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  • 99
    Thermo Fisher red blood cell lysis buffer
    Deletion of Vhlh in liver leukocytes. a By immunohistochemistry, GFP is prominent in the collecting ducts in kidney of the reporter strain HOXB7-GFP-Cre; Vhlh fl/fl [ 25 , 29 ]. b , c GFP is also detectable in single cells interspersed within the liver parenchyma ( b1 ) or within hemangiomas ( c2 , 3 ). d Isolation of leukocytes from livers of HOXB7-Cre; Vhlh fl/fl mice. Liver <t>cell</t> suspensions were prepared by mincing livers, digesting with collagenase, and lysing <t>red</t> <t>blood</t> cells with red blood cell <t>lysis</t> <t>buffer.</t> Liver cell suspensions were then stained with the pan-leukocyte marker CD45 and propidium iodide (live/dead stain). Live CD45+ cells were isolated with FACS; debris and doublets were gated out. After the sort, CD45+ fractions were > 90 % pure. e By PCR, Vhlh inactivation was detected in CD45+ leukocyte fraction of 5 independent samples (1–5). Samples 3–5 were additionally examined for the presence of floxed alleles (the amount of DNA samples 1 and 2 were insufficient for additional PCR tests). As control, PCR was performed without template (no template control) or with genomic DNA from a HOXB7-Cre; Vhlh fl/fl knockout kidney (positive control, which contains both Vhlh -inactivated and wild-type cells)
    Red Blood Cell Lysis Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1835 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore red blood cell lysis buffer
    PDK1 knockout in the haematopoietic system blocks the development of mature T and B cells. PDK1 fl/fl /Vav-Cre +ve mice were found to have an increased spleen size ( A ) and weight ( B ) relative to PDK1 +/+ /Vav-Cre +ve control mice. Post <t>lysis</t> of <t>red</t> <t>blood</t> cells however spleen <t>cell</t> numbers were similar between the PDK1 fl/fl /Vav-Cre +ve mice ( n =15) and PDK1 +/+ /Vav-Cre +ve ( n =18), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =6) control genotypes ( C ). Error bars represent standard deviation. H E staining of the spleens showed that PDK1 knockout resulted in a disruption of the white pulp ( D ). Analysis of T- and B-cell populations in the spleen by FACS ( E , F ) demonstrated that PDK1 fl/fl /Vav-Cre +ve mice ( n =7) had negligible numbers of mature T and B cells relative to PDK1 +/+ /Vav-Cre +ve ( n =6), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =4) control genotypes. Error bars represent the standard deviation of 4–7 mice per genotype. In ( B , C and E ) a P -value (Student's t -test) of
    Red Blood Cell Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen dneasy blood and tissue kit
    Agarose gel electrophoresis of purified enterococcal <t>DNA.</t> (A) Purified genomic DNA of E. faecium NCTC 12202. Lane 2, cells not exposed to metal surfaces; lane 3, cells exposed to stainless steel for 2 h; lane 4, cells exposed to copper for 2 h; lane 5, cells exposed to copper for 4 h. (B) Purified genomic DNA of clinical isolates E. faecium 5 (lanes 7 and 8) and E. faecalis 2 (lanes 9 and 10) exposed to stainless steel (lanes 7 and 9) or copper (lanes 8 and 10) for 2 h at 22°C. (C) Purified plasmid DNA of E. faecium NCTC 12202 not exposed to metal (lane 11) or exposed to stainless steel (lane 12) or copper (lane 13) for 2 h at 22°C. Control lanes are Bioline Hyperladder I (lanes 1) and Hyperladder II (lanes 6). Genomic DNA was purified using the Qiagen <t>DNeasy</t> Blood and Tissue kit (2% agarose) and the Qiaprep Spin Miniprep kit for plasmid DNA (0.9% agarose).
    Dneasy Blood And Tissue Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 54227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiaamp dna blood mini kit
    PCR of different HLA targets using gDNA prepared using the PES membrane or <t>QIAamp</t> 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 <t>DNA</t> 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).
    Qiaamp Dna Blood Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 23148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson sheep blood
    PCR of different HLA targets using gDNA prepared using the PES membrane or <t>QIAamp</t> 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 <t>DNA</t> 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).
    Sheep Blood, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 3472 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher sheep blood
    PCR of different HLA targets using gDNA prepared using the PES membrane or <t>QIAamp</t> 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 <t>DNA</t> 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).
    Sheep Blood, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2673 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen puregene blood kit
    PCR of different HLA targets using gDNA prepared using the PES membrane or <t>QIAamp</t> 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 <t>DNA</t> 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).
    Puregene Blood Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiaamp dna blood midi kit
    Comparison of PCR products obtained with <t>DNA</t> purified blood by each extraction method with primers of CRYGD gene. Electrophoresed on 1.5% agarose. A: modified salting‐out method, B: modified <t>QIAamp</t> ® DNA Blood Midi Kit, C: QIAamp ® DNA Blood Midi Kit. Lane 10:50‐bp DNA ladder (Fermentas); Lane 11: negative control
    Qiaamp Dna Blood Midi Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen paxgene blood rna kit
    Humoral responses induced by iFMDV, Ad5-FMDV, and Ad5-FMDV+ISA206VG vaccine in sheep. a Experimental design: Three groups of 10 sheep each were immunized with iFDMV (intramuscular route), or Ad5-FMDV with or without ISA206VG (half intramuscular and half subcutaneous route). Blood was collected on <t>PAXgene®</t> Blood <t>RNA</t> tubes at T0H, T4H, and T24H and processed to RNA-seq. Sera were collected at T0H and every 2 weeks for 365 days. b The mean percent inhibition values provided by the PrioCHECK ELISA in each group at each time point are plotted over time (365 days), and the standard errors of the mean are shown above the mean (10 sheep per group). Differences between the areas under the curve over time of the three groups are statistically significant ( p
    Paxgene Blood Rna Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiaamp rna blood mini kit
