isolation, FACS, Purification, Polymerase Chain Reaction, Nested PCR, Translocation Assay, Clone Assay, Sequencing

    32) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    33) Product Images from "Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR"

    Article Title: Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.11.5273-5276.2003

    Amplification of a serial dilution of the EUROHEP standard with HBV-specific primers in a nested PCR following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. (A) Chemagen extracts. (B) QIAGEN extracts. Lane M shows a 100-bp ladder, and lane C shows results for the negative control. Lanes 1 through 10 show results for different viral loads expressed in numbers of copies per ml: 1, 4 × 10 5 ; 2, 4 × 10 4 ; 3, 4 × 10 3 ; 4, 4 × 10 2 ; 5, 2 × 10 2 ; 6, 1 × 10 2 ; 7, 8 × 10 1 ; 8, 4 × 10 1 ; 9, 2 × 10 1 ; 10, 1 × 10 1 .
    Figure Legend Snippet: Amplification of a serial dilution of the EUROHEP standard with HBV-specific primers in a nested PCR following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. (A) Chemagen extracts. (B) QIAGEN extracts. Lane M shows a 100-bp ladder, and lane C shows results for the negative control. Lanes 1 through 10 show results for different viral loads expressed in numbers of copies per ml: 1, 4 × 10 5 ; 2, 4 × 10 4 ; 3, 4 × 10 3 ; 4, 4 × 10 2 ; 5, 2 × 10 2 ; 6, 1 × 10 2 ; 7, 8 × 10 1 ; 8, 4 × 10 1 ; 9, 2 × 10 1 ; 10, 1 × 10 1 .

    Techniques Used: Amplification, Serial Dilution, Nested PCR, Negative Control

    Amplification of a serial dilution of the standard plasmid pCR-gpB by real-time PCR with CMV-specific primers (amplicon, 254 bp) on the LightCycler instrument (software, version 3.5; analysis method, fit points with manual noise band adjustment, hybridization probe format) following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. pCR-gpB concentrations (from left to right) were 2 × 10 5 , 2 × 10 4 , 2 × 10 3 , and 2 × 10 2 copies per ml of serum. Continuous line, Chemagen extracts; broken line, QIAGEN extracts. Cycle number, cycle number of the amplification reaction; F2/F1, quotient of fluorescence channel F2 (hybridization probe) and fluorescence channel F1 (fluorescein).
    Figure Legend Snippet: Amplification of a serial dilution of the standard plasmid pCR-gpB by real-time PCR with CMV-specific primers (amplicon, 254 bp) on the LightCycler instrument (software, version 3.5; analysis method, fit points with manual noise band adjustment, hybridization probe format) following extraction with the Chemagic DNA/RNA kit and the QIAamp DNA Blood Mini kit. pCR-gpB concentrations (from left to right) were 2 × 10 5 , 2 × 10 4 , 2 × 10 3 , and 2 × 10 2 copies per ml of serum. Continuous line, Chemagen extracts; broken line, QIAGEN extracts. Cycle number, cycle number of the amplification reaction; F2/F1, quotient of fluorescence channel F2 (hybridization probe) and fluorescence channel F1 (fluorescein).

    Techniques Used: Amplification, Serial Dilution, Plasmid Preparation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Software, Hybridization, Fluorescence

    34) Product Images from "Mycobacterium tuberculosis co-infection is associated with increased surrogate marker of the HIV reservoir"

    Article Title: Mycobacterium tuberculosis co-infection is associated with increased surrogate marker of the HIV reservoir