    Humoral responses induced by iFMDV, Ad5-FMDV, and Ad5-FMDV+ISA206VG vaccine in sheep. a Experimental design: Three groups of 10 sheep each were immunized with iFDMV (intramuscular route), or Ad5-FMDV with or without ISA206VG (half intramuscular and half subcutaneous route). Blood was collected on <t>PAXgene®</t> Blood <t>RNA</t> tubes at T0H, T4H, and T24H and processed to RNA-seq. Sera were collected at T0H and every 2 weeks for 365 days. b The mean percent inhibition values provided by the PrioCHECK ELISA in each group at each time point are plotted over time (365 days), and the standard errors of the mean are shown above the mean (10 sheep per group). Differences between the areas under the curve over time of the three groups are statistically significant ( p
    Qiaamp Rna Blood Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2674 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen blood and tissue kit
    Humoral responses induced by iFMDV, Ad5-FMDV, and Ad5-FMDV+ISA206VG vaccine in sheep. a Experimental design: Three groups of 10 sheep each were immunized with iFDMV (intramuscular route), or Ad5-FMDV with or without ISA206VG (half intramuscular and half subcutaneous route). Blood was collected on <t>PAXgene®</t> Blood <t>RNA</t> tubes at T0H, T4H, and T24H and processed to RNA-seq. Sera were collected at T0H and every 2 weeks for 365 days. b The mean percent inhibition values provided by the PrioCHECK ELISA in each group at each time point are plotted over time (365 days), and the standard errors of the mean are shown above the mean (10 sheep per group). Differences between the areas under the curve over time of the three groups are statistically significant ( p
    Blood And Tissue Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2058 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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.
    Dna Blood Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1954 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    tiangen biotech co tianamp blood dna kit
    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 <t>DNA</t> isolation kit; Kit 2 is the QIAamp DNA Blood Mini Kit; Kit 3 is the QIAamp UCP Pathogen Mini Kit; Kit 4 is the <t>TIANamp</t> 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.
    Tianamp Blood Dna Kit, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 1905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen qiaamp dna blood maxi kit
    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 <t>DNA</t> isolation kit; Kit 2 is the QIAamp DNA Blood Mini Kit; Kit 3 is the QIAamp UCP Pathogen Mini Kit; Kit 4 is the <t>TIANamp</t> 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.
    Qiaamp Dna Blood Maxi Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1625 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PreAnalytiX paxgene blood rna tubes
    Sample collection, processing and <t>RNA</t> extraction: Whole blood was collected in triplicate followed by peripheral blood mononuclear cells (PBMCs) separation in a subset of samples. The following sample collection tubes were used for the study: RNAgard ® , <t>PAXgene</t> ® RNA, EDTA, ACD-A, and CPT tubes. The PBMC separation was done using standard procedures for CPT tubes, and magnetic bead, LeukoLOCK ™ and LSM methods. The samples were then stored at either 4°C (Cold) or -80°C (Frozen) and shipped overnight (o/n) for follow-up RNA extraction. Next, they were treated with one or more of several different RNA extraction procedures: Biomaxi Precip Buffer/ PAXgene ® Blood miRNA, PAXgene ® Blood miRNA, TRIzol ® LS, ACK Lysing Buffer/ Qiagen miRNeasy, and TRIzol ® Reagent manufacturer’s protocol.
    Paxgene Blood Rna Tubes, supplied by PreAnalytiX, used in various techniques. Bioz Stars score: 93/100, based on 1783 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher columbia blood agar
    Sample collection, processing and <t>RNA</t> extraction: Whole blood was collected in triplicate followed by peripheral blood mononuclear cells (PBMCs) separation in a subset of samples. The following sample collection tubes were used for the study: RNAgard ® , <t>PAXgene</t> ® RNA, EDTA, ACD-A, and CPT tubes. The PBMC separation was done using standard procedures for CPT tubes, and magnetic bead, LeukoLOCK ™ and LSM methods. The samples were then stored at either 4°C (Cold) or -80°C (Frozen) and shipped overnight (o/n) for follow-up RNA extraction. Next, they were treated with one or more of several different RNA extraction procedures: Biomaxi Precip Buffer/ PAXgene ® Blood miRNA, PAXgene ® Blood miRNA, TRIzol ® LS, ACK Lysing Buffer/ Qiagen miRNeasy, and TRIzol ® Reagent manufacturer’s protocol.
    Columbia Blood Agar, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sheep blood agar
    Sample collection, processing and <t>RNA</t> extraction: Whole blood was collected in triplicate followed by peripheral blood mononuclear cells (PBMCs) separation in a subset of samples. The following sample collection tubes were used for the study: RNAgard ® , <t>PAXgene</t> ® RNA, EDTA, ACD-A, and CPT tubes. The PBMC separation was done using standard procedures for CPT tubes, and magnetic bead, LeukoLOCK ™ and LSM methods. The samples were then stored at either 4°C (Cold) or -80°C (Frozen) and shipped overnight (o/n) for follow-up RNA extraction. Next, they were treated with one or more of several different RNA extraction procedures: Biomaxi Precip Buffer/ PAXgene ® Blood miRNA, PAXgene ® Blood miRNA, TRIzol ® LS, ACK Lysing Buffer/ Qiagen miRNeasy, and TRIzol ® Reagent manufacturer’s protocol.
    Sheep Blood Agar, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1081 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Deletion of Vhlh in liver leukocytes. a By immunohistochemistry, GFP is prominent in the collecting ducts in kidney of the reporter strain HOXB7-GFP-Cre; Vhlh fl/fl [ 25 , 29 ]. b , c GFP is also detectable in single cells interspersed within the liver parenchyma ( b1 ) or within hemangiomas ( c2 , 3 ). d Isolation of leukocytes from livers of HOXB7-Cre; Vhlh fl/fl mice. Liver cell suspensions were prepared by mincing livers, digesting with collagenase, and lysing red blood cells with red blood cell lysis buffer. Liver cell suspensions were then stained with the pan-leukocyte marker CD45 and propidium iodide (live/dead stain). Live CD45+ cells were isolated with FACS; debris and doublets were gated out. After the sort, CD45+ fractions were > 90 % pure. e By PCR, Vhlh inactivation was detected in CD45+ leukocyte fraction of 5 independent samples (1–5). Samples 3–5 were additionally examined for the presence of floxed alleles (the amount of DNA samples 1 and 2 were insufficient for additional PCR tests). As control, PCR was performed without template (no template control) or with genomic DNA from a HOXB7-Cre; Vhlh fl/fl knockout kidney (positive control, which contains both Vhlh -inactivated and wild-type cells)

    Journal: BMC Cancer

    Article Title: Inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) in granulocytes contributes to development of liver hemangiomas in a mouse model

    doi: 10.1186/s12885-016-2802-3

    Figure Lengend Snippet: Deletion of Vhlh in liver leukocytes. a By immunohistochemistry, GFP is prominent in the collecting ducts in kidney of the reporter strain HOXB7-GFP-Cre; Vhlh fl/fl [ 25 , 29 ]. b , c GFP is also detectable in single cells interspersed within the liver parenchyma ( b1 ) or within hemangiomas ( c2 , 3 ). d Isolation of leukocytes from livers of HOXB7-Cre; Vhlh fl/fl mice. Liver cell suspensions were prepared by mincing livers, digesting with collagenase, and lysing red blood cells with red blood cell lysis buffer. Liver cell suspensions were then stained with the pan-leukocyte marker CD45 and propidium iodide (live/dead stain). Live CD45+ cells were isolated with FACS; debris and doublets were gated out. After the sort, CD45+ fractions were > 90 % pure. e By PCR, Vhlh inactivation was detected in CD45+ leukocyte fraction of 5 independent samples (1–5). Samples 3–5 were additionally examined for the presence of floxed alleles (the amount of DNA samples 1 and 2 were insufficient for additional PCR tests). As control, PCR was performed without template (no template control) or with genomic DNA from a HOXB7-Cre; Vhlh fl/fl knockout kidney (positive control, which contains both Vhlh -inactivated and wild-type cells)

    Article Snippet: Red blood cell lysis buffer, Fc-block and antibodies for flow cytometry (except anti-CD45 antibody) were obtained from eBioscience (San Diego, CA, USA).

    Techniques: Immunohistochemistry, Isolation, Mouse Assay, Lysis, Staining, Marker, FACS, Polymerase Chain Reaction, Knock-Out, Positive Control

    Extramedullary erythropoiesis in Vhlh knockout mice. a Erythrocyte progenitors are increased by ~10-fold in livers of HOXB7-Cre; Vhlh fl/fl knockout mice. Liver cell suspensions were prepared by mincing livers, followed by collagenase digestion and treatment with red blood cell lysis buffer (see Methods ). Flow cytometric quantification of erythrocyte progenitors (TER119 + CD71+) was performed. Shown are representative FACS-plots ( left panels ) and quantification ( right panel ). Gate was set on live CD45+ cells, and doublets were gated out. b Quantification of erythroid progenitors with colony forming unit assay. Liver cell suspensions from HOXB7-Cre; Vhlh fl/fl (Vhlh -/- ), HOXB7-Cre; Vhlh fl/fl ; Hif2α fl/+ (Vhlh -/- ; Hif2α +/- ), HOXB7-Cre; Vhlh fl/fl ; Hif2α fl/fl (Vhlh -/- ; Hif2α -/- ), and Vhlh fl/fl (WT) mice were prepared and cultured as described in Methods . Left panel: Representative image of erythroid progenitor colonies (BFU-E) from a Vhlh knockout mouse. Right panel: Quantification of BFU-Es. While Vhlh knockout increased the number of BFU-E compared to that obtained from liver cell suspensions of Cre negative mice (WT), no difference was seen in the number of BFU-Es from WT and HOXB7-Cre Vhlh-Hif-2α double knockouts. One Vhlh -/- and Hif-2α single allele knockout animal of the genotype HOXB7-Cre; Vhlh fl/fl ; Hif-2α fl/+ (Vhlh -/- ; Hif2α +/- ) was used as a reference, which showed increased BFU-E number as in the Vhlh knockout. Mice were > 3 months old. p-values were calculated using Student’s t -test with Welch’s correction. *: statistically significant ( p

    Journal: BMC Cancer

    Article Title: Inactivation of the tumor suppressor gene von Hippel-Lindau (VHL) in granulocytes contributes to development of liver hemangiomas in a mouse model

    doi: 10.1186/s12885-016-2802-3

    Figure Lengend Snippet: Extramedullary erythropoiesis in Vhlh knockout mice. a Erythrocyte progenitors are increased by ~10-fold in livers of HOXB7-Cre; Vhlh fl/fl knockout mice. Liver cell suspensions were prepared by mincing livers, followed by collagenase digestion and treatment with red blood cell lysis buffer (see Methods ). Flow cytometric quantification of erythrocyte progenitors (TER119 + CD71+) was performed. Shown are representative FACS-plots ( left panels ) and quantification ( right panel ). Gate was set on live CD45+ cells, and doublets were gated out. b Quantification of erythroid progenitors with colony forming unit assay. Liver cell suspensions from HOXB7-Cre; Vhlh fl/fl (Vhlh -/- ), HOXB7-Cre; Vhlh fl/fl ; Hif2α fl/+ (Vhlh -/- ; Hif2α +/- ), HOXB7-Cre; Vhlh fl/fl ; Hif2α fl/fl (Vhlh -/- ; Hif2α -/- ), and Vhlh fl/fl (WT) mice were prepared and cultured as described in Methods . Left panel: Representative image of erythroid progenitor colonies (BFU-E) from a Vhlh knockout mouse. Right panel: Quantification of BFU-Es. While Vhlh knockout increased the number of BFU-E compared to that obtained from liver cell suspensions of Cre negative mice (WT), no difference was seen in the number of BFU-Es from WT and HOXB7-Cre Vhlh-Hif-2α double knockouts. One Vhlh -/- and Hif-2α single allele knockout animal of the genotype HOXB7-Cre; Vhlh fl/fl ; Hif-2α fl/+ (Vhlh -/- ; Hif2α +/- ) was used as a reference, which showed increased BFU-E number as in the Vhlh knockout. Mice were > 3 months old. p-values were calculated using Student’s t -test with Welch’s correction. *: statistically significant ( p

    Article Snippet: Red blood cell lysis buffer, Fc-block and antibodies for flow cytometry (except anti-CD45 antibody) were obtained from eBioscience (San Diego, CA, USA).