    Journal: AIDS Research and Therapy

    doi: 10.1186/s12981-020-00320-0

    Correlations between the levels of on-ART HIV DNA and factors (baseline viral load, on-ART CD4 cell count and on-ART CD4/CD8 ratio). The correlation analyzed between the levels of on-ART HIV DNA and baseline viral load ( a ), on-ART CD4 cell count ( b ), and on-ART CD4/CD8 ratio ( c ) in mono-HIV infected group. Similarly, the correlation analyzed between the levels of on-ART HIV DNA and baseline viral load ( d ), on-ART CD4 cell count ( e ), and on-ART CD4/CD8 ratio ( f ) in HIV/Tb co-infected group. All the data analyzed by non-parametric test. The blue dot represents mono-HIV infected patients and the orange dot represents HIV/Tb co-infected patients
    Figure Legend Snippet: Correlations between the levels of on-ART HIV DNA and factors (baseline viral load, on-ART CD4 cell count and on-ART CD4/CD8 ratio). The correlation analyzed between the levels of on-ART HIV DNA and baseline viral load ( a ), on-ART CD4 cell count ( b ), and on-ART CD4/CD8 ratio ( c ) in mono-HIV infected group. Similarly, the correlation analyzed between the levels of on-ART HIV DNA and baseline viral load ( d ), on-ART CD4 cell count ( e ), and on-ART CD4/CD8 ratio ( f ) in HIV/Tb co-infected group. All the data analyzed by non-parametric test. The blue dot represents mono-HIV infected patients and the orange dot represents HIV/Tb co-infected patients

    Techniques Used: Cell Counting, Infection

    Correlations of plasma IDO activities with the levels of on-ART HIV DNA. The plasma IDO activities in the two groups at baseline ( a ), and after ART ( b ). The correlations between the levels of on-ART HIV DNA and the plasma IDO activities both at baseline and after ART in the mono-HIV infected group and HIV/Tb co-infected group, respectively ( c–f ). All the data analyzed by non-parametric test. The blue dot represents mono-HIV infected patients and the orange dot represents HIV/Tb co-infected patients
    Figure Legend Snippet: Correlations of plasma IDO activities with the levels of on-ART HIV DNA. The plasma IDO activities in the two groups at baseline ( a ), and after ART ( b ). The correlations between the levels of on-ART HIV DNA and the plasma IDO activities both at baseline and after ART in the mono-HIV infected group and HIV/Tb co-infected group, respectively ( c–f ). All the data analyzed by non-parametric test. The blue dot represents mono-HIV infected patients and the orange dot represents HIV/Tb co-infected patients

    Techniques Used: Infection

    Correlations of plasma IL-7 concentrations with the levels of on-ART HIV DNA. The plasma IL-7 concentrations in the two groups group at baseline ( a ), and after ART ( b ). The correlations between the levels of on-ART HIV DNA and the plasma IL-7 concentrations both at baseline and after ART in the mono-HIV infected group and HIV/Tb co-infected group, respectively ( c−f ). All the data analyzed by non-parametric test. The blue dot represents mono-HIV infected patients and the orange dot represents HIV/Tb co-infected patients
    Figure Legend Snippet: Correlations of plasma IL-7 concentrations with the levels of on-ART HIV DNA. The plasma IL-7 concentrations in the two groups group at baseline ( a ), and after ART ( b ). The correlations between the levels of on-ART HIV DNA and the plasma IL-7 concentrations both at baseline and after ART in the mono-HIV infected group and HIV/Tb co-infected group, respectively ( c−f ). All the data analyzed by non-parametric test. The blue dot represents mono-HIV infected patients and the orange dot represents HIV/Tb co-infected patients

    Techniques Used: Infection

    The levels of HIV DNA differ between mono-HIV infected patients and HIV/Tb co-infected patients. a The levels of HIV DNA in the mono-HIV infected group and HIV/Tb co-infected group at baseline. b, c Change in the level of HIV DNA in each group after more than 1year’s ART. d The levels of HIV DNA in the mono-HIV infected group and HIV/Tb co-infected group after ART. All the data analyzed by non-parametric test
    Figure Legend Snippet: The levels of HIV DNA differ between mono-HIV infected patients and HIV/Tb co-infected patients. a The levels of HIV DNA in the mono-HIV infected group and HIV/Tb co-infected group at baseline. b, c Change in the level of HIV DNA in each group after more than 1year’s ART. d The levels of HIV DNA in the mono-HIV infected group and HIV/Tb co-infected group after ART. All the data analyzed by non-parametric test

    Techniques Used: Infection

    35) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    36) Product Images from "Mitochondrial DNA Leakage Caused by Streptococcus pneumoniae Hydrogen Peroxide Promotes Type I IFN Expression in Lung Cells"

    Article Title: Mitochondrial DNA Leakage Caused by Streptococcus pneumoniae Hydrogen Peroxide Promotes Type I IFN Expression in Lung Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.00630

    mtDNA leakage caused by S. pn -secreted H 2 O 2 was involved in IFN-I induction. (A) A549 cells were infected with D39 (MOI = 200) at indicated time points, DNA in the cytosolic fraction was isolated, and the copy number of mtDNA (mtDNA sequences as primers) was measured and normalized with the copy number of nuclear DNA (nuclear DNA sequences as primers). (B) A549 cells were stimulated with D39 with or without catalase (Cat) and D39Δ spxB (MOI = 200), as well as 1 mM H 2 O 2 for 2 h, DNA in the cytosolic fraction was isolated, and the copy number of mtDNA was measured and normalized with GAPDH. (C) A549 cells were transfected with cytosolic DNA isolated from different stimulations, including D39 with or without catalase (Cat) and D39Δ spxB , as well as H 2 O 2 , IFNβ mRNA level were determined by real-time PCR. POLY (dA: dT) (2 μg/ml) was applied as positive control. (D) mtDNA in A549 cells were evaluated by real-time PCR after being treated with EtBr (300 ng/ml) for 5 days (left panel). IFNβ mRNA level in A549 cells treated with 1 mM H 2 O 2 or D39 (MOI = 200) were determined by real-time PCR (right panel). NC, negative control. All data were presented as means ± SD from three independent experiments. ∗ P
    Figure Legend Snippet: mtDNA leakage caused by S. pn -secreted H 2 O 2 was involved in IFN-I induction. (A) A549 cells were infected with D39 (MOI = 200) at indicated time points, DNA in the cytosolic fraction was isolated, and the copy number of mtDNA (mtDNA sequences as primers) was measured and normalized with the copy number of nuclear DNA (nuclear DNA sequences as primers). (B) A549 cells were stimulated with D39 with or without catalase (Cat) and D39Δ spxB (MOI = 200), as well as 1 mM H 2 O 2 for 2 h, DNA in the cytosolic fraction was isolated, and the copy number of mtDNA was measured and normalized with GAPDH. (C) A549 cells were transfected with cytosolic DNA isolated from different stimulations, including D39 with or without catalase (Cat) and D39Δ spxB , as well as H 2 O 2 , IFNβ mRNA level were determined by real-time PCR. POLY (dA: dT) (2 μg/ml) was applied as positive control. (D) mtDNA in A549 cells were evaluated by real-time PCR after being treated with EtBr (300 ng/ml) for 5 days (left panel). IFNβ mRNA level in A549 cells treated with 1 mM H 2 O 2 or D39 (MOI = 200) were determined by real-time PCR (right panel). NC, negative control. All data were presented as means ± SD from three independent experiments. ∗ P

    Techniques Used: Infection, Isolation, Transfection, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    37) Product Images from "Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications"

    Article Title: Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-239

    Internal control amplification . Mean Ct value of PhHV PCR of the 4 materials (A, B, C and D) for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
    Figure Legend Snippet: Internal control amplification . Mean Ct value of PhHV PCR of the 4 materials (A, B, C and D) for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

    Techniques Used: Amplification, Polymerase Chain Reaction, DNA Extraction

    Assessment of maximum amplicon length: PCR product yield . Mean relative yield (%) of 200, 400 and 600 bp PCR products of 4 materials (A, B, C and D). The prot. K + QIAamp extraction's yield was set at 100%. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
    Figure Legend Snippet: Assessment of maximum amplicon length: PCR product yield . Mean relative yield (%) of 200, 400 and 600 bp PCR products of 4 materials (A, B, C and D). The prot. K + QIAamp extraction's yield was set at 100%. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

    Techniques Used: Amplification, Polymerase Chain Reaction, DNA Extraction

    SNP analysis using real time PCR . A . Two representative component plots generated after proteinase K treatment followed by EasyMAG extraction from colon tissue B and real time amplification using SNP assay rs1350138. The light grey line indicates allele 1 (VIC label), the dark grey line indicates allele 2 (FAM label). B . Mean Ct value of SNP rs2043731 and rs1350138 of the 4 materials (A, B, C and D) in JBZ 4× mastermix or ABI 2× mastermix for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.
    Figure Legend Snippet: SNP analysis using real time PCR . A . Two representative component plots generated after proteinase K treatment followed by EasyMAG extraction from colon tissue B and real time amplification using SNP assay rs1350138. The light grey line indicates allele 1 (VIC label), the dark grey line indicates allele 2 (FAM label). B . Mean Ct value of SNP rs2043731 and rs1350138 of the 4 materials (A, B, C and D) in JBZ 4× mastermix or ABI 2× mastermix for the different extraction methods. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G.