    Techniques: Knock-Out, Mouse Assay, Lysis, Flow Cytometry, FACS, Colony-forming Unit Assay, Cell Culture

    PDK1 knockout in the haematopoietic system blocks the development of mature T and B cells. PDK1 fl/fl /Vav-Cre +ve mice were found to have an increased spleen size ( A ) and weight ( B ) relative to PDK1 +/+ /Vav-Cre +ve control mice. Post lysis of red blood cells however spleen cell numbers were similar between the PDK1 fl/fl /Vav-Cre +ve mice ( n =15) and PDK1 +/+ /Vav-Cre +ve ( n =18), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =6) control genotypes ( C ). Error bars represent standard deviation. H E staining of the spleens showed that PDK1 knockout resulted in a disruption of the white pulp ( D ). Analysis of T- and B-cell populations in the spleen by FACS ( E , F ) demonstrated that PDK1 fl/fl /Vav-Cre +ve mice ( n =7) had negligible numbers of mature T and B cells relative to PDK1 +/+ /Vav-Cre +ve ( n =6), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =4) control genotypes. Error bars represent the standard deviation of 4–7 mice per genotype. In ( B , C and E ) a P -value (Student's t -test) of

    Journal: The EMBO Journal

    Article Title: PDK1 regulates VDJ recombination, cell-cycle exit and survival during B-cell development

    doi: 10.1038/emboj.2013.40

    Figure Lengend Snippet: PDK1 knockout in the haematopoietic system blocks the development of mature T and B cells. PDK1 fl/fl /Vav-Cre +ve mice were found to have an increased spleen size ( A ) and weight ( B ) relative to PDK1 +/+ /Vav-Cre +ve control mice. Post lysis of red blood cells however spleen cell numbers were similar between the PDK1 fl/fl /Vav-Cre +ve mice ( n =15) and PDK1 +/+ /Vav-Cre +ve ( n =18), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =6) control genotypes ( C ). Error bars represent standard deviation. H E staining of the spleens showed that PDK1 knockout resulted in a disruption of the white pulp ( D ). Analysis of T- and B-cell populations in the spleen by FACS ( E , F ) demonstrated that PDK1 fl/fl /Vav-Cre +ve mice ( n =7) had negligible numbers of mature T and B cells relative to PDK1 +/+ /Vav-Cre +ve ( n =6), PDK1 +/+ /Vav-Cre −ve ( n =6) and PDK1 fl/fl /Vav-Cre −ve ( n =4) control genotypes. Error bars represent the standard deviation of 4–7 mice per genotype. In ( B , C and E ) a P -value (Student's t -test) of

    Article Snippet: Isolation of B-cell progenitors and cell culture Single cell bone marrow suspensions were treated with red blood cell lysis buffer (Sigma) and then Gr1, CD11b, Ter119 and CD3-positive cells were depleted using MACS columns.

    Techniques: Knock-Out, Mouse Assay, Lysis, Standard Deviation, Staining, FACS

    Agarose gel electrophoresis of purified enterococcal DNA. (A) Purified genomic DNA of E. faecium NCTC 12202. Lane 2, cells not exposed to metal surfaces; lane 3, cells exposed to stainless steel for 2 h; lane 4, cells exposed to copper for 2 h; lane 5, cells exposed to copper for 4 h. (B) Purified genomic DNA of clinical isolates E. faecium 5 (lanes 7 and 8) and E. faecalis 2 (lanes 9 and 10) exposed to stainless steel (lanes 7 and 9) or copper (lanes 8 and 10) for 2 h at 22°C. (C) Purified plasmid DNA of E. faecium NCTC 12202 not exposed to metal (lane 11) or exposed to stainless steel (lane 12) or copper (lane 13) for 2 h at 22°C. Control lanes are Bioline Hyperladder I (lanes 1) and Hyperladder II (lanes 6). Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit (2% agarose) and the Qiaprep Spin Miniprep kit for plasmid DNA (0.9% agarose).

    Journal: Applied and Environmental Microbiology

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿

    doi: 10.1128/AEM.03050-09

    Figure Lengend Snippet: Agarose gel electrophoresis of purified enterococcal DNA. (A) Purified genomic DNA of E. faecium NCTC 12202. Lane 2, cells not exposed to metal surfaces; lane 3, cells exposed to stainless steel for 2 h; lane 4, cells exposed to copper for 2 h; lane 5, cells exposed to copper for 4 h. (B) Purified genomic DNA of clinical isolates E. faecium 5 (lanes 7 and 8) and E. faecalis 2 (lanes 9 and 10) exposed to stainless steel (lanes 7 and 9) or copper (lanes 8 and 10) for 2 h at 22°C. (C) Purified plasmid DNA of E. faecium NCTC 12202 not exposed to metal (lane 11) or exposed to stainless steel (lane 12) or copper (lane 13) for 2 h at 22°C. Control lanes are Bioline Hyperladder I (lanes 1) and Hyperladder II (lanes 6). Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit (2% agarose) and the Qiaprep Spin Miniprep kit for plasmid DNA (0.9% agarose).

    Article Snippet: Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit, which cuts the bacterial genome into approximately 50-kb fragments before separation by agarose gel electrophoresis.

    Techniques: Agarose Gel Electrophoresis, Purification, Plasmid Preparation

    Agarose gel electrophoresis of DNA isolated from cadaver tissues using the DNeasy Blood Tissue Kit. MW = Quick-load 1 kb DNA Ladder (New England BioLabs). Lane 1: Liver without RNAse treatment. Lane 2: Heart without RNAse treatment. Lane 3: Liver with RNAse treatment. Lane 4: Heart with RNAse treatment. Lane 5: Skeletal Muscle with RNAse treatment. Lane 6: Skin with RNAse treatment

    Journal: BMC Medical Genomics

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum

    doi: 10.1186/s12920-016-0223-4

    Figure Lengend Snippet: Agarose gel electrophoresis of DNA isolated from cadaver tissues using the DNeasy Blood Tissue Kit. MW = Quick-load 1 kb DNA Ladder (New England BioLabs). Lane 1: Liver without RNAse treatment. Lane 2: Heart without RNAse treatment. Lane 3: Liver with RNAse treatment. Lane 4: Heart with RNAse treatment. Lane 5: Skeletal Muscle with RNAse treatment. Lane 6: Skin with RNAse treatment

    Article Snippet: Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.).