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

    Assessment of maximum amplicon length: visualisation on gel . Gel image of 200-400-600 bp multiplex PCR-products of representative tissue B. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G. Neg = ultrapure water in PCR, Pos = QIAamp extracted DNA from EDTA-blood.
    Figure Legend Snippet: Assessment of maximum amplicon length: visualisation on gel . Gel image of 200-400-600 bp multiplex PCR-products of representative tissue B. The +/ and -/ indicate the use of proteinase K digestion or no digestion, respectively. The different extraction methods are indicated by: heat-treatment =/-, QIAamp DNA extraction =/Q, EasyMAG DNA extraction =/EM, Gentra DNA extraction =/G. Neg = ultrapure water in PCR, Pos = QIAamp extracted DNA from EDTA-blood.

    Techniques Used: Amplification, Multiplex Assay, Polymerase Chain Reaction, DNA Extraction

    38) Product Images from "Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome"

    Article Title: Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome

    Journal: International Journal of Medical Sciences

    doi:

    Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.
    Figure Legend Snippet: Recovery of PCR product in DNA samples isolated from serum specimens using QIAamp DNA blood midi kit, protein A and phenol-chloroform extraction method. 1 - molecular size standards; 2, 6 and 10 - PCR-positive serum from patient M; 3, 7 and 11 - PCR-positive serum from patient P; 4, 8 and 12 - PCR negative serum from patient S; 5, 9 and 13 - extraction control; 14 - negative control; 15 - positive control.

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

    39) Product Images from "Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots"

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    Journal: Biology Methods & Protocols

    doi: 10.1093/biomethods/bpaa009

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.
    Figure Legend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Techniques Used:

    40) Product Images from "Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR"

    Article Title: Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2011.07.006

    The proportion of positive EBV PCR reactions for blood samples with various EBV DNA load values. Solid symbols, QIAsymphony-extracted samples; open symbols, QIAamp-extracted samples. The proportion values were obtained from extraction of six replicates
    Figure Legend Snippet: The proportion of positive EBV PCR reactions for blood samples with various EBV DNA load values. Solid symbols, QIAsymphony-extracted samples; open symbols, QIAamp-extracted samples. The proportion values were obtained from extraction of six replicates

    Techniques Used: Polymerase Chain Reaction

    Related Articles

    DNA Extraction:

    Article Title: A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification
    Article Snippet: Equal volumes of these dilution series were further used for genomic DNA extraction by microwave irradiation and, subsequently, for standard PCR and tailored LAMP assays. .. 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. .. Next, three standard operation procedures (SOPs) were established for microwave irradiation based DNA extraction (MDA oven, model number: MW17M70G-AU, 230 V, 50 HZ).

    Irradiation:

    Article Title: A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification
    Article Snippet: Equal volumes of these dilution series were further used for genomic DNA extraction by microwave irradiation and, subsequently, for standard PCR and tailored LAMP assays. .. 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. .. Next, three standard operation procedures (SOPs) were established for microwave irradiation based DNA extraction (MDA oven, model number: MW17M70G-AU, 230 V, 50 HZ).

    Cell Culture:

    Article Title: A reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification
    Article Snippet: Equal volumes of these dilution series were further used for genomic DNA extraction by microwave irradiation and, subsequently, for standard PCR and tailored LAMP assays. .. 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. .. Next, three standard operation procedures (SOPs) were established for microwave irradiation based DNA extraction (MDA oven, model number: MW17M70G-AU, 230 V, 50 HZ).

    other:

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots
    Article Snippet: The control Chelex® protocol yielded 590% more DNA than the QIAamp® DNA Blood Mini Kit .

    Isolation:

    Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood
    Article Snippet: Two hundred microliters of Tris-EDTA (10 mM Tris-Cl and 1 mM EDTA, pH 8.0) was added to the tube containing the membrane and incubated at room temperature for 2 h with occasional finger tapping ( , Step 13) to elute the membrane-bound gDNA. .. Among the various commonly available commercial kits for gDNA isolation, the kit from Qiagen has been shown to have columns with superior binding capability., Hence, the QIAamp DNA Blood Mini Kit (Qiagen) was chosen for comparison of the efficiency of the gDNA isolation with the described method here. ..

    Article Title: Aberrant ZNF423 impedes B cell differentiation and is linked to adverse outcome of ETV6-RUNX1 negative B precursor acute lymphoblastic leukemia
    Article Snippet: .. Genomic DNA was isolated from initial ALL samples and matched MNC from remission BM using the QIAGEN DNA Blood Mini kit following the manufacturer’s instructions. .. Sample preparation, hybridization, and staining were performed according to the manufacturer’s standard protocol for Affymetrix GenomeWide Human SNP 6.0 microarrays.

    Binding Assay:

    Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood
    Article Snippet: Two hundred microliters of Tris-EDTA (10 mM Tris-Cl and 1 mM EDTA, pH 8.0) was added to the tube containing the membrane and incubated at room temperature for 2 h with occasional finger tapping ( , Step 13) to elute the membrane-bound gDNA. .. Among the various commonly available commercial kits for gDNA isolation, the kit from Qiagen has been shown to have columns with superior binding capability., Hence, the QIAamp DNA Blood Mini Kit (Qiagen) was chosen for comparison of the efficiency of the gDNA isolation with the described method here. ..

    Amplification:

    Article Title: Inflammatory Genital Infections Mitigate a Severe Genetic Bottleneck in Heterosexual Transmission of Subtype A and C HIV-1
    Article Snippet: For individuals identified as antibody negative and p24 antigen positive, the estimated date of infection was determined as 22 days prior to that date (Fiebig stage II), and finally for individuals identified as viral RNA positive and antibody/p24 antigen negative, the estimated date of infection was determined as 17 days prior to this sample date (Fiebig stage I). .. End-Point Dilution, Single Genome Amplification of HIV-1 env Genes For amplification of proviral HIV-1 env genes, genomic DNA was extracted from uncultured peripheral blood mononuclear cells (PBMC) using the Qiagen DNA Blood Mini Kit. .. For amplification of plasma HIV-1 env genes, RNA was purified from plasma samples using the Qiagen Viral Isolation Kit and cDNA prepared using the SuperScript III reverse transcriptase according to the manufacturer's instructions.

    Article Title: JAK2V617F Allele Burden Measurement in Peripheral Blood of Iranian Patients with Myeloproliferative Neoplasms and Effect of Hydroxyurea on JAK2V617F Allele Burden
    Article Snippet: The patients were selected from Hematology-Oncology and BMT Research Center at Shariati and Imam Khomeini Hospital affiliated with Tehran University of Medical Science. .. The study was approved by our institutional review board and written informed consent was obtained from all patients. (Ethical code: ir.tums.horcsct.1394.103.7) JAK2 V617F screening by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) Genomic DNA was prepared from leukocytes using the DNA blood mini kit (Qiagen, Germany). .. Mutation analysis of the JAK2 V617F was initially performed using ARMS-PCR.

    Two Tailed Test:

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots
    Article Snippet: Total gEq recovered for all conditions was calculated as the concentration of the sample by beta-globin qPCR multiplied by the final elution volume. .. We compared the mean efficiency of the Chelex® control method to the QIAamp® DNA Blood Mini Kit using a two-tailed Student’s t -test. ..

    Mutagenesis:

    Article Title: JAK2V617F Allele Burden Measurement in Peripheral Blood of Iranian Patients with Myeloproliferative Neoplasms and Effect of Hydroxyurea on JAK2V617F Allele Burden
    Article Snippet: The patients were selected from Hematology-Oncology and BMT Research Center at Shariati and Imam Khomeini Hospital affiliated with Tehran University of Medical Science. .. The study was approved by our institutional review board and written informed consent was obtained from all patients. (Ethical code: ir.tums.horcsct.1394.103.7) JAK2 V617F screening by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) Genomic DNA was prepared from leukocytes using the DNA blood mini kit (Qiagen, Germany). .. Mutation analysis of the JAK2 V617F was initially performed using ARMS-PCR.