    Techniques: Agarose Gel Electrophoresis, Isolation

    Agarose gel electrophoresis of DNA isolated from cadaver tissues. MW = GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Lane 1: Heart DNA extracted with DNeasy Blood Tissue Kit. Lane 2: Liver DNA extracted with DNeasy Blood Tissue Kit. Lane 3: Heart DNA extracted with FFPE kit. Lane 4: Liver DNA extracted with FFPE kit

    Journal: BMC Medical Genomics

    Article Title: The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum

    doi: 10.1186/s12920-016-0223-4

    Figure Lengend Snippet: Agarose gel electrophoresis of DNA isolated from cadaver tissues. MW = GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Lane 1: Heart DNA extracted with DNeasy Blood Tissue Kit. Lane 2: Liver DNA extracted with DNeasy Blood Tissue Kit. Lane 3: Heart DNA extracted with FFPE kit. Lane 4: Liver DNA extracted with FFPE kit

    Article Snippet: Samples of tissue (1 cm3 ) were finely minced using a scalpel blade and then subjected to DNA isolation using either the QIAamp DNA FFPE Tissue Kit or the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc.).

    Techniques: Agarose Gel Electrophoresis, Isolation, Formalin-fixed Paraffin-Embedded

    DNeasy treated samples. (a) Two LightCycler PCR runs, first one with standard dilution series of human Hep2 DNA before (flat negative curves) and after DNeasy (indicated with squares and triangles), showed on the left and second with DNA free water before (marked with squares) and after DNeasy (triangles) on the right. (b) Melting curve analysis of DNeasy treated H 2 O (triangles), Hep2 DNA (squares) and clinical sample.

    Journal: BMC Microbiology

    Article Title: Development of real-time PCR for detection of Mycoplasma hominis

    doi: 10.1186/1471-2180-4-35

    Figure Lengend Snippet: DNeasy treated samples. (a) Two LightCycler PCR runs, first one with standard dilution series of human Hep2 DNA before (flat negative curves) and after DNeasy (indicated with squares and triangles), showed on the left and second with DNA free water before (marked with squares) and after DNeasy (triangles) on the right. (b) Melting curve analysis of DNeasy treated H 2 O (triangles), Hep2 DNA (squares) and clinical sample.

    Article Snippet: Twenty-five μl of each dilution and DNA free double distilled water were then treated with DNeasy™ Tissue Kit (QIAGEN GmbH, Hilden, Germany) procedure.

    Techniques: Polymerase Chain Reaction

    Procoagulant activities of DNA, polyP, and silica particles. (A-D) Shown are plasma clot times versus concentration, with horizontal dashed lines showing the mean clot time without activator (± standard error of the mean as horizontal dotted lines). (A) Clot times with: HEK 293 cell DNA isolated with DNeasy Blood Tissue (open diamond); HEK 293 cell DNA isolated with phenol/chloroform (solid diamond); NET-derived DNA isolated with DNeasy Blood Tissue kit (open inverted triangle); or NET-derived DNA isolated with phenol/chloroform (solid inverted triangle). Each data set represents the mean clot time for 3 separate purifications as detailed in supplemental Figure 2A (HEK293 cell DNA) and supplemental Figure 2B (NET DNA). (B) Clot times with silica particles: glass milk (solid triangle) or homogenized DNeasy column matrix (open triangle). (C) Clot times with water elutions from 3 different lots of DNeasy columns (open triangle) or 2 different lots of Econospin columns (open circle). On the x-axis, fold concentration refers to the concentration relative to the volume eluted from the Qiagen column, with 1 equaling the original elution volume. (D) Clot times of λ phage DNA before (blue square) or after (open square) repurification on DNeasy columns; or of short-chain polyP before (blue circle) or after (open circle) repurification on DNeasy columns. The purification data sets represent mean clot times for 2 different purifications as detailed in supplemental Figure 4. (E) Clot times of various samples before (red bars) or after (blue bars) digestion with a combination of Benzonase and calf intestine alkaline phosphatase. Samples include buffer control (no activator), 20 µg/mL HEK 293 cell DNA purified using DNeasy, 20 µg/mL NET DNA purified using DNeasy, water applied to DNeasy column as described in panel C (water elution), or 2 µg/mL long-chain polyP. Digestion of DNA by Benzonase and polyP by phosphatase was confirmed by gel electrophoresis (supplemental Figure 1). (F) Clot times of various samples before (red bars) or after (blue bars) acid hydrolysis. Samples include buffer control (no activator), 20 µg/mL HEK 293 cell DNA purified using DNeasy, 20 µg/mL NET DNA purified using DNeasy, water applied to DNeasy column (water elution), 2 µg/mL long-chain polyP, or 5 µg/mL polyguanylate (polyG). (G) Clot times in the presence of varying concentrations of histones with (open triangle) or without (solid triangle) a 10-fold concentrated water elution (prepared as described for panel C). (H) Tissue factor–triggered clot times in the presence of varying concentrations of histones.

    Journal: Blood

    Article Title: Silica particles contribute to the procoagulant activity of DNA and polyphosphate isolated using commercial kits

    doi: 10.1182/blood-2017-03-772848

    Figure Lengend Snippet: Procoagulant activities of DNA, polyP, and silica particles. (A-D) Shown are plasma clot times versus concentration, with horizontal dashed lines showing the mean clot time without activator (± standard error of the mean as horizontal dotted lines). (A) Clot times with: HEK 293 cell DNA isolated with DNeasy Blood Tissue (open diamond); HEK 293 cell DNA isolated with phenol/chloroform (solid diamond); NET-derived DNA isolated with DNeasy Blood Tissue kit (open inverted triangle); or NET-derived DNA isolated with phenol/chloroform (solid inverted triangle). Each data set represents the mean clot time for 3 separate purifications as detailed in supplemental Figure 2A (HEK293 cell DNA) and supplemental Figure 2B (NET DNA). (B) Clot times with silica particles: glass milk (solid triangle) or homogenized DNeasy column matrix (open triangle). (C) Clot times with water elutions from 3 different lots of DNeasy columns (open triangle) or 2 different lots of Econospin columns (open circle). On the x-axis, fold concentration refers to the concentration relative to the volume eluted from the Qiagen column, with 1 equaling the original elution volume. (D) Clot times of λ phage DNA before (blue square) or after (open square) repurification on DNeasy columns; or of short-chain polyP before (blue circle) or after (open circle) repurification on DNeasy columns. The purification data sets represent mean clot times for 2 different purifications as detailed in supplemental Figure 4. (E) Clot times of various samples before (red bars) or after (blue bars) digestion with a combination of Benzonase and calf intestine alkaline phosphatase. Samples include buffer control (no activator), 20 µg/mL HEK 293 cell DNA purified using DNeasy, 20 µg/mL NET DNA purified using DNeasy, water applied to DNeasy column as described in panel C (water elution), or 2 µg/mL long-chain polyP. Digestion of DNA by Benzonase and polyP by phosphatase was confirmed by gel electrophoresis (supplemental Figure 1). (F) Clot times of various samples before (red bars) or after (blue bars) acid hydrolysis. Samples include buffer control (no activator), 20 µg/mL HEK 293 cell DNA purified using DNeasy, 20 µg/mL NET DNA purified using DNeasy, water applied to DNeasy column (water elution), 2 µg/mL long-chain polyP, or 5 µg/mL polyguanylate (polyG). (G) Clot times in the presence of varying concentrations of histones with (open triangle) or without (solid triangle) a 10-fold concentrated water elution (prepared as described for panel C). (H) Tissue factor–triggered clot times in the presence of varying concentrations of histones.

    Article Snippet: Briefly, DNA was purified from HEK 293 cells or human NETs by using DNeasy Blood and Tissue kits (Qiagen) or phenol/chloroform extraction.

    Techniques: Concentration Assay, Isolation, Derivative Assay, Purification, Nucleic Acid Electrophoresis

    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).

    Journal: Journal of Biomolecular Techniques : JBT

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

    doi: 10.7171/jbt.18-2902-001

    Figure Lengend 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).

    Article Snippet: 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.

    Techniques: 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.

    Journal: Journal of Biomolecular Techniques : JBT

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

    doi: 10.7171/jbt.18-2902-001

    Figure Lengend 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.

    Article Snippet: 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.

    Techniques: 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).

    Journal: Journal of Biomolecular Techniques : JBT

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

    doi: 10.7171/jbt.18-2902-001

    Figure Lengend 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).

    Article Snippet: 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.

    Techniques: Agarose Gel Electrophoresis, Marker

    Detection limit of the TaqMan assay for Y. enterocolitica in blood samples. Different amounts of Y. enterocolitica cells were spiked into blood, and the total DNA was isolated as described in Materials and Methods. (A) Bacteria were spiked into 200 μl of blood, and DNA was isolated by the QIAamp blood kit in a final volume of 100 μl. The corresponding numbers of cells spiked are as follows: plot 1, none (unseeded blood); plot 2, 30 cells/ml (1.2 cell equivalent/50 μl of PCR mixture); plot 3, 60 cells/ml (2.4 cell equivalent/50 μl of PCR mixture); plot 4, 300 cells/ml; plot 5, 3,000 cells/ml; and plot 6, 30,000 cells/ml. The gel inset shows the products obtained, and lanes 1 to 6 correspond to plots 1 to 6. Lane 7 is a no-template control. (B) Bacteria were spiked into 100 μl of blood, and DNA was extracted by the Dynal DNA Direct kit in a final volume of 75 μl. The corresponding cell numbers are as follows: plot 1, none (unseeded blood); plot 2, 30 cells/ml (1.2 cell equivalent/PCR mixture); plot 3, 300 CFU/ml (12 cell equivalents/PCR mixture); plot 4, 3,000 cells; and plot 5, 30,000 cells. The gel inset represents the products obtained, and lanes 1 to 5 correspond to plots 1 to 5. M is the 50-bp molecular size DNA ladder, and the arrow indicates the 201-bp product.

    Journal: Journal of Clinical Microbiology

    Article Title: Rapid Identification of Yersinia enterocolitica in Blood by the 5? Nuclease PCR Assay

    doi:

    Figure Lengend Snippet: Detection limit of the TaqMan assay for Y. enterocolitica in blood samples. Different amounts of Y. enterocolitica cells were spiked into blood, and the total DNA was isolated as described in Materials and Methods. (A) Bacteria were spiked into 200 μl of blood, and DNA was isolated by the QIAamp blood kit in a final volume of 100 μl. The corresponding numbers of cells spiked are as follows: plot 1, none (unseeded blood); plot 2, 30 cells/ml (1.2 cell equivalent/50 μl of PCR mixture); plot 3, 60 cells/ml (2.4 cell equivalent/50 μl of PCR mixture); plot 4, 300 cells/ml; plot 5, 3,000 cells/ml; and plot 6, 30,000 cells/ml. The gel inset shows the products obtained, and lanes 1 to 6 correspond to plots 1 to 6. Lane 7 is a no-template control. (B) Bacteria were spiked into 100 μl of blood, and DNA was extracted by the Dynal DNA Direct kit in a final volume of 75 μl. The corresponding cell numbers are as follows: plot 1, none (unseeded blood); plot 2, 30 cells/ml (1.2 cell equivalent/PCR mixture); plot 3, 300 CFU/ml (12 cell equivalents/PCR mixture); plot 4, 3,000 cells; and plot 5, 30,000 cells. The gel inset represents the products obtained, and lanes 1 to 5 correspond to plots 1 to 5. M is the 50-bp molecular size DNA ladder, and the arrow indicates the 201-bp product.

    Article Snippet: Chromosomal DNA was prepared from spiked blood by the QIAamp blood kit (Qiagen Corp., Santa Clarita, Calif.) or the Dynabeads DNA Direct system II kit (Dynal, Oslo, Norway).

    Techniques: TaqMan Assay, Isolation, Polymerase Chain Reaction

    Concentrations of phage 933W evaluated by measurement of numbers of stx 2 GC/μl from phage DNA extracted using conventional phage DNA extraction (Phage Ext) methods and commercial DNA extraction kits (QIAamp DNA Blood minikit, QIAamp DNA Stool

    Journal: Applied and Environmental Microbiology

    Article Title: Improving Detection of Shiga Toxin-Producing Escherichia coli by Molecular Methods by Reducing the Interference of Free Shiga Toxin-Encoding Bacteriophages

    doi: 10.1128/AEM.02941-14

    Figure Lengend Snippet: Concentrations of phage 933W evaluated by measurement of numbers of stx 2 GC/μl from phage DNA extracted using conventional phage DNA extraction (Phage Ext) methods and commercial DNA extraction kits (QIAamp DNA Blood minikit, QIAamp DNA Stool

    Article Snippet: In addition, phage 933W DNA was extracted using three commercial DNA extraction methods applied for bacterial or total DNA extraction, according to the manufacturers' instructions, as follows: QIAamp DNA Blood minikit (Qiagen GmbH, Hilden, Germany), QIAamp DNA Stool minikit (Qiagen), and NucliSENS miniMAG (bioMérieux España, Madrid, Spain).

    Techniques: DNA Extraction

    Comparison of PCR products obtained with DNA purified blood by each extraction method with primers of CRYGD gene. Electrophoresed on 1.5% agarose. A: modified salting‐out method, B: modified QIAamp ® DNA Blood Midi Kit, C: QIAamp ® DNA Blood Midi Kit. Lane 10:50‐bp DNA ladder (Fermentas); Lane 11: negative control

    Journal: Journal of Clinical Laboratory Analysis

    Article Title: A method for improving the efficiency of DNA extraction from clotted blood samples, et al. A method for improving the efficiency of DNA extraction from clotted blood samples

    doi: 10.1002/jcla.22892

    Figure Lengend Snippet: Comparison of PCR products obtained with DNA purified blood by each extraction method with primers of CRYGD gene. Electrophoresed on 1.5% agarose. A: modified salting‐out method, B: modified QIAamp ® DNA Blood Midi Kit, C: QIAamp ® DNA Blood Midi Kit. Lane 10:50‐bp DNA ladder (Fermentas); Lane 11: negative control

    Article Snippet: The quality of the DNA was fairly uniform in both modified salting‐out and modified QIAamp® DNA Blood Midi Kit methods.

    Techniques: Polymerase Chain Reaction, Purification, Modification, Salting Out, Negative Control

    Comparison of genomic DNA purified from clotted blood by each extraction method on 2% agarose gel. Stained with DNA Green Viewer™. A: modified salting‐out method, B: modified QIAamp ® DNA Blood Midi Kit, C: QIAamp ® DNA Blood Midi Kit. Lane 11:50‐bp DNA ladder (Fermentas)

    Journal: Journal of Clinical Laboratory Analysis

    Article Title: A method for improving the efficiency of DNA extraction from clotted blood samples, et al. A method for improving the efficiency of DNA extraction from clotted blood samples

    doi: 10.1002/jcla.22892

    Figure Lengend Snippet: Comparison of genomic DNA purified from clotted blood by each extraction method on 2% agarose gel. Stained with DNA Green Viewer™. A: modified salting‐out method, B: modified QIAamp ® DNA Blood Midi Kit, C: QIAamp ® DNA Blood Midi Kit. Lane 11:50‐bp DNA ladder (Fermentas)

    Article Snippet: The quality of the DNA was fairly uniform in both modified salting‐out and modified QIAamp® DNA Blood Midi Kit methods.

    Techniques: Purification, Agarose Gel Electrophoresis, Staining, Modification, Salting Out

    Humoral responses induced by iFMDV, Ad5-FMDV, and Ad5-FMDV+ISA206VG vaccine in sheep. a Experimental design: Three groups of 10 sheep each were immunized with iFDMV (intramuscular route), or Ad5-FMDV with or without ISA206VG (half intramuscular and half subcutaneous route). Blood was collected on PAXgene® Blood RNA tubes at T0H, T4H, and T24H and processed to RNA-seq. Sera were collected at T0H and every 2 weeks for 365 days. b The mean percent inhibition values provided by the PrioCHECK ELISA in each group at each time point are plotted over time (365 days), and the standard errors of the mean are shown above the mean (10 sheep per group). Differences between the areas under the curve over time of the three groups are statistically significant ( p

    Journal: NPJ Vaccines

    Article Title: The antibody response induced FMDV vaccines in sheep correlates with early transcriptomic responses in blood

    doi: 10.1038/s41541-019-0151-3

    Figure Lengend Snippet: Humoral responses induced by iFMDV, Ad5-FMDV, and Ad5-FMDV+ISA206VG vaccine in sheep. a Experimental design: Three groups of 10 sheep each were immunized with iFDMV (intramuscular route), or Ad5-FMDV with or without ISA206VG (half intramuscular and half subcutaneous route). Blood was collected on PAXgene® Blood RNA tubes at T0H, T4H, and T24H and processed to RNA-seq. Sera were collected at T0H and every 2 weeks for 365 days. b The mean percent inhibition values provided by the PrioCHECK ELISA in each group at each time point are plotted over time (365 days), and the standard errors of the mean are shown above the mean (10 sheep per group). Differences between the areas under the curve over time of the three groups are statistically significant ( p

    Article Snippet: RNA extraction, quality check, and sequencing RNA was extracted from the PAXgene® Blood RNA tubes using the PAXgene® Blood RNA kit and purified with the RNeasy MinElute Cleanup kit (Qiagen).

    Techniques: RNA Sequencing Assay, Inhibition, Enzyme-linked Immunosorbent Assay

    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

    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.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-31200-3

    Figure Lengend 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.

    Article Snippet: These kits included the BiOstic bacteremia DNA isolation kit (MOBIO, Carlsbad, CA, USA), QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), QIAamp UCP Pathogen Mini Kit (Qiagen, Hilden, Germany), TIANamp Blood DNA Kit (Tiangen, Beijing, China) and QIAamp cador Pathogen Mini Kit (Qiagen, Hilden, Germany).

    Techniques: DNA Extraction

    Sample collection, processing and RNA extraction: Whole blood was collected in triplicate followed by peripheral blood mononuclear cells (PBMCs) separation in a subset of samples. The following sample collection tubes were used for the study: RNAgard ® , PAXgene ® RNA, EDTA, ACD-A, and CPT tubes. The PBMC separation was done using standard procedures for CPT tubes, and magnetic bead, LeukoLOCK ™ and LSM methods. The samples were then stored at either 4°C (Cold) or -80°C (Frozen) and shipped overnight (o/n) for follow-up RNA extraction. Next, they were treated with one or more of several different RNA extraction procedures: Biomaxi Precip Buffer/ PAXgene ® Blood miRNA, PAXgene ® Blood miRNA, TRIzol ® LS, ACK Lysing Buffer/ Qiagen miRNeasy, and TRIzol ® Reagent manufacturer’s protocol.

    Journal: PLoS ONE

    Article Title: Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods

    doi: 10.1371/journal.pone.0225137

    Figure Lengend Snippet: Sample collection, processing and RNA extraction: Whole blood was collected in triplicate followed by peripheral blood mononuclear cells (PBMCs) separation in a subset of samples. The following sample collection tubes were used for the study: RNAgard ® , PAXgene ® RNA, EDTA, ACD-A, and CPT tubes. The PBMC separation was done using standard procedures for CPT tubes, and magnetic bead, LeukoLOCK ™ and LSM methods. The samples were then stored at either 4°C (Cold) or -80°C (Frozen) and shipped overnight (o/n) for follow-up RNA extraction. Next, they were treated with one or more of several different RNA extraction procedures: Biomaxi Precip Buffer/ PAXgene ® Blood miRNA, PAXgene ® Blood miRNA, TRIzol ® LS, ACK Lysing Buffer/ Qiagen miRNeasy, and TRIzol ® Reagent manufacturer’s protocol.

    Article Snippet: PAXgene® blood RNA tubes: PAXgene® blood miRNA kit Whole blood collected in PAXgene® Blood RNA tubes was stored at -80°C for 1 to 2 days after being received, and then was thawed and incubated overnight at room temperature to ensure complete lysis of blood cells and maximize the mRNA yield.

    Techniques: RNA Extraction, Cycling Probe Technology

    Two dimensional Sammon projection using the top 50% of most variable transcripts (25370) for whole blood (red) and PBMC (black). There is a clear distinction in clusters for PBMCs and whole blood sample processing as marked by a black tilted line. All whole blood samples are shown on left side and PBMC samples are on the right side of the plot. The (F) and (C) denotes the frozen and cold conditions for the sample collection and storage. The description of all the legends is given here. PBMCs samples: ACD_Magnetic - blood collected in ACD-A tubes and separated using magnetic method followed by extraction using TRIzol ® method. CPT —blood collected in CPT tubes and PBMCs separation using manufacturer recommendations followed by extraction using TRIzol ® method. EDTA_LSM - blood collected in EDTA tube and PBMC separation using LSM followed by extraction using TRIzol ® method. EDTA_Leukolock - blood collected in EDTA tube, PBMCs separated using LeukoLOCK ™ method followed by extraction using TRIzol ® method. Whole blood samples: ACD - blood collected in ACD-A tube followed by RNA extraction using TRIzol ® LS method. ACD_RBC lysis- blood collected in ACD-A tube followed by RBC lysis using ACK buffer and RNA extraction using miRNAeasy extraction kit. EDTA_RBC lysis -blood collected in EDTA tube followed by RBC lysis using ACK buffer and RNA extraction using miRNAeasy extraction kit. EDTA - blood collected in EDTA tube followed by RNA extraction using TRIzol ® LS method. PAXgene - blood collected in PAXgene ® tube followed by RNA extraction using PAXgene ® blood miRNA kit. RNAgard— blood collected in RNAgard ® tube followed by RNA extraction using Biomaxi ™ Precip Buffer and PAXgene ® Blood miRNA kit.

    Journal: PLoS ONE

    Article Title: Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods

    doi: 10.1371/journal.pone.0225137

    Figure Lengend Snippet: Two dimensional Sammon projection using the top 50% of most variable transcripts (25370) for whole blood (red) and PBMC (black). There is a clear distinction in clusters for PBMCs and whole blood sample processing as marked by a black tilted line. All whole blood samples are shown on left side and PBMC samples are on the right side of the plot. The (F) and (C) denotes the frozen and cold conditions for the sample collection and storage. The description of all the legends is given here. PBMCs samples: ACD_Magnetic - blood collected in ACD-A tubes and separated using magnetic method followed by extraction using TRIzol ® method. CPT —blood collected in CPT tubes and PBMCs separation using manufacturer recommendations followed by extraction using TRIzol ® method. EDTA_LSM - blood collected in EDTA tube and PBMC separation using LSM followed by extraction using TRIzol ® method. EDTA_Leukolock - blood collected in EDTA tube, PBMCs separated using LeukoLOCK ™ method followed by extraction using TRIzol ® method. Whole blood samples: ACD - blood collected in ACD-A tube followed by RNA extraction using TRIzol ® LS method. ACD_RBC lysis- blood collected in ACD-A tube followed by RBC lysis using ACK buffer and RNA extraction using miRNAeasy extraction kit. EDTA_RBC lysis -blood collected in EDTA tube followed by RBC lysis using ACK buffer and RNA extraction using miRNAeasy extraction kit. EDTA - blood collected in EDTA tube followed by RNA extraction using TRIzol ® LS method. PAXgene - blood collected in PAXgene ® tube followed by RNA extraction using PAXgene ® blood miRNA kit. RNAgard— blood collected in RNAgard ® tube followed by RNA extraction using Biomaxi ™ Precip Buffer and PAXgene ® Blood miRNA kit.

    Article Snippet: PAXgene® blood RNA tubes: PAXgene® blood miRNA kit Whole blood collected in PAXgene® Blood RNA tubes was stored at -80°C for 1 to 2 days after being received, and then was thawed and incubated overnight at room temperature to ensure complete lysis of blood cells and maximize the mRNA yield.

    Techniques: Cycling Probe Technology, RNA Extraction, Lysis