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    Qiagen dna blood mini kit
    Transmission of multiple variants. Aligned nucleotide sequences for linked donors (green circles) and linked recipients (blue circles) were used to generate neighbor-joining trees for individual transmission pairs (A) RW57 and (B) ZM229. Horizontal branch lengths are drawn to scale with scale bar representing 1% divergence. Open circles represent sequences derived from uncultured <t>PBMC</t> <t>DNA</t> and closed circles represent sequences derived from plasma RNA. PBMC samples were unavailable for ZM229; therefore, only plasma sequences were derived for this transmission pair. Asterisks indicate branches with bootstrap values greater than 0.99.
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    Transmission of multiple variants. Aligned nucleotide sequences for linked donors (green circles) and linked recipients (blue circles) were used to generate neighbor-joining trees for individual transmission pairs (A) RW57 and (B) ZM229. Horizontal branch lengths are drawn to scale with scale bar representing 1% divergence. Open circles represent sequences derived from uncultured PBMC DNA and closed circles represent sequences derived from plasma RNA. PBMC samples were unavailable for ZM229; therefore, only plasma sequences were derived for this transmission pair. Asterisks indicate branches with bootstrap values greater than 0.99.

    Journal: PLoS Pathogens

    Article Title: Inflammatory Genital Infections Mitigate a Severe Genetic Bottleneck in Heterosexual Transmission of Subtype A and C HIV-1

    doi: 10.1371/journal.ppat.1000274

    Figure Lengend Snippet: Transmission of multiple variants. Aligned nucleotide sequences for linked donors (green circles) and linked recipients (blue circles) were used to generate neighbor-joining trees for individual transmission pairs (A) RW57 and (B) ZM229. Horizontal branch lengths are drawn to scale with scale bar representing 1% divergence. Open circles represent sequences derived from uncultured PBMC DNA and closed circles represent sequences derived from plasma RNA. PBMC samples were unavailable for ZM229; therefore, only plasma sequences were derived for this transmission pair. Asterisks indicate branches with bootstrap values greater than 0.99.

    Article Snippet: End-Point Dilution, Single Genome Amplification of HIV-1 env Genes For amplification of proviral HIV-1 env genes, genomic DNA was extracted from uncultured peripheral blood mononuclear cells (PBMC) using the Qiagen DNA Blood Mini Kit.

    Techniques: Transmission Assay, Derivative Assay

    ZNF423 expression is regulated by DNA methylation. (A) Transactivation of ZNF423 isoform-specific promoters in dependence of central CGI. Reporter gene assay using ZNF423 promoters (α and β) with or without CGI after transfection in 293T cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing indicated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (B) Methylation map of CpGs in the central CGI at the ZNF423 locus. Genomic DNA from primary ALL ( n = 58), matched BM MNCs in complete continuous remission (MNC [CCR]; n = 58), H1, HES2 ESC lines, and normal hematopoietic cells at various stages of differentiation ( n = 42) from healthy donors were sequenced after bisulfite conversion. Each row represents one cytosine in a CG dinucleotide of the analyzed sequence. For cell lineage abbreviations, refer to Figure 1 . Significance is calculated by Student’s t test, comparing primary ALL to immunologically characterized control samples (in brackets; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Color code represents degree of methylation in percent. White boxes indicate no sequencing data available. (C) ZNF423 promoter activity in dependence of CPG mutational status. Disruption of CpGs was performed by site-directed mutagenesis as indicated by red marks. Combined deletion mutant (pCpGL-CGI-del_comb_α-prom) represents deletions at CGI positions 102, 183, 220, 250. Wild-type and mutated plasmids were treated with DNA-methylase M.SssI and transfected into 293T. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing M.SssI-treated wild-type and mutated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (D) DNA methylation pattern of central CGI. Diagram was created with BiQ Analyzer software from Max-Planck-Institute of Informatics. Drawing is to scale. White lollipop, unmethylated; black lollipop, methylated. Marked CpGs refer to heatmap in Fig. 4 B . (E and F) Expression of ZNF423 transcripts upon DNA demethylation and BMP2 stimulation. SEM cells (E) and CD34 + cord blood cells (F) were treated with 3 µM 5A2D for 24, 48, and 72 h. In SEM cells, additional BMP2 treatment was performed 6 h before lysis. Relative fold induction was measured by qPCR (2 −ΔΔCt ) using a ZNF423 isoform-specific primer design in SEM cells. mRNA levels were normalized to B2M and solvent control. Error bars represent SD out of three technical replicates. Significance is calculated using 2 −ΔCt values by Student’s t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant). Data were reproduced in three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Aberrant ZNF423 impedes B cell differentiation and is linked to adverse outcome of ETV6-RUNX1 negative B precursor acute lymphoblastic leukemia

    doi: 10.1084/jem.20130497

    Figure Lengend Snippet: ZNF423 expression is regulated by DNA methylation. (A) Transactivation of ZNF423 isoform-specific promoters in dependence of central CGI. Reporter gene assay using ZNF423 promoters (α and β) with or without CGI after transfection in 293T cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing indicated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (B) Methylation map of CpGs in the central CGI at the ZNF423 locus. Genomic DNA from primary ALL ( n = 58), matched BM MNCs in complete continuous remission (MNC [CCR]; n = 58), H1, HES2 ESC lines, and normal hematopoietic cells at various stages of differentiation ( n = 42) from healthy donors were sequenced after bisulfite conversion. Each row represents one cytosine in a CG dinucleotide of the analyzed sequence. For cell lineage abbreviations, refer to Figure 1 . Significance is calculated by Student’s t test, comparing primary ALL to immunologically characterized control samples (in brackets; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Color code represents degree of methylation in percent. White boxes indicate no sequencing data available. (C) ZNF423 promoter activity in dependence of CPG mutational status. Disruption of CpGs was performed by site-directed mutagenesis as indicated by red marks. Combined deletion mutant (pCpGL-CGI-del_comb_α-prom) represents deletions at CGI positions 102, 183, 220, 250. Wild-type and mutated plasmids were treated with DNA-methylase M.SssI and transfected into 293T. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD from three technical replicates. Significance is calculated by Student’s t test, comparing M.SssI-treated wild-type and mutated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (D) DNA methylation pattern of central CGI. Diagram was created with BiQ Analyzer software from Max-Planck-Institute of Informatics. Drawing is to scale. White lollipop, unmethylated; black lollipop, methylated. Marked CpGs refer to heatmap in Fig. 4 B . (E and F) Expression of ZNF423 transcripts upon DNA demethylation and BMP2 stimulation. SEM cells (E) and CD34 + cord blood cells (F) were treated with 3 µM 5A2D for 24, 48, and 72 h. In SEM cells, additional BMP2 treatment was performed 6 h before lysis. Relative fold induction was measured by qPCR (2 −ΔΔCt ) using a ZNF423 isoform-specific primer design in SEM cells. mRNA levels were normalized to B2M and solvent control. Error bars represent SD out of three technical replicates. Significance is calculated using 2 −ΔCt values by Student’s t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant). Data were reproduced in three independent experiments.

    Article Snippet: Genomic DNA was isolated from initial ALL samples and matched MNC from remission BM using the QIAGEN DNA Blood Mini kit following the manufacturer’s instructions.

    Techniques: Expressing, DNA Methylation Assay, Reporter Gene Assay, Transfection, Luciferase, Activity Assay, Methylation, Sequencing, Mutagenesis, Software, Lysis, Real-time Polymerase Chain Reaction

    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

    Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Journal: Biology Methods & Protocols

    Article Title: Optimization of Chelex 100 resin-based extraction of genomic DNA from dried blood spots

    doi: 10.1093/biomethods/bpaa009

    Figure Lengend Snippet: Control Chelex ® 100 resin-based protocol versus QIAamp DNA Blood Mini Kit for the extraction of gDNA from DBS. QIAamp DNA Blood Mini Kit was performed with a first (Ex 1, El 1) and second elution (Ex 1, El 2). Following this, the DBS was processed through the kit a second time (Ex 2, El 1 and Ex 2, El 2). The total Qiagen yield was calculated by adding together the DNA yield from both extractions and both elutions. Replicates of five were conducted for each condition in a single experiment. Data are represented as mean efficiency ± standard error.

    Article Snippet: The control Chelex® protocol yielded 590% more DNA than the QIAamp® DNA Blood Mini Kit .

    Techniques: