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  • 99
    Thermo Fisher pcr buffer
    Pcr Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 28996 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore redtaq readymix pcr reaction mix
    Redtaq Readymix Pcr Reaction Mix, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Bio-Rad pcr reactions
    Expression of E1 during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad <t>QX200</t> droplet digital <t>PCR</t> system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: E1A ≤ 0.001; E1B-19k
    Pcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 15744 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad pcr reaction
    Agarose gel electrophoresis of <t>DNA</t> amplified from various genomic DNA extracts of Korean native honey (KNH) and European honey (EH) in duplex <t>PCR</t> with the KNH-specific primers, C6-F and C5-R, and the EH-specific primers, M2-F and M2-R.
    Pcr Reaction, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 6953 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad qrt pcr reactions
    ROS content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots inoculated with Rhizobium tropici . Measurements were done at initial time (0 h) and 3, 6, 12, 24 and 48 h after inoculation with R. tropici. (A) Histological (fluorescence) detection of ROS accumulation in inoculated root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by <t>qRT-PCR,</t> of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in inoculated roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.
    Qrt Pcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 3672 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eppendorf AG pcr reactions
    Retinoic acid-related orphan receptor gamma t-independent intestinal graft-versus-host disease (GvHD) is controlled by basic leucine zipper transcription factor ATF-like (BATF). (A) Transcript levels of Batf in colonic tissue of allo-HSCT BALB/c mice with severe signs of intestinal GvHD following transplantation of allogeneic CD3 + T cells derived from of Batf −/− and <t>Rorc</t> −/− mice were measured by quantitative real-time <t>PCR</t> (qPCR) at day 15 ( Batf −/− n = 12; Rorc −/− n = 2) and day 30 ( Batf −/− n = 12; Rorc −/− n = 4). Data represent pooled data from two independent experiments. Gene expression levels detected in colonic tissues of no T cells (noT) controls at day 15 were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level detected within noT controls. (B) Transcript levels of Rorc in colonic tissue of irradiated BALB/c mice transplanted with T cell depleted bone marrow alone (noT, n = 11) or followed by injection of allogeneic CD3 + T cells of wild-type (WT) ( n = 12) mice were measured by qPCR at day 30. (C) Transcript levels of RORC in colonic tissue biopsies from allo-HCT patients were measured by qPCR. Samples were grouped into indicated categories based on the absence (−GvHD; n = 30) or presence (+GvHD; n = 22) of GvHD-associated histopathological lesions or by the absence (−apoptosis; n = 32) or presence (+apoptosis; n = 20) of GvHD-related epithelial cell apoptosis. Data are shown as normalized relative RORC expression levels calculated from a standard curve. (D–G) Experimental GvHD induction was achieved as described in Figures 1 and 2 . To this end, allogeneic hematopoietic stem cell transplantation BALB/c mice were transplanted with allogeneic CD3 + T cells derived from WT ( n = 4), Rorc −/− ( n = 3), or Rorc −/− Batf −/− ( n = 3) mice or received no T cells (noT) as a control ( n = 4). (D) Mice were assessed three times a week for signs and severity of systemic GvHD. Data were analyzed by two-way ANOVA testing followed by Bonferroni’s multiple comparisons posttest. **** p
    Pcr Reactions, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 94/100, based on 3494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr reaction
    Retinoic acid-related orphan receptor gamma t-independent intestinal graft-versus-host disease (GvHD) is controlled by basic leucine zipper transcription factor ATF-like (BATF). (A) Transcript levels of Batf in colonic tissue of allo-HSCT BALB/c mice with severe signs of intestinal GvHD following transplantation of allogeneic CD3 + T cells derived from of Batf −/− and <t>Rorc</t> −/− mice were measured by quantitative real-time <t>PCR</t> (qPCR) at day 15 ( Batf −/− n = 12; Rorc −/− n = 2) and day 30 ( Batf −/− n = 12; Rorc −/− n = 4). Data represent pooled data from two independent experiments. Gene expression levels detected in colonic tissues of no T cells (noT) controls at day 15 were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level detected within noT controls. (B) Transcript levels of Rorc in colonic tissue of irradiated BALB/c mice transplanted with T cell depleted bone marrow alone (noT, n = 11) or followed by injection of allogeneic CD3 + T cells of wild-type (WT) ( n = 12) mice were measured by qPCR at day 30. (C) Transcript levels of RORC in colonic tissue biopsies from allo-HCT patients were measured by qPCR. Samples were grouped into indicated categories based on the absence (−GvHD; n = 30) or presence (+GvHD; n = 22) of GvHD-associated histopathological lesions or by the absence (−apoptosis; n = 32) or presence (+apoptosis; n = 20) of GvHD-related epithelial cell apoptosis. Data are shown as normalized relative RORC expression levels calculated from a standard curve. (D–G) Experimental GvHD induction was achieved as described in Figures 1 and 2 . To this end, allogeneic hematopoietic stem cell transplantation BALB/c mice were transplanted with allogeneic CD3 + T cells derived from WT ( n = 4), Rorc −/− ( n = 3), or Rorc −/− Batf −/− ( n = 3) mice or received no T cells (noT) as a control ( n = 4). (D) Mice were assessed three times a week for signs and severity of systemic GvHD. Data were analyzed by two-way ANOVA testing followed by Bonferroni’s multiple comparisons posttest. **** p
    Pcr Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Stratagene pcr reactions
    Knockdown of MLH1 or <t>MLH3</t> significantly reduces GAA•TTC expansion rate in human cells. Four independent FRDA model lines were transduced with shRNA knockdown lentiviral pools targeting the indicated MutL subunits and an empty vector control virus (pLKO). ( A ) Representative gel image of <t>PCR</t> products measuring GAA•TTC expansion. T 0 is the day of transduction. PCR product sizes reflect 3X(number of repeats) plus 669 bp of flanking sequence. Lane M is the 1 Kb Plus DNA ladder showing 2, 1.6 and 1 Kb bands. ( B ) Mean repeats gained over four weeks. Knockdown of MLH1 and MLH3 reduced expansion compared to the empty vector control virus (pLKO). MLH1sh was significantly different ( P = 0.0009) as was MLH3sh ( P = 0.00045) whereas PMS2sh did not reach significance ( P = 0.053). Error bars show SEM.
    Pcr Reactions, supplied by Stratagene, used in various techniques. Bioz Stars score: 94/100, based on 3387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MJ Research pcr reactions
    Agarose gel (1%) of <t>PCR</t> products for analysis of SXT element and class 1 integron showing amplification with (A) primer pair SXT-F/SXT-R for detection of SXT integrase; (B) primer pair L2/L3 for detection of 5′ CS of class 1 integron; (C) primer pair qacEΔ1-F/Sul1-B for amplification of 3′ CS of class 1 integron; (D) primer pair In-F/In-B for amplification of variable regions associated with class 1 integrons. All the <t>DNA</t> samples used as templates are indicated on top of each lane. V. cholerae MO10 and V. fluvialis BD146 were used as controls for various PCR assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.
    Pcr Reactions, supplied by MJ Research, used in various techniques. Bioz Stars score: 93/100, based on 2430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eppendorf AG pcr reaction
    Agarose gel (1%) of <t>PCR</t> products for analysis of SXT element and class 1 integron showing amplification with (A) primer pair SXT-F/SXT-R for detection of SXT integrase; (B) primer pair L2/L3 for detection of 5′ CS of class 1 integron; (C) primer pair qacEΔ1-F/Sul1-B for amplification of 3′ CS of class 1 integron; (D) primer pair In-F/In-B for amplification of variable regions associated with class 1 integrons. All the <t>DNA</t> samples used as templates are indicated on top of each lane. V. cholerae MO10 and V. fluvialis BD146 were used as controls for various PCR assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.
    Pcr Reaction, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 94/100, based on 1566 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phire plant direct pcr kit without sampling tools
    Agarose gel (1%) of <t>PCR</t> products for analysis of SXT element and class 1 integron showing amplification with (A) primer pair SXT-F/SXT-R for detection of SXT integrase; (B) primer pair L2/L3 for detection of 5′ CS of class 1 integron; (C) primer pair qacEΔ1-F/Sul1-B for amplification of 3′ CS of class 1 integron; (D) primer pair In-F/In-B for amplification of variable regions associated with class 1 integrons. All the <t>DNA</t> samples used as templates are indicated on top of each lane. V. cholerae MO10 and V. fluvialis BD146 were used as controls for various PCR assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.
    Phire Plant Direct Pcr Kit Without Sampling Tools, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Stratagene pcr reaction
    TSC22D4 deficiency counteracts diabetic hyperglycaemia and insulin resistance. ( a ) Glucose tolerance test in control or TSC22D4 shRNA adenovirus–injected db/db mice 1 week after injection. Glucose was injected i.p. at a concentration of 1 g glucose kg −1 body weight (means±s.e.m., n ≥6). ( b ) Serum glucose levels in same mice as in a after 16 h fasting and refeeding. ( c ) Serum insulin levels in same mice as in a . ( d ) Serum C-Peptide levels in same mice as in a after 16 h fasting and refeeding. ( e ) Representative western blot of liver extracts from control (NC shRNA) or TSC22D4 (TSC shRNA) shRNA adenovirus–injected db/db mice; same mice as in a using indicated antibodies. Blot representative of 6 similar samples. ( f ) Glucose tolerance test in control or TSC22D4 shRNA adenovirus–injected NZO mice 1 week after injection. Glucose was i.p. injected at a concentration of 1 g glucose kg −1 body weight (means±s.e.m., n ≥6). ( g ) Serum glucose levels in same mice as in f after 16 h fasting and refeeding. ( h ) HOMA-IR index in control or TSC22D4 miRNA AAV–injected (at 5 weeks of age) db/db mice 4 weeks after miRNA injection (means±s.e.m., n ≥6). ( i ) Serum insulin levels in same mice as in h 10 weeks after miRNA injection. ( j ) Glucose tolerance test in the same mice as in h 4 weeks after miRNA injection. Glucose was injected i.p at a concentration of 1 g glucose kg −1 body weight. ( k ) Weekly serum glucose quantification of same mice as in h . ( l – n ) Quantitative <t>PCR</t> analysis of phosphoenolpyruvate carboxykinase ( Pepck1 ), peroxisome proliferator-activated receptor gamma coactivator 1-alpha ( Pgc-1alpha ), glucose-6 phosphatase ( G6Pase ), carnitine palmitoyltransferase I ( Cpt1 ), acyl-CoA synthetase long-chain family member 1 ( Acsl1 ), peroxisome proliferator-activated receptor alpha ( PPARalpha ), acetyl-CoA carboxylase alpha ( Acaca ), fatty acid synthase ( Fasn ), stearoyl-coA desaturase-1 ( Scd1 ), sterol regulatory element-binding protein 1 ( Srebp1 ) in livers of same mice as in h 10 weeks after miRNA injection. Error bars in l − n represent standard deviation (s.d.). Statistical analysis: Student's t -test, * P ≤0.05; ** P ≤0.01; *** P ≤0.001.
    Pcr Reaction, supplied by Stratagene, used in various techniques. Bioz Stars score: 94/100, based on 1480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad quantitative pcr reactions
    TSC22D4 deficiency counteracts diabetic hyperglycaemia and insulin resistance. ( a ) Glucose tolerance test in control or TSC22D4 shRNA adenovirus–injected db/db mice 1 week after injection. Glucose was injected i.p. at a concentration of 1 g glucose kg −1 body weight (means±s.e.m., n ≥6). ( b ) Serum glucose levels in same mice as in a after 16 h fasting and refeeding. ( c ) Serum insulin levels in same mice as in a . ( d ) Serum C-Peptide levels in same mice as in a after 16 h fasting and refeeding. ( e ) Representative western blot of liver extracts from control (NC shRNA) or TSC22D4 (TSC shRNA) shRNA adenovirus–injected db/db mice; same mice as in a using indicated antibodies. Blot representative of 6 similar samples. ( f ) Glucose tolerance test in control or TSC22D4 shRNA adenovirus–injected NZO mice 1 week after injection. Glucose was i.p. injected at a concentration of 1 g glucose kg −1 body weight (means±s.e.m., n ≥6). ( g ) Serum glucose levels in same mice as in f after 16 h fasting and refeeding. ( h ) HOMA-IR index in control or TSC22D4 miRNA AAV–injected (at 5 weeks of age) db/db mice 4 weeks after miRNA injection (means±s.e.m., n ≥6). ( i ) Serum insulin levels in same mice as in h 10 weeks after miRNA injection. ( j ) Glucose tolerance test in the same mice as in h 4 weeks after miRNA injection. Glucose was injected i.p at a concentration of 1 g glucose kg −1 body weight. ( k ) Weekly serum glucose quantification of same mice as in h . ( l – n ) Quantitative <t>PCR</t> analysis of phosphoenolpyruvate carboxykinase ( Pepck1 ), peroxisome proliferator-activated receptor gamma coactivator 1-alpha ( Pgc-1alpha ), glucose-6 phosphatase ( G6Pase ), carnitine palmitoyltransferase I ( Cpt1 ), acyl-CoA synthetase long-chain family member 1 ( Acsl1 ), peroxisome proliferator-activated receptor alpha ( PPARalpha ), acetyl-CoA carboxylase alpha ( Acaca ), fatty acid synthase ( Fasn ), stearoyl-coA desaturase-1 ( Scd1 ), sterol regulatory element-binding protein 1 ( Srebp1 ) in livers of same mice as in h 10 weeks after miRNA injection. Error bars in l − n represent standard deviation (s.d.). Statistical analysis: Student's t -test, * P ≤0.05; ** P ≤0.01; *** P ≤0.001.
    Quantitative Pcr Reactions, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 1141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Meridian Life Science pcr reaction
    Linear correlation between percent T509 allele in Blumeria graminis f. sp. hordei genomic <t>DNA</t> samples quantified by digital <t>PCR</t> and the percentage of the triazole resistant isolate Per (T509) in a genomic DNA mixture of Per and the triazole sensitive isolate Wagga (S509). Each point represents the average of triplicate chips.
    Pcr Reaction, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 92/100, based on 1350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr reactions
    Linear correlation between percent T509 allele in Blumeria graminis f. sp. hordei genomic <t>DNA</t> samples quantified by digital <t>PCR</t> and the percentage of the triazole resistant isolate Per (T509) in a genomic DNA mixture of Per and the triazole sensitive isolate Wagga (S509). Each point represents the average of triplicate chips.
    Pcr Reactions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc pcr reaction
    Overview of the Long-16S method. (A) 16S rRNA gene template molecules are tagged with unique tags via two single rounds of annealing and extension with tagged forward and reverse primers containing random tags (see Fig. 2 ), that also contain <t>Illumina</t> adapter sequences. After removal of tagged primers, tagged templates are amplified via <t>PCR</t> using primers complementary to the adapter sequences. Libraries from one or more samples can then be pooled and sequenced on the MiSeq. Blue triangles and yellow stars indicate random tags of 10 nt. (B) Full-length 16S rRNA gene amplicon Illumina libraries are tagmented using the standard Nextera method, and two pools of products are amplified which contain either the left end of the tagged amplicons and an internal region, or the right end of the amplicon and an internal region. This procedure adds Nextera adapters for sequencing at the internal end of the fragments. (C) Both full-length and tagmented libraries are paired end sequenced, and the unique molecular tags are used to computationally group sequences from the same progenitor 16S rRNA gene molecule for assembly of near full-length sequences.
    Pcr Reaction, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 926 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies pcr reactions
    <t>qRT-PCR</t> analysis of <t>JAK2</t> and LMO2 expression. ( a ) The mean mRNA values of JAK2 and LMO2 in T-LBLs harboring JAK2 genetic alterations were normalized to those of a pool of fetal thymuses. Data represent three independent replicates. Error bars represent the s.d.; * P
    Pcr Reactions, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 2105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad qrt pcr reaction
    In vitro alcohol exposure of human monocytes does not inhibit TLR3-TRIF cytokine/chemokine expression Adherence isolated human monocytes from healthy donors were treated with 25 μg/ml polyI:C or 25 mM alcohol (EtOH) for 2 hours or pre-exposed to 25 mM alcohol (EtOH) for 1, 5 or 24 hours followed by polyI:C stimulation for 2 hours. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of polyI:C stimulation were analyzed by <t>qRT-PCR.</t> Fold change in expression of genes was calculated with respect to untreated. (D) Secreted RANTES protein after overnight (18–22 hours) polyI:C stimulation was analyzed by ELISA. Data summarize mean ± SD of 6 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p
    Qrt Pcr Reaction, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 848 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of E1 during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: E1A ≤ 0.001; E1B-19k

    Journal: PLoS ONE

    Article Title: Temporal dynamics of adenovirus 5 gene expression in normal human cells

    doi: 10.1371/journal.pone.0211192

    Figure Lengend Snippet: Expression of E1 during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: E1A ≤ 0.001; E1B-19k

    Article Snippet: PCR reactions were carried out using EvaGreen for QX200 master mix according to manufacturer’s directions using a QX200 digital droplet PCR instrument (BioRad).

    Techniques: Expressing, Infection, Digital PCR, Real-time Polymerase Chain Reaction, Standard Deviation

    Expression of E2 , E3 , and E4 during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at an MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: DBP

    Journal: PLoS ONE

    Article Title: Temporal dynamics of adenovirus 5 gene expression in normal human cells

    doi: 10.1371/journal.pone.0211192

    Figure Lengend Snippet: Expression of E2 , E3 , and E4 during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at an MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: DBP

    Article Snippet: PCR reactions were carried out using EvaGreen for QX200 master mix according to manufacturer’s directions using a QX200 digital droplet PCR instrument (BioRad).

    Techniques: Expressing, Infection, Digital PCR, Real-time Polymerase Chain Reaction, Standard Deviation

    Expression of VAII RNA during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the p ≤ 0.0027. All time-points between 17 and 36 hours were statistically significant.

    Journal: PLoS ONE

    Article Title: Temporal dynamics of adenovirus 5 gene expression in normal human cells

    doi: 10.1371/journal.pone.0211192

    Figure Lengend Snippet: Expression of VAII RNA during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the p ≤ 0.0027. All time-points between 17 and 36 hours were statistically significant.

    Article Snippet: PCR reactions were carried out using EvaGreen for QX200 master mix according to manufacturer’s directions using a QX200 digital droplet PCR instrument (BioRad).

    Techniques: Expressing, Infection, Digital PCR, Real-time Polymerase Chain Reaction, Standard Deviation

    Analysis of viral genome copy number in the first 48 hours of infection with HAdV5. (A) Arrested IMR-90 cells were infected with dl 309 at a MOI of 50. Viral DNA was extracted at the indicated time points, and absolute quantification of viral genomes using E4orf3 primers was measured using the BioRad QX200 droplet digital PCR system. Viral genomes are plotted on per cell basis. Error bars represent standard deviation of biological replicates, n = 2. Bar with asterisk represents changes above mock level that are statistically significant with p ≤ 0.0001. (B) Sample droplet distribution from one of the biological replicates for 29 and 30 hour time points from (A).

    Journal: PLoS ONE

    Article Title: Temporal dynamics of adenovirus 5 gene expression in normal human cells

    doi: 10.1371/journal.pone.0211192

    Figure Lengend Snippet: Analysis of viral genome copy number in the first 48 hours of infection with HAdV5. (A) Arrested IMR-90 cells were infected with dl 309 at a MOI of 50. Viral DNA was extracted at the indicated time points, and absolute quantification of viral genomes using E4orf3 primers was measured using the BioRad QX200 droplet digital PCR system. Viral genomes are plotted on per cell basis. Error bars represent standard deviation of biological replicates, n = 2. Bar with asterisk represents changes above mock level that are statistically significant with p ≤ 0.0001. (B) Sample droplet distribution from one of the biological replicates for 29 and 30 hour time points from (A).

    Article Snippet: PCR reactions were carried out using EvaGreen for QX200 master mix according to manufacturer’s directions using a QX200 digital droplet PCR instrument (BioRad).

    Techniques: Infection, Digital PCR, Standard Deviation

    Expression of viral late genes during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: 52k EP ≤ 0.001; pIIIa ≤ 0.0006; Penton base ≤ 0.0001; Hexon ≤ 0.0002; 100k HAP ≤ 0.0007; Fiber ≤ 0.0004. All time-points between 17 and 36 hours were statistically significant.

    Journal: PLoS ONE

    Article Title: Temporal dynamics of adenovirus 5 gene expression in normal human cells

    doi: 10.1371/journal.pone.0211192

    Figure Lengend Snippet: Expression of viral late genes during the first 36 hours after infection with HAdV5. Arrested IMR-90 cells were infected with dl 309 at a MOI of 50 and total RNA was extracted at the indicated time points using Nucleozol reagent, treated with DNase I and reverse-transcribed using VILO. Transcript levels of the indicated viral mRNAs were quantified using the BioRad QX200 droplet digital PCR system for the first 16 hours, and using BioRad CFX96 real-time PCR instrument for 17 to 36 hours, and plotted as percentage of GAPDH expression. Right column shows droplet distribution at the time when transcripts increase above background levels. Error bars represent standard deviation of biological replicates, n = 3. Bar with asterisk represents changes above mock level that are statistically significant with the following p values: 52k EP ≤ 0.001; pIIIa ≤ 0.0006; Penton base ≤ 0.0001; Hexon ≤ 0.0002; 100k HAP ≤ 0.0007; Fiber ≤ 0.0004. All time-points between 17 and 36 hours were statistically significant.

    Article Snippet: PCR reactions were carried out using EvaGreen for QX200 master mix according to manufacturer’s directions using a QX200 digital droplet PCR instrument (BioRad).

    Techniques: Expressing, Infection, Digital PCR, Real-time Polymerase Chain Reaction, Standard Deviation

    AZD5363-induced upregulation of IGF-IR, IGF-I and IGF-II is dependent on ER and FoxO3 . A ) LTED cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. T47D cells were serum starved for 24 hours and then treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. RNA was extracted, reverse transcribed to cDNA and analyzed by real-time PCR. Threshold cycle values were normalized for actin. qPCR reactions with no input RNA were used as negative controls. Data are presented as fold versus control; each bar, mean ± SEM ( n = 4; * P

    Journal: Breast Cancer Research : BCR

    Article Title: Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation

    doi: 10.1186/bcr3449

    Figure Lengend Snippet: AZD5363-induced upregulation of IGF-IR, IGF-I and IGF-II is dependent on ER and FoxO3 . A ) LTED cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. T47D cells were serum starved for 24 hours and then treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. RNA was extracted, reverse transcribed to cDNA and analyzed by real-time PCR. Threshold cycle values were normalized for actin. qPCR reactions with no input RNA were used as negative controls. Data are presented as fold versus control; each bar, mean ± SEM ( n = 4; * P

    Article Snippet: Using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), 1 µg of RNA was reverse transcribed to cDNA and real-time PCR reactions were conducted in 96-well plates using the iCycler iQ (Bio-Rad) and primers obtained from SABiosciences (Qiagen).

    Techniques: Droplet Countercurrent Chromatography, Real-time Polymerase Chain Reaction

    Combined inhibition of AKT and ER suppresses hormone-independent tumor growth . A ) LTED cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. RNA was extracted, reverse transcribed to cDNA and analyzed by real-time PCR. Threshold cycle values were normalized for actin. Data are presented as fold versus control; each bar, mean ± SEM ( n = 2; * P

    Journal: Breast Cancer Research : BCR

    Article Title: Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation

    doi: 10.1186/bcr3449

    Figure Lengend Snippet: Combined inhibition of AKT and ER suppresses hormone-independent tumor growth . A ) LTED cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. RNA was extracted, reverse transcribed to cDNA and analyzed by real-time PCR. Threshold cycle values were normalized for actin. Data are presented as fold versus control; each bar, mean ± SEM ( n = 2; * P

    Article Snippet: Using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), 1 µg of RNA was reverse transcribed to cDNA and real-time PCR reactions were conducted in 96-well plates using the iCycler iQ (Bio-Rad) and primers obtained from SABiosciences (Qiagen).

    Techniques: Inhibition, Droplet Countercurrent Chromatography, Real-time Polymerase Chain Reaction

    Inhibition of AKT results in feedback upregulation of RTKs . A ) LTED cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. RNA was extracted, reverse transcribed to cDNA and analyzed by real-time PCR. Threshold cycle values were normalized for actin. Average fold changes over control (Con) were calculated and used to generate a heatmap. B ) LTED cells were treated for 24 hours in 10% DCC-FBS ± 15 µM AZD5363. Protein lysates were analyzed by immunoblot. Densitometric analysis was performed and the ratio of InsR:actin is shown below the InsR blots. C ) LTED cells in 10% DCC-FBS were treated ± 2 µM AZD5363 for 0 to 24 hours. Cell lysates (0.5 mg) were prepared and analyzed by phospho-RTK arrays. D ) Mice bearing MCF-7 xenografts ≥150 mm 3 were treated with vehicle or AZD5363 (150 mg/kg bid p.o.) for one or three days. Xenografts were harvested four hours after the last dose; tumor lysates were prepared and analyzed by immunoblot. Densitometric analysis was performed and a graphical representation of the average RTK:actin levels is shown below the blot. E ) Xenografts from D) were homogenized and RNA was extracted and analyzed by real-time PCR as in A. Data are presented as fold versus control; each bar, mean ± SEM ( n = 3; * P

    Journal: Breast Cancer Research : BCR

    Article Title: Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation

    doi: 10.1186/bcr3449

    Figure Lengend Snippet: Inhibition of AKT results in feedback upregulation of RTKs . A ) LTED cells were treated with 10% DCC-FBS ± 2 µM AZD5363 for 24 hours. RNA was extracted, reverse transcribed to cDNA and analyzed by real-time PCR. Threshold cycle values were normalized for actin. Average fold changes over control (Con) were calculated and used to generate a heatmap. B ) LTED cells were treated for 24 hours in 10% DCC-FBS ± 15 µM AZD5363. Protein lysates were analyzed by immunoblot. Densitometric analysis was performed and the ratio of InsR:actin is shown below the InsR blots. C ) LTED cells in 10% DCC-FBS were treated ± 2 µM AZD5363 for 0 to 24 hours. Cell lysates (0.5 mg) were prepared and analyzed by phospho-RTK arrays. D ) Mice bearing MCF-7 xenografts ≥150 mm 3 were treated with vehicle or AZD5363 (150 mg/kg bid p.o.) for one or three days. Xenografts were harvested four hours after the last dose; tumor lysates were prepared and analyzed by immunoblot. Densitometric analysis was performed and a graphical representation of the average RTK:actin levels is shown below the blot. E ) Xenografts from D) were homogenized and RNA was extracted and analyzed by real-time PCR as in A. Data are presented as fold versus control; each bar, mean ± SEM ( n = 3; * P

    Article Snippet: Using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), 1 µg of RNA was reverse transcribed to cDNA and real-time PCR reactions were conducted in 96-well plates using the iCycler iQ (Bio-Rad) and primers obtained from SABiosciences (Qiagen).

    Techniques: Inhibition, Droplet Countercurrent Chromatography, Real-time Polymerase Chain Reaction, Mouse Assay

    IFN-αβ production following DNA vaccination is Irf7 dependent WT, Sting −/− , Irf7 −/− , and DKO mice were vaccinated intramuscularly with pH1HA vaccine, and the site of injection was harvested 6 or 12 hours later. Total RNA was isolated from tissue biopsies and subjected to rt-PCR for ( A ) IFN-α, ( B ) IFN-β, and ( C ) IP10. Data are the averages ± SEM of 5 mice per group. Values are relative to naïve mouse gene expression. *p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A cGAS-independent STING-IRF7 pathway mediates the immunogenicity of DNA vaccines

    doi: 10.4049/jimmunol.1501836

    Figure Lengend Snippet: IFN-αβ production following DNA vaccination is Irf7 dependent WT, Sting −/− , Irf7 −/− , and DKO mice were vaccinated intramuscularly with pH1HA vaccine, and the site of injection was harvested 6 or 12 hours later. Total RNA was isolated from tissue biopsies and subjected to rt-PCR for ( A ) IFN-α, ( B ) IFN-β, and ( C ) IP10. Data are the averages ± SEM of 5 mice per group. Values are relative to naïve mouse gene expression. *p

    Article Snippet: IFN-α and IFN-β rt-PCR was performed on RNA isolated from immortalized BMDM. rt-PCR reactions were performed on a Bio-Rad CFX-96 cycler.

    Techniques: Mouse Assay, Injection, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing

    Irf3 is required for DNA vaccine-induced IFN-αβ production in vitro Irf +/+ , Irf3 −/− , and DKO BMDMs were transfected with poly (dA:dT) or pH1HA plasmid for 18 hours. ( A ) IFN-α and ( B ) IFN-β levels in transfected BMDM were quantified by rt-PCR. ( C ) Secreted IFN-β in the culture supernatants was analyzed by ELISA. Data are presented as mean ± SEM from 3 independent experiments. *p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A cGAS-independent STING-IRF7 pathway mediates the immunogenicity of DNA vaccines

    doi: 10.4049/jimmunol.1501836

    Figure Lengend Snippet: Irf3 is required for DNA vaccine-induced IFN-αβ production in vitro Irf +/+ , Irf3 −/− , and DKO BMDMs were transfected with poly (dA:dT) or pH1HA plasmid for 18 hours. ( A ) IFN-α and ( B ) IFN-β levels in transfected BMDM were quantified by rt-PCR. ( C ) Secreted IFN-β in the culture supernatants was analyzed by ELISA. Data are presented as mean ± SEM from 3 independent experiments. *p

    Article Snippet: IFN-α and IFN-β rt-PCR was performed on RNA isolated from immortalized BMDM. rt-PCR reactions were performed on a Bio-Rad CFX-96 cycler.

    Techniques: In Vitro, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Decreases in cGas −/− IFN-αβ production following DNA vaccination are transient WT and cGas −/− mice were vaccinated intramuscularly with the pH1HA vaccine, and the site of injection was harvested either 6 or 12 hours later. Total RNA was isolated from tissue biopsies and subjected to rt-PCR for ( A ) IFN-α, ( B ) IFN-β, and ( C ) IP10. Data are the averages ± SEM of 5 mice per group. Values are relative to naïve mouse gene expression. *p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: A cGAS-independent STING-IRF7 pathway mediates the immunogenicity of DNA vaccines

    doi: 10.4049/jimmunol.1501836

    Figure Lengend Snippet: Decreases in cGas −/− IFN-αβ production following DNA vaccination are transient WT and cGas −/− mice were vaccinated intramuscularly with the pH1HA vaccine, and the site of injection was harvested either 6 or 12 hours later. Total RNA was isolated from tissue biopsies and subjected to rt-PCR for ( A ) IFN-α, ( B ) IFN-β, and ( C ) IP10. Data are the averages ± SEM of 5 mice per group. Values are relative to naïve mouse gene expression. *p

    Article Snippet: IFN-α and IFN-β rt-PCR was performed on RNA isolated from immortalized BMDM. rt-PCR reactions were performed on a Bio-Rad CFX-96 cycler.

    Techniques: Mouse Assay, Injection, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing

    (A) Identification of new origins of replication, JunB and 5S rDNA in HT1080 cells. A real-time PCR-based nascent DNA enrichment assay ( A ) and ChIP assays with antibodies against components of pre-replication complex ( B , C ). cMyc origin was included as a positive control. Nascent DNA was normalized with JunB-nonORI (JunB-5). Precipitated chromatins for all antibodies were normalized with IgG. ( D, E ) Mapping of replication origin sites upstream the JunB gene in different human cell lines using nsDNA assay. Positions of primers used to map replication initiation sites within the JunB locus. A set of primers separated by 1.2 to 4.8 kb were chosen for mapping. Positions of primers sequences on the chromosome 19 in hg19 are shown in Supplementary Table S1. nsDNA enrichment in the JunB regions in different human cell lines. In each cell line, a profile of nascent DNA enrichment around JunB was characterized. In all analyzed cell lines, except for MCF-7 and NCI:OV:ADR-RES, the highest peak of nsDNA abundance corresponds to the JunB-2 sequence. In MCF-7 and NCI:OV:ADR-RES cells, origin of replication is mapped to the JunB-1 and JunB-3 sequences, respectively. Student's t -test was used: P

    Journal: Nucleic Acids Research

    Article Title: Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

    doi: 10.1093/nar/gku835

    Figure Lengend Snippet: (A) Identification of new origins of replication, JunB and 5S rDNA in HT1080 cells. A real-time PCR-based nascent DNA enrichment assay ( A ) and ChIP assays with antibodies against components of pre-replication complex ( B , C ). cMyc origin was included as a positive control. Nascent DNA was normalized with JunB-nonORI (JunB-5). Precipitated chromatins for all antibodies were normalized with IgG. ( D, E ) Mapping of replication origin sites upstream the JunB gene in different human cell lines using nsDNA assay. Positions of primers used to map replication initiation sites within the JunB locus. A set of primers separated by 1.2 to 4.8 kb were chosen for mapping. Positions of primers sequences on the chromosome 19 in hg19 are shown in Supplementary Table S1. nsDNA enrichment in the JunB regions in different human cell lines. In each cell line, a profile of nascent DNA enrichment around JunB was characterized. In all analyzed cell lines, except for MCF-7 and NCI:OV:ADR-RES, the highest peak of nsDNA abundance corresponds to the JunB-2 sequence. In MCF-7 and NCI:OV:ADR-RES cells, origin of replication is mapped to the JunB-1 and JunB-3 sequences, respectively. Student's t -test was used: P

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control, Sequencing

    Replication timing of two different HORs, D5Z1 and D5Z2, on chromosome 5. Replication timing was measured by sorting BrdU-labeled cells into different S-phase fractions followed by BrdU-IPs. BrdU-labeled cells were stained with Hoechst 33342 and sorted to six fractions by FACS: G1, S1, S2, S3, S4 and G2/M. ( A, B ) Replication timing of β-globin and collagen origins. They replicate in early and late S phase, correspondingly. ( C, D ) Enrichment of BrdU-labeled alpha-satellite DNA in fractions identified by PCR using specific primers for D5Z1 and D5Z2 HORs. Statistically significant difference was observed between S2 and S3 phases for D5Z1 and D5Z2 in three experiments ( t -test, N = 3, P

    Journal: Nucleic Acids Research

    Article Title: Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

    doi: 10.1093/nar/gku835

    Figure Lengend Snippet: Replication timing of two different HORs, D5Z1 and D5Z2, on chromosome 5. Replication timing was measured by sorting BrdU-labeled cells into different S-phase fractions followed by BrdU-IPs. BrdU-labeled cells were stained with Hoechst 33342 and sorted to six fractions by FACS: G1, S1, S2, S3, S4 and G2/M. ( A, B ) Replication timing of β-globin and collagen origins. They replicate in early and late S phase, correspondingly. ( C, D ) Enrichment of BrdU-labeled alpha-satellite DNA in fractions identified by PCR using specific primers for D5Z1 and D5Z2 HORs. Statistically significant difference was observed between S2 and S3 phases for D5Z1 and D5Z2 in three experiments ( t -test, N = 3, P

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: Labeling, Staining, FACS, Polymerase Chain Reaction

    Replication timing of alphoid arrays. ( A ) Replication timing was measured by sorting BrdU-labeled cells into different S-phase fractions followed by BrdU-IPs. BrdU-labeled cells were stained with Hoechst 33342 and sorted to six fractions by FACS: G1, S1, S2, S3, S4 and G2/M. ( B–D ) Replication timing of JunB , 5S rDNA and cMyc sites. They replicate early in S phase. ( E ) Enrichment of BrdU-labeled alpha-satellite DNA in fractions identified by PCR using primers for the alpha-satellite consensus sequence. This experiment suggests late replication of alphoid DNA.

    Journal: Nucleic Acids Research

    Article Title: Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

    doi: 10.1093/nar/gku835

    Figure Lengend Snippet: Replication timing of alphoid arrays. ( A ) Replication timing was measured by sorting BrdU-labeled cells into different S-phase fractions followed by BrdU-IPs. BrdU-labeled cells were stained with Hoechst 33342 and sorted to six fractions by FACS: G1, S1, S2, S3, S4 and G2/M. ( B–D ) Replication timing of JunB , 5S rDNA and cMyc sites. They replicate early in S phase. ( E ) Enrichment of BrdU-labeled alpha-satellite DNA in fractions identified by PCR using primers for the alpha-satellite consensus sequence. This experiment suggests late replication of alphoid DNA.

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: Labeling, Staining, FACS, Polymerase Chain Reaction, Sequencing

    Mapping of replication initiation events within the alphoid tetO -HAC sequence. ( A ) Schematic of the HAC organization. The circular alphoid tetO -HAC was assembled from input DNA comprising ∼10 kb of RCA vector sequence (BAC/YAC) containing the Bsr gene and ∼40 kb of the synthetic tetO-alphoid array. The synthetic array is composed by tetO alphoid monomer (in green) arrange within every other monomer from chromosome 17 comprising CENP-B box (in gray). FISH demonstrates the presence of an autonomous form of alphoid tetO -HAC in HT1080 cells. The HAC was visualized using a vector probe (RCA) (in red) and tetO-alphoid probe (in green). ( B) DNA fiber-FISH analysis using a YAC/BAC vector probe demonstrating a regular structure of the HAC, i.e. a 10 kb YAC/BAC vector signals (in red) are flanked with a 40 kb alphoid tetO array (in green). ( C ) Real-time PCR-based nascent DNA enrichment assay to map origins of replication along the HAC sequence. ( D, E ) ChIP-qPCR with antibodies against Orc2 and Treslin shows enrichment of ORC on the Bsr sequences. JUNB-2 and cMyc were used as positive controls. ( F ) Chromatin profiling of the alphoid tetO -HAC shows H3K4me2 accumulation on the Bsr sequences. JUNB-2 and cMyc were used as positive controls. ( G ) ChIP analysis of H3K27me3 chromatin in the HAC. Student's t -test was used: P

    Journal: Nucleic Acids Research

    Article Title: Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

    doi: 10.1093/nar/gku835

    Figure Lengend Snippet: Mapping of replication initiation events within the alphoid tetO -HAC sequence. ( A ) Schematic of the HAC organization. The circular alphoid tetO -HAC was assembled from input DNA comprising ∼10 kb of RCA vector sequence (BAC/YAC) containing the Bsr gene and ∼40 kb of the synthetic tetO-alphoid array. The synthetic array is composed by tetO alphoid monomer (in green) arrange within every other monomer from chromosome 17 comprising CENP-B box (in gray). FISH demonstrates the presence of an autonomous form of alphoid tetO -HAC in HT1080 cells. The HAC was visualized using a vector probe (RCA) (in red) and tetO-alphoid probe (in green). ( B) DNA fiber-FISH analysis using a YAC/BAC vector probe demonstrating a regular structure of the HAC, i.e. a 10 kb YAC/BAC vector signals (in red) are flanked with a 40 kb alphoid tetO array (in green). ( C ) Real-time PCR-based nascent DNA enrichment assay to map origins of replication along the HAC sequence. ( D, E ) ChIP-qPCR with antibodies against Orc2 and Treslin shows enrichment of ORC on the Bsr sequences. JUNB-2 and cMyc were used as positive controls. ( F ) Chromatin profiling of the alphoid tetO -HAC shows H3K4me2 accumulation on the Bsr sequences. JUNB-2 and cMyc were used as positive controls. ( G ) ChIP analysis of H3K27me3 chromatin in the HAC. Student's t -test was used: P

    Article Snippet: The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad, USA).

    Techniques: HAC Assay, Sequencing, Plasmid Preparation, BAC Assay, Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Agarose gel electrophoresis of DNA amplified from various genomic DNA extracts of Korean native honey (KNH) and European honey (EH) in duplex PCR with the KNH-specific primers, C6-F and C5-R, and the EH-specific primers, M2-F and M2-R.

    Journal: Korean Journal for Food Science of Animal Resources

    Article Title: Detection of Korean Native Honey and European Honey by Using Duplex Polymerase Chain Reaction and Immunochromatographic Assay

    doi: 10.5851/kosfa.2017.37.4.599

    Figure Lengend Snippet: Agarose gel electrophoresis of DNA amplified from various genomic DNA extracts of Korean native honey (KNH) and European honey (EH) in duplex PCR with the KNH-specific primers, C6-F and C5-R, and the EH-specific primers, M2-F and M2-R.

    Article Snippet: The PCR reaction was performed in the DNA Engine Gradient Cycler (Bio-Rad, USA).

    Techniques: Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction

    Agarose gel electrophoresis of DNA amplified from genomic DNA extracts of the mixtures of Korean native honey (KNH) and European honey (EH) in duplex PCR with the KNH-specific primers, C6-F and C5-R, and the EH-specific primers, M5-F and M5-R.

    Journal: Korean Journal for Food Science of Animal Resources

    Article Title: Detection of Korean Native Honey and European Honey by Using Duplex Polymerase Chain Reaction and Immunochromatographic Assay

    doi: 10.5851/kosfa.2017.37.4.599

    Figure Lengend Snippet: Agarose gel electrophoresis of DNA amplified from genomic DNA extracts of the mixtures of Korean native honey (KNH) and European honey (EH) in duplex PCR with the KNH-specific primers, C6-F and C5-R, and the EH-specific primers, M5-F and M5-R.

    Article Snippet: The PCR reaction was performed in the DNA Engine Gradient Cycler (Bio-Rad, USA).

    Techniques: Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction

    Agarose gel electrophoresis of DNA amplified from genomic DNA extracts of ten Korean native honey (KNH) products in duplex PCR with the KNH- specific primers, C6-F and C5-R and the European honey-specific primers, M5-F and M5-R.

    Journal: Korean Journal for Food Science of Animal Resources

    Article Title: Detection of Korean Native Honey and European Honey by Using Duplex Polymerase Chain Reaction and Immunochromatographic Assay

    doi: 10.5851/kosfa.2017.37.4.599

    Figure Lengend Snippet: Agarose gel electrophoresis of DNA amplified from genomic DNA extracts of ten Korean native honey (KNH) products in duplex PCR with the KNH- specific primers, C6-F and C5-R and the European honey-specific primers, M5-F and M5-R.

    Article Snippet: The PCR reaction was performed in the DNA Engine Gradient Cycler (Bio-Rad, USA).

    Techniques: Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction

    Immunochromatographic assay of DNA amplified from genomic DNA extracts of the mixtures of Korean native honey (KNH) and European honey (EH) in duplex PCR with the KNH-specific primers, C6-F and C5-R and the EH-specific primers, M5-F and M5-R.

    Journal: Korean Journal for Food Science of Animal Resources

    Article Title: Detection of Korean Native Honey and European Honey by Using Duplex Polymerase Chain Reaction and Immunochromatographic Assay

    doi: 10.5851/kosfa.2017.37.4.599

    Figure Lengend Snippet: Immunochromatographic assay of DNA amplified from genomic DNA extracts of the mixtures of Korean native honey (KNH) and European honey (EH) in duplex PCR with the KNH-specific primers, C6-F and C5-R and the EH-specific primers, M5-F and M5-R.

    Article Snippet: The PCR reaction was performed in the DNA Engine Gradient Cycler (Bio-Rad, USA).

    Techniques: Immunochromatographic Assay, Amplification, Polymerase Chain Reaction

    Immunochromatographic assay of DNA amplified from genomic DNA extracts of seven Korean native honey (KNH) products in duplex PCR with the KNH-specific primers, C6-F and C5-R, and the European honey(EH)-specific primers, M5-F and M5-R.

    Journal: Korean Journal for Food Science of Animal Resources

    Article Title: Detection of Korean Native Honey and European Honey by Using Duplex Polymerase Chain Reaction and Immunochromatographic Assay

    doi: 10.5851/kosfa.2017.37.4.599

    Figure Lengend Snippet: Immunochromatographic assay of DNA amplified from genomic DNA extracts of seven Korean native honey (KNH) products in duplex PCR with the KNH-specific primers, C6-F and C5-R, and the European honey(EH)-specific primers, M5-F and M5-R.

    Article Snippet: The PCR reaction was performed in the DNA Engine Gradient Cycler (Bio-Rad, USA).

    Techniques: Immunochromatographic Assay, Amplification, Polymerase Chain Reaction

    Agarose gel electrophoresis of DNA amplified from genomic DNA extracts of the mixtures of Korean native honey (KNH) and European honey (EH) in duplex PCR with the KNH-specific primers, C6-F and C5-R, and the EH-specific primers, M2-F and M2-R.

    Journal: Korean Journal for Food Science of Animal Resources

    Article Title: Detection of Korean Native Honey and European Honey by Using Duplex Polymerase Chain Reaction and Immunochromatographic Assay

    doi: 10.5851/kosfa.2017.37.4.599

    Figure Lengend Snippet: Agarose gel electrophoresis of DNA amplified from genomic DNA extracts of the mixtures of Korean native honey (KNH) and European honey (EH) in duplex PCR with the KNH-specific primers, C6-F and C5-R, and the EH-specific primers, M2-F and M2-R.

    Article Snippet: The PCR reaction was performed in the DNA Engine Gradient Cycler (Bio-Rad, USA).

    Techniques: Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction

    Inflammation, oxidative stress, and cardiac remodeling in p47 phox−/− mice in response to chronic Trypanosoma cruzi infection. P47 phox−/− mice were infected with T cruzi (2000 parasites/mouse) and euthanized 120 days postinfection. Shown are (A) plasma levels of nitrite (a), LDH (b), and MPO (c), myocardial levels of MPO (d), and (B) H E staining of heart tissue sections from normal and chronically infected mice. C, Real‐time RT‐PCR determination of mRNA levels for cytokines as detailed in Figure 4 . D, Western blotting for myocardial level of oxidative stress. E, Real‐time PCR determination of Cyt b– and CO II–encoding regions of mitochondrial DNA. F, Combined MPF and SHG microscopic images of normal and chronically infected p47 phox−/− mice. 3‐NT indicates 3‐nitrotyrosine; Carb, carbonyls; Carbp, plasma carbonyls; CO II, cytochrome oxidase II; Cyt b, cytochrome b; GPx, glutathione peroxidase; H E, hematoxylin and eosin; IFN‐γ, interferon‐γ; IL, interleukin; LDH p , plasma levels of lactate dehydrogenase; MDA, malondialdehyde; MnSOD tg , Mn 2+ superoxide dismutase; MPF, multiphoton fluorescence; MPO, myocardial levels of myeloperoxidase; MPO p , plasma levels of myeloperoxidase; • NO p , plasma levels of nitric oxide; RT‐PCR, reverse‐transcriptase polymerase chain reaction; SHG, second harmonic generation; Tc , Trypanosoma cruzi ; TGF‐β, transforming growth factor–β; TNF, tumor necrosis factor; wt, wild type.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: MnSODtg Mice Control Myocardial Inflammatory and Oxidative Stress and Remodeling Responses Elicited in Chronic Chagas Disease

    doi: 10.1161/JAHA.113.000302

    Figure Lengend Snippet: Inflammation, oxidative stress, and cardiac remodeling in p47 phox−/− mice in response to chronic Trypanosoma cruzi infection. P47 phox−/− mice were infected with T cruzi (2000 parasites/mouse) and euthanized 120 days postinfection. Shown are (A) plasma levels of nitrite (a), LDH (b), and MPO (c), myocardial levels of MPO (d), and (B) H E staining of heart tissue sections from normal and chronically infected mice. C, Real‐time RT‐PCR determination of mRNA levels for cytokines as detailed in Figure 4 . D, Western blotting for myocardial level of oxidative stress. E, Real‐time PCR determination of Cyt b– and CO II–encoding regions of mitochondrial DNA. F, Combined MPF and SHG microscopic images of normal and chronically infected p47 phox−/− mice. 3‐NT indicates 3‐nitrotyrosine; Carb, carbonyls; Carbp, plasma carbonyls; CO II, cytochrome oxidase II; Cyt b, cytochrome b; GPx, glutathione peroxidase; H E, hematoxylin and eosin; IFN‐γ, interferon‐γ; IL, interleukin; LDH p , plasma levels of lactate dehydrogenase; MDA, malondialdehyde; MnSOD tg , Mn 2+ superoxide dismutase; MPF, multiphoton fluorescence; MPO, myocardial levels of myeloperoxidase; MPO p , plasma levels of myeloperoxidase; • NO p , plasma levels of nitric oxide; RT‐PCR, reverse‐transcriptase polymerase chain reaction; SHG, second harmonic generation; Tc , Trypanosoma cruzi ; TGF‐β, transforming growth factor–β; TNF, tumor necrosis factor; wt, wild type.

    Article Snippet: Total DNA (100 ng) was submitted to a real‐time PCR reaction on an iCycler thermal cycler with SYBR Green Supermix (Bio‐Rad) and Tc 18SrDNA‐specific primers.

    Techniques: Mouse Assay, Infection, Staining, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Preservation of mitochondrial structure and DNA content in chronically infected MnSOD tg mice. A, Representative micrographs of heart tissue sections from chronically infected mice, submitted to transmission electron microscopy (magnification, 9400×). B, Percentage of changes in mitochondrial DNA content in cardiac biopsies of mice during the chronic disease phase was determined by real‐time PCR amplification of Cyt b (a) and CO II (b) regions of mitochondrial DNA. Data were normalized to β‐globin encoded by nuclear DNA. CO II indicates cytochrome oxidase II; Cyt b, cytochrome b; GPx, glutathione peroxidase; MnSOD tg , Mn 2+ superoxide dismutase; Tc , Trypanosoma cruzi .

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: MnSODtg Mice Control Myocardial Inflammatory and Oxidative Stress and Remodeling Responses Elicited in Chronic Chagas Disease

    doi: 10.1161/JAHA.113.000302

    Figure Lengend Snippet: Preservation of mitochondrial structure and DNA content in chronically infected MnSOD tg mice. A, Representative micrographs of heart tissue sections from chronically infected mice, submitted to transmission electron microscopy (magnification, 9400×). B, Percentage of changes in mitochondrial DNA content in cardiac biopsies of mice during the chronic disease phase was determined by real‐time PCR amplification of Cyt b (a) and CO II (b) regions of mitochondrial DNA. Data were normalized to β‐globin encoded by nuclear DNA. CO II indicates cytochrome oxidase II; Cyt b, cytochrome b; GPx, glutathione peroxidase; MnSOD tg , Mn 2+ superoxide dismutase; Tc , Trypanosoma cruzi .

    Article Snippet: Total DNA (100 ng) was submitted to a real‐time PCR reaction on an iCycler thermal cycler with SYBR Green Supermix (Bio‐Rad) and Tc 18SrDNA‐specific primers.

    Techniques: Preserving, Infection, Mouse Assay, Transmission Assay, Electron Microscopy, Real-time Polymerase Chain Reaction, Amplification

    Parasite burden. C57BL/6 mice (wild type, MnSOD tg , GPx −/− ) were infected with Trypanosoma cruzi , and peripheral blood, and heart tissues were collected days 120 postinfection. Shown are the (A) blood and (B) tissue levels of parasite burden, determined by real‐time PCR amplification of the Tc 18SrDNA sequence. Results were normalized to murine GAPDH DNA and represent fold change in Tc DNA levels in chronically infected mice compared with that noted in matched normal controls. In all figures, data are presented as means±SDs (n=8/group). Significance is shown as *normal vs infected, # wild type vs genetically modified and is represented by * # P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: MnSODtg Mice Control Myocardial Inflammatory and Oxidative Stress and Remodeling Responses Elicited in Chronic Chagas Disease

    doi: 10.1161/JAHA.113.000302

    Figure Lengend Snippet: Parasite burden. C57BL/6 mice (wild type, MnSOD tg , GPx −/− ) were infected with Trypanosoma cruzi , and peripheral blood, and heart tissues were collected days 120 postinfection. Shown are the (A) blood and (B) tissue levels of parasite burden, determined by real‐time PCR amplification of the Tc 18SrDNA sequence. Results were normalized to murine GAPDH DNA and represent fold change in Tc DNA levels in chronically infected mice compared with that noted in matched normal controls. In all figures, data are presented as means±SDs (n=8/group). Significance is shown as *normal vs infected, # wild type vs genetically modified and is represented by * # P

    Article Snippet: Total DNA (100 ng) was submitted to a real‐time PCR reaction on an iCycler thermal cycler with SYBR Green Supermix (Bio‐Rad) and Tc 18SrDNA‐specific primers.

    Techniques: Mouse Assay, Infection, Real-time Polymerase Chain Reaction, Amplification, Sequencing, Genetically Modified

    ROS content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots inoculated with Rhizobium tropici . Measurements were done at initial time (0 h) and 3, 6, 12, 24 and 48 h after inoculation with R. tropici. (A) Histological (fluorescence) detection of ROS accumulation in inoculated root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in inoculated roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: ROS content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots inoculated with Rhizobium tropici . Measurements were done at initial time (0 h) and 3, 6, 12, 24 and 48 h after inoculation with R. tropici. (A) Histological (fluorescence) detection of ROS accumulation in inoculated root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in inoculated roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Expressing, Fluorescence, Quantitative RT-PCR

    Expression pattern of miR398 and target genes CSD1 and Nod19 in tissues from common bean plants under copper deficiency (CuD) or copper toxicity (CuT). (A) miR398 levels in roots, nodules and leaves of plants grown under control (C) or stress (CuD or CuT) conditions were detected by Northern blot analysis using U6snRNA as loading control. Signal intensity of the hybridization bands was calculated and the expression ratio (stress:control) was obtained. Relative expression of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod19 (red) in roots, nodules and leaves of plants grown under CuD (light colors) or CuT (dark colors) as determined by qRT-PCR. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Expression pattern of miR398 and target genes CSD1 and Nod19 in tissues from common bean plants under copper deficiency (CuD) or copper toxicity (CuT). (A) miR398 levels in roots, nodules and leaves of plants grown under control (C) or stress (CuD or CuT) conditions were detected by Northern blot analysis using U6snRNA as loading control. Signal intensity of the hybridization bands was calculated and the expression ratio (stress:control) was obtained. Relative expression of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod19 (red) in roots, nodules and leaves of plants grown under CuD (light colors) or CuT (dark colors) as determined by qRT-PCR. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Expressing, Northern Blot, Hybridization, Quantitative RT-PCR

    Effect of miR398b transient over-expression in Nicotiana benthamiana leaves infected with Sclerotinia sclerotiorum . N. benthamiana leaves were infiltrated with water (Control) or with A. tumefaciens bearing EV or OE398 plasmids and miR398b expression level was determined 3d after infiltration (A) . Subsequently, infiltrated leaves (EV or OE398) were inoculated with S. sclerotiorum . Characteristic fungal lesions (B) quantified by measuring the infection halo; asterisk: Student's t test, P ≤0.01 (C) and miR398b expression levels determined by qRT-PCR (D) at 48 h after fungal infection.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Effect of miR398b transient over-expression in Nicotiana benthamiana leaves infected with Sclerotinia sclerotiorum . N. benthamiana leaves were infiltrated with water (Control) or with A. tumefaciens bearing EV or OE398 plasmids and miR398b expression level was determined 3d after infiltration (A) . Subsequently, infiltrated leaves (EV or OE398) were inoculated with S. sclerotiorum . Characteristic fungal lesions (B) quantified by measuring the infection halo; asterisk: Student's t test, P ≤0.01 (C) and miR398b expression levels determined by qRT-PCR (D) at 48 h after fungal infection.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Over Expression, Infection, Expressing, Quantitative RT-PCR

    Effect of miR398b over-expression in transgenic roots from composite plants grown under CuD. Composite plants were obtained through A. rhizogenes transformation with EV or with OE398 plasmid, these were grown in control (sufficient nutrient) condition (C) or in CuD stress condition. ( A ) Relative expression of miR398b (blue) and of ( B ) target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from the EV roots grown in the C condition that was set to 1. ( C ) Anthocyanin contents in root crown of composite plants. ( D ) Expression ratio (CuD:C) of copper-stress responsive genes: Fe superoxide dismutase (FSD, yellow), high affinity Cu transporter (COPT, purple) and ferric chelate reductase (FRO, brown). Values represent the average ± SD from three biological replicates.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Effect of miR398b over-expression in transgenic roots from composite plants grown under CuD. Composite plants were obtained through A. rhizogenes transformation with EV or with OE398 plasmid, these were grown in control (sufficient nutrient) condition (C) or in CuD stress condition. ( A ) Relative expression of miR398b (blue) and of ( B ) target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from the EV roots grown in the C condition that was set to 1. ( C ) Anthocyanin contents in root crown of composite plants. ( D ) Expression ratio (CuD:C) of copper-stress responsive genes: Fe superoxide dismutase (FSD, yellow), high affinity Cu transporter (COPT, purple) and ferric chelate reductase (FRO, brown). Values represent the average ± SD from three biological replicates.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Over Expression, Transgenic Assay, Transformation Assay, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Reactive oxygen species (ROS) content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots exposed to high Cu (CuT). Measurements were done at initial time (0 h) and after 12, 24 and 48 h of high Cu (70 µM CuSO 4 ) application. (A) Histological (fluorescence) detection of ROS accumulation in CuT stressed root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in CuT-stressed roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Reactive oxygen species (ROS) content and expression pattern of miR398 and target genes CSD1 and Nod19 in roots exposed to high Cu (CuT). Measurements were done at initial time (0 h) and after 12, 24 and 48 h of high Cu (70 µM CuSO 4 ) application. (A) Histological (fluorescence) detection of ROS accumulation in CuT stressed root tips using 2′,7′- dichlorodihydrofluorescein diacetate (H 2 DCF-DA). The values in parenthesis indicate the average integrated fluorescence intensity per unit area of root tissue ±SD. Asterisk: Student's t test, P ≤0.05. Relative expression, determined by qRT-PCR, of (B) miR398b (blue) and of (C) target genes CSD1 (green) and Nod1 9 (red) in CuT-stressed roots at the indicated time points. Values were normalized to the value from the C condition that was set to 1 as indicated with a dashed line. Values represent the average ± SD from three biological replicates.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Expressing, Fluorescence, Quantitative RT-PCR

    Expression pattern of miR398b and target genes CSD1 and Nod19 in common bean leaves infected with Sclerotinia sclerotiorum . (A) Mock (left) or S. sclerotiorum infected (right) common bean leaves after 24 h. (B) Relative expression of miR398b (blue) and of target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from mock that was set to 1 as indicated with a dashed line.

    Journal: PLoS ONE

    Article Title: Regulation of Copper Homeostasis and Biotic Interactions by MicroRNA 398b in Common Bean

    doi: 10.1371/journal.pone.0084416

    Figure Lengend Snippet: Expression pattern of miR398b and target genes CSD1 and Nod19 in common bean leaves infected with Sclerotinia sclerotiorum . (A) Mock (left) or S. sclerotiorum infected (right) common bean leaves after 24 h. (B) Relative expression of miR398b (blue) and of target genes CSD1 (green) and Nod19 (red) determined by qRT-PCR; values were normalized to the value from mock that was set to 1 as indicated with a dashed line.

    Article Snippet: All qRT-PCR reactions were made by duplicate in iCycler BioRad equipment and at least two independent experiments were performed.

    Techniques: Expressing, Infection, Quantitative RT-PCR

    Retinoic acid-related orphan receptor gamma t-independent intestinal graft-versus-host disease (GvHD) is controlled by basic leucine zipper transcription factor ATF-like (BATF). (A) Transcript levels of Batf in colonic tissue of allo-HSCT BALB/c mice with severe signs of intestinal GvHD following transplantation of allogeneic CD3 + T cells derived from of Batf −/− and Rorc −/− mice were measured by quantitative real-time PCR (qPCR) at day 15 ( Batf −/− n = 12; Rorc −/− n = 2) and day 30 ( Batf −/− n = 12; Rorc −/− n = 4). Data represent pooled data from two independent experiments. Gene expression levels detected in colonic tissues of no T cells (noT) controls at day 15 were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level detected within noT controls. (B) Transcript levels of Rorc in colonic tissue of irradiated BALB/c mice transplanted with T cell depleted bone marrow alone (noT, n = 11) or followed by injection of allogeneic CD3 + T cells of wild-type (WT) ( n = 12) mice were measured by qPCR at day 30. (C) Transcript levels of RORC in colonic tissue biopsies from allo-HCT patients were measured by qPCR. Samples were grouped into indicated categories based on the absence (−GvHD; n = 30) or presence (+GvHD; n = 22) of GvHD-associated histopathological lesions or by the absence (−apoptosis; n = 32) or presence (+apoptosis; n = 20) of GvHD-related epithelial cell apoptosis. Data are shown as normalized relative RORC expression levels calculated from a standard curve. (D–G) Experimental GvHD induction was achieved as described in Figures 1 and 2 . To this end, allogeneic hematopoietic stem cell transplantation BALB/c mice were transplanted with allogeneic CD3 + T cells derived from WT ( n = 4), Rorc −/− ( n = 3), or Rorc −/− Batf −/− ( n = 3) mice or received no T cells (noT) as a control ( n = 4). (D) Mice were assessed three times a week for signs and severity of systemic GvHD. Data were analyzed by two-way ANOVA testing followed by Bonferroni’s multiple comparisons posttest. **** p

    Journal: Frontiers in Immunology

    Article Title: Targeting Inflammatory T Helper Cells via Retinoic Acid-Related Orphan Receptor Gamma t Is Ineffective to Prevent Allo-Response-Driven Colitis

    doi: 10.3389/fimmu.2018.01138

    Figure Lengend Snippet: Retinoic acid-related orphan receptor gamma t-independent intestinal graft-versus-host disease (GvHD) is controlled by basic leucine zipper transcription factor ATF-like (BATF). (A) Transcript levels of Batf in colonic tissue of allo-HSCT BALB/c mice with severe signs of intestinal GvHD following transplantation of allogeneic CD3 + T cells derived from of Batf −/− and Rorc −/− mice were measured by quantitative real-time PCR (qPCR) at day 15 ( Batf −/− n = 12; Rorc −/− n = 2) and day 30 ( Batf −/− n = 12; Rorc −/− n = 4). Data represent pooled data from two independent experiments. Gene expression levels detected in colonic tissues of no T cells (noT) controls at day 15 were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level detected within noT controls. (B) Transcript levels of Rorc in colonic tissue of irradiated BALB/c mice transplanted with T cell depleted bone marrow alone (noT, n = 11) or followed by injection of allogeneic CD3 + T cells of wild-type (WT) ( n = 12) mice were measured by qPCR at day 30. (C) Transcript levels of RORC in colonic tissue biopsies from allo-HCT patients were measured by qPCR. Samples were grouped into indicated categories based on the absence (−GvHD; n = 30) or presence (+GvHD; n = 22) of GvHD-associated histopathological lesions or by the absence (−apoptosis; n = 32) or presence (+apoptosis; n = 20) of GvHD-related epithelial cell apoptosis. Data are shown as normalized relative RORC expression levels calculated from a standard curve. (D–G) Experimental GvHD induction was achieved as described in Figures 1 and 2 . To this end, allogeneic hematopoietic stem cell transplantation BALB/c mice were transplanted with allogeneic CD3 + T cells derived from WT ( n = 4), Rorc −/− ( n = 3), or Rorc −/− Batf −/− ( n = 3) mice or received no T cells (noT) as a control ( n = 4). (D) Mice were assessed three times a week for signs and severity of systemic GvHD. Data were analyzed by two-way ANOVA testing followed by Bonferroni’s multiple comparisons posttest. **** p

    Article Snippet: Gene expression of RORC and 18S ribosomal RNA was analyzed by quantitative PCR reactions that were performed on a Mastercyler Ep Realplex (Eppendorf) using a QuantiFast SYBR Green PCR Kit (Qiagen).

    Techniques: Mouse Assay, Transplantation Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Irradiation, Injection

    Retinoic acid-related orphan receptor gamma t (RORγt)-dependent T helper (Th17) cells are dispensable for acute intestinal graft-versus-host disease (GvHD). (A) Naïve CD4 + T cells were isolated from the spleen by negative selection using magnetic microbeads and in vitro polarized into inflammatory Th17 cells by co-culturing anti-CD3/anti-CD28 activated T cells in the presence of anti-IFNγ antibodies and either without (−) or with (+) recombinant interleukin (IL)-1β, IL-6, and IL-23. After 5 days, T cells were harvested, RNA was isolated, and transcribed into cDNA followed by quantitative real-time PCR (qPCR) analyses of Il23r transcript levels. Gene expression levels detected within T cells cultured under drift conditions were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level of this control. Data are combined from two individual experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons posttest. ** p

    Journal: Frontiers in Immunology

    Article Title: Targeting Inflammatory T Helper Cells via Retinoic Acid-Related Orphan Receptor Gamma t Is Ineffective to Prevent Allo-Response-Driven Colitis

    doi: 10.3389/fimmu.2018.01138

    Figure Lengend Snippet: Retinoic acid-related orphan receptor gamma t (RORγt)-dependent T helper (Th17) cells are dispensable for acute intestinal graft-versus-host disease (GvHD). (A) Naïve CD4 + T cells were isolated from the spleen by negative selection using magnetic microbeads and in vitro polarized into inflammatory Th17 cells by co-culturing anti-CD3/anti-CD28 activated T cells in the presence of anti-IFNγ antibodies and either without (−) or with (+) recombinant interleukin (IL)-1β, IL-6, and IL-23. After 5 days, T cells were harvested, RNA was isolated, and transcribed into cDNA followed by quantitative real-time PCR (qPCR) analyses of Il23r transcript levels. Gene expression levels detected within T cells cultured under drift conditions were arbitrarily set down to 1 and all other gene expression levels were normalized to the expression level of this control. Data are combined from two individual experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons posttest. ** p

    Article Snippet: Gene expression of RORC and 18S ribosomal RNA was analyzed by quantitative PCR reactions that were performed on a Mastercyler Ep Realplex (Eppendorf) using a QuantiFast SYBR Green PCR Kit (Qiagen).

    Techniques: Isolation, Selection, In Vitro, Recombinant, Real-time Polymerase Chain Reaction, Expressing, Cell Culture

    Genetic depletion of retinoic acid-related orphan receptor gamma t-dependent T helper cells preserves the granulocyte-macrophage colony-stimulating factor (GM-CSF) expressing donor T cell pool. Experimental graft-versus-host disease (GvHD) induction was performed as described in Figures 1 and 2 . (A) Gene expression levels in total colonic tissue of wild-type (WT) and Il23r −/− donor T cell receiving mice of Ifng, Csf2 (WT n = 14; Il23r −/− n = 15) and Il17a (WT n = 9; Il23r −/− n = 5) transcripts were analyzed by quantitative real-time PCR (qPCR). (B) Gene expression levels in total colonic tissue of WT and Rorc −/− donor T cell receiving mice of Ifng, Csf2 (WT n = 17; Rorc −/− n = 14) and Il17a (WT n = 7; Rorc −/− n = 8) transcripts were analyzed and quantitated by qPCR around day 30, i.e., when allogeneic hematopoietic stem cell transplantation mice displayed fully established signs of intestinal GvHD. Data represent pooled data from two (A) and at least three (B) individual experiments, respectively. (C,D) In addition, lamina propria mononuclear cells of WT ( n = 6) and Il23r −/− ( n = 6) T cell receiving mice (C) or WT ( n = 4) and Rorc −/− ( n = 3) T cell receiving mice (D) were assessed by flow cytometry for the frequencies and absolute numbers of GM-CSF-, IFNγ- or interleukin 17a (IL-17a)-producing CD4 + donor T cells, respectively, employing intracellular cytokine staining techniques. Displayed data are derived from one representative of two independent experiments and were analyzed by Student’s t -test. * p

    Journal: Frontiers in Immunology

    Article Title: Targeting Inflammatory T Helper Cells via Retinoic Acid-Related Orphan Receptor Gamma t Is Ineffective to Prevent Allo-Response-Driven Colitis

    doi: 10.3389/fimmu.2018.01138

    Figure Lengend Snippet: Genetic depletion of retinoic acid-related orphan receptor gamma t-dependent T helper cells preserves the granulocyte-macrophage colony-stimulating factor (GM-CSF) expressing donor T cell pool. Experimental graft-versus-host disease (GvHD) induction was performed as described in Figures 1 and 2 . (A) Gene expression levels in total colonic tissue of wild-type (WT) and Il23r −/− donor T cell receiving mice of Ifng, Csf2 (WT n = 14; Il23r −/− n = 15) and Il17a (WT n = 9; Il23r −/− n = 5) transcripts were analyzed by quantitative real-time PCR (qPCR). (B) Gene expression levels in total colonic tissue of WT and Rorc −/− donor T cell receiving mice of Ifng, Csf2 (WT n = 17; Rorc −/− n = 14) and Il17a (WT n = 7; Rorc −/− n = 8) transcripts were analyzed and quantitated by qPCR around day 30, i.e., when allogeneic hematopoietic stem cell transplantation mice displayed fully established signs of intestinal GvHD. Data represent pooled data from two (A) and at least three (B) individual experiments, respectively. (C,D) In addition, lamina propria mononuclear cells of WT ( n = 6) and Il23r −/− ( n = 6) T cell receiving mice (C) or WT ( n = 4) and Rorc −/− ( n = 3) T cell receiving mice (D) were assessed by flow cytometry for the frequencies and absolute numbers of GM-CSF-, IFNγ- or interleukin 17a (IL-17a)-producing CD4 + donor T cells, respectively, employing intracellular cytokine staining techniques. Displayed data are derived from one representative of two independent experiments and were analyzed by Student’s t -test. * p

    Article Snippet: Gene expression of RORC and 18S ribosomal RNA was analyzed by quantitative PCR reactions that were performed on a Mastercyler Ep Realplex (Eppendorf) using a QuantiFast SYBR Green PCR Kit (Qiagen).

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Transplantation Assay, Flow Cytometry, Cytometry, Staining, Derivative Assay

    Knockdown of MLH1 or MLH3 significantly reduces GAA•TTC expansion rate in human cells. Four independent FRDA model lines were transduced with shRNA knockdown lentiviral pools targeting the indicated MutL subunits and an empty vector control virus (pLKO). ( A ) Representative gel image of PCR products measuring GAA•TTC expansion. T 0 is the day of transduction. PCR product sizes reflect 3X(number of repeats) plus 669 bp of flanking sequence. Lane M is the 1 Kb Plus DNA ladder showing 2, 1.6 and 1 Kb bands. ( B ) Mean repeats gained over four weeks. Knockdown of MLH1 and MLH3 reduced expansion compared to the empty vector control virus (pLKO). MLH1sh was significantly different ( P = 0.0009) as was MLH3sh ( P = 0.00045) whereas PMS2sh did not reach significance ( P = 0.053). Error bars show SEM.

    Journal: Nucleic Acids Research

    Article Title: GAA•TTC repeat expansion in human cells is mediated by mismatch repair complex MutLγ and depends upon the endonuclease domain in MLH3 isoform one

    doi: 10.1093/nar/gky143

    Figure Lengend Snippet: Knockdown of MLH1 or MLH3 significantly reduces GAA•TTC expansion rate in human cells. Four independent FRDA model lines were transduced with shRNA knockdown lentiviral pools targeting the indicated MutL subunits and an empty vector control virus (pLKO). ( A ) Representative gel image of PCR products measuring GAA•TTC expansion. T 0 is the day of transduction. PCR product sizes reflect 3X(number of repeats) plus 669 bp of flanking sequence. Lane M is the 1 Kb Plus DNA ladder showing 2, 1.6 and 1 Kb bands. ( B ) Mean repeats gained over four weeks. Knockdown of MLH1 and MLH3 reduced expansion compared to the empty vector control virus (pLKO). MLH1sh was significantly different ( P = 0.0009) as was MLH3sh ( P = 0.00045) whereas PMS2sh did not reach significance ( P = 0.053). Error bars show SEM.

    Article Snippet: PCR reactions for MLH3 isoforms consisted of 50 μl reactions with 2 μl of cDNA template, 200 nM primers, 250 μM each dNTP (Stratagene) and 2.5 U Paq5000 DNA polymerase (Stratagene).

    Techniques: Transduction, shRNA, Plasmid Preparation, Polymerase Chain Reaction, Sequencing

    Splice switching oligonucleotides (SSOs) mask the acceptor or donor region of MLH3 exon 7 in pre-mRNA to promote exon skipping. ( A ) Design strategy. Morpholino antisense oligonucleotides were designed to bind the intron–exon junction in MLH3 pre-mRNA at either the ‘acceptor’ region (exon 6 side) or the ‘donor’ region (exon 8 side) of MLH3 exon 7. These SSOs were used to induce skipping of exon 7 and preferential production of MLH3 splice isoform 2 (iso2). ( B ) Gel image shows an example of SSO mediated splice switching as revealed by RT-PCR 48 h after treatment. RT-PCR products spanning exon 6 to exon 8 produced a 434 or 362 bp product from MLH3 isoform 1 or isoform 2, respectively (arrows at right). FRDA rapid expansion model cells in culture were treated with acceptor, donor or an equimolar mixture of both SSOs at the indicated total SSO concentrations. Lane M is the 1 Kb Plus DNA ladder showing 500, 400 and 300 bp.

    Journal: Nucleic Acids Research

    Article Title: GAA•TTC repeat expansion in human cells is mediated by mismatch repair complex MutLγ and depends upon the endonuclease domain in MLH3 isoform one

    doi: 10.1093/nar/gky143

    Figure Lengend Snippet: Splice switching oligonucleotides (SSOs) mask the acceptor or donor region of MLH3 exon 7 in pre-mRNA to promote exon skipping. ( A ) Design strategy. Morpholino antisense oligonucleotides were designed to bind the intron–exon junction in MLH3 pre-mRNA at either the ‘acceptor’ region (exon 6 side) or the ‘donor’ region (exon 8 side) of MLH3 exon 7. These SSOs were used to induce skipping of exon 7 and preferential production of MLH3 splice isoform 2 (iso2). ( B ) Gel image shows an example of SSO mediated splice switching as revealed by RT-PCR 48 h after treatment. RT-PCR products spanning exon 6 to exon 8 produced a 434 or 362 bp product from MLH3 isoform 1 or isoform 2, respectively (arrows at right). FRDA rapid expansion model cells in culture were treated with acceptor, donor or an equimolar mixture of both SSOs at the indicated total SSO concentrations. Lane M is the 1 Kb Plus DNA ladder showing 500, 400 and 300 bp.

    Article Snippet: PCR reactions for MLH3 isoforms consisted of 50 μl reactions with 2 μl of cDNA template, 200 nM primers, 250 μM each dNTP (Stratagene) and 2.5 U Paq5000 DNA polymerase (Stratagene).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Produced

    Weekly treatment with SSOs slows GAA•TTC expansion in non-dividing FRDA patient-derived fibroblasts. ( A ) Gel image shows an example of SSO mediated splice switching as revealed by RT-PCR 48 h after a treatment for FRDA patient derived fibroblasts. RT-PCR products spanning exon 6 to exon 8 produced a 434 or 362 bp product from MLH3 isoform 1 or isoform 2, respectively. Primary FRDA patient-derived fibroblasts GM04078, FA4111 and FA6196 were transduced with lentiviral vectors expressing MSH3 to accelerate expansion ( 5 ) and allowed to reach confluency, which stopped cell division by contact inhibition. The confluent cells were then treated once a week with 750 nM SSOs for 6 weeks. Lane M is the 1 Kb Plus DNA ladder showing 500, 400 and 300 bp. ( B ) Gel images show PCR sizing of GAA•TTC repeats for three different FRDA patient derived fibroblasts after six weeks in culture with and without SSO treatment. One set of cells was taken up at the time of the first treatment in the experiment (T 0 ) to provide a baseline repeat size. The T 0 samples were loaded to flank the control and SSO treated 6-week samples. Alongside the gel images ‘L’ represents the longer and ‘S’ the shorter allele for each patient with number indicating the nominal size of the alleles originally reported for these cells. The primers used add 498 bp to the size of the GAA•TTC repeat. M is a 1 Kb Plus DNA ladder showing 2 Kb and 1.6 Kb bands as well as 1 Kb and 850 bp for FA6196.

    Journal: Nucleic Acids Research

    Article Title: GAA•TTC repeat expansion in human cells is mediated by mismatch repair complex MutLγ and depends upon the endonuclease domain in MLH3 isoform one

    doi: 10.1093/nar/gky143

    Figure Lengend Snippet: Weekly treatment with SSOs slows GAA•TTC expansion in non-dividing FRDA patient-derived fibroblasts. ( A ) Gel image shows an example of SSO mediated splice switching as revealed by RT-PCR 48 h after a treatment for FRDA patient derived fibroblasts. RT-PCR products spanning exon 6 to exon 8 produced a 434 or 362 bp product from MLH3 isoform 1 or isoform 2, respectively. Primary FRDA patient-derived fibroblasts GM04078, FA4111 and FA6196 were transduced with lentiviral vectors expressing MSH3 to accelerate expansion ( 5 ) and allowed to reach confluency, which stopped cell division by contact inhibition. The confluent cells were then treated once a week with 750 nM SSOs for 6 weeks. Lane M is the 1 Kb Plus DNA ladder showing 500, 400 and 300 bp. ( B ) Gel images show PCR sizing of GAA•TTC repeats for three different FRDA patient derived fibroblasts after six weeks in culture with and without SSO treatment. One set of cells was taken up at the time of the first treatment in the experiment (T 0 ) to provide a baseline repeat size. The T 0 samples were loaded to flank the control and SSO treated 6-week samples. Alongside the gel images ‘L’ represents the longer and ‘S’ the shorter allele for each patient with number indicating the nominal size of the alleles originally reported for these cells. The primers used add 498 bp to the size of the GAA•TTC repeat. M is a 1 Kb Plus DNA ladder showing 2 Kb and 1.6 Kb bands as well as 1 Kb and 850 bp for FA6196.

    Article Snippet: PCR reactions for MLH3 isoforms consisted of 50 μl reactions with 2 μl of cDNA template, 200 nM primers, 250 μM each dNTP (Stratagene) and 2.5 U Paq5000 DNA polymerase (Stratagene).

    Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Produced, Transduction, Expressing, Inhibition, Polymerase Chain Reaction

    Agarose gel (1%) of PCR products for analysis of SXT element and class 1 integron showing amplification with (A) primer pair SXT-F/SXT-R for detection of SXT integrase; (B) primer pair L2/L3 for detection of 5′ CS of class 1 integron; (C) primer pair qacEΔ1-F/Sul1-B for amplification of 3′ CS of class 1 integron; (D) primer pair In-F/In-B for amplification of variable regions associated with class 1 integrons. All the DNA samples used as templates are indicated on top of each lane. V. cholerae MO10 and V. fluvialis BD146 were used as controls for various PCR assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.

    Journal: Frontiers in Microbiology

    Article Title: Role of integrons, plasmids and SXT elements in multidrug resistance of Vibrio cholerae and Providencia vermicola obtained from a clinical isolate of diarrhea

    doi: 10.3389/fmicb.2015.00057

    Figure Lengend Snippet: Agarose gel (1%) of PCR products for analysis of SXT element and class 1 integron showing amplification with (A) primer pair SXT-F/SXT-R for detection of SXT integrase; (B) primer pair L2/L3 for detection of 5′ CS of class 1 integron; (C) primer pair qacEΔ1-F/Sul1-B for amplification of 3′ CS of class 1 integron; (D) primer pair In-F/In-B for amplification of variable regions associated with class 1 integrons. All the DNA samples used as templates are indicated on top of each lane. V. cholerae MO10 and V. fluvialis BD146 were used as controls for various PCR assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.

    Article Snippet: In PCR for Providencia sp. (using Provi_F and Provi_R to detect both P. rettgeri and P. vermicola ), annealing was carried out at 62∘ C for 1 min and polymerization was carried out at 72∘ C for 1.5 min, while in PCR for P. vermicola (using Provi_F and P_Vermi_R to detect only P. vermicola and not P. rettgeri ), annealing was carried out at 68∘ C for 1 min and polymerization was carried out at 72∘ C for 1.5 min. PCR reactions were performed using a PTC-225 DNA Engine TetradTM Cycler (MJ Research Inc., Waltham, MA, USA).

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

    Identification and differentiation of constituent bacteria of a clinical isolate of diarrhea. Agarose gel (1%) analysis of (A) PCR products for confirming V. cholerae species with primer pair OmpW- F/OmpW-R; (B) PFGE of DNA from Vc IDH02365 and P. vermicola Pv NBA2365 digested with Sfi I restriction enzyme; (C) RAPD profile of clinical isolates with 1281 and 1283 primers; and (D) PCR with species-specific primers for P. rettgeri/P. vermicola and only P. vermicola. All the DNA samples used as templates are indicated on top of each lane. The clinical isolates of V. cholerae IDH01526, IDH01572, IDH01581, N16961, MO10, P. vermicola BAB 812, P. rettgeri MTCC8099 and mixture of Vc IDH02365 and Pv NBA2365 as obtained from the original stab culture (mixed) were used as controls for various assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.

    Journal: Frontiers in Microbiology

    Article Title: Role of integrons, plasmids and SXT elements in multidrug resistance of Vibrio cholerae and Providencia vermicola obtained from a clinical isolate of diarrhea

    doi: 10.3389/fmicb.2015.00057

    Figure Lengend Snippet: Identification and differentiation of constituent bacteria of a clinical isolate of diarrhea. Agarose gel (1%) analysis of (A) PCR products for confirming V. cholerae species with primer pair OmpW- F/OmpW-R; (B) PFGE of DNA from Vc IDH02365 and P. vermicola Pv NBA2365 digested with Sfi I restriction enzyme; (C) RAPD profile of clinical isolates with 1281 and 1283 primers; and (D) PCR with species-specific primers for P. rettgeri/P. vermicola and only P. vermicola. All the DNA samples used as templates are indicated on top of each lane. The clinical isolates of V. cholerae IDH01526, IDH01572, IDH01581, N16961, MO10, P. vermicola BAB 812, P. rettgeri MTCC8099 and mixture of Vc IDH02365 and Pv NBA2365 as obtained from the original stab culture (mixed) were used as controls for various assays. Marker positions in kb have been indicated in the left of each panel. Arrows indicate the position of relevant amplicons for each PCR.

    Article Snippet: In PCR for Providencia sp. (using Provi_F and Provi_R to detect both P. rettgeri and P. vermicola ), annealing was carried out at 62∘ C for 1 min and polymerization was carried out at 72∘ C for 1.5 min, while in PCR for P. vermicola (using Provi_F and P_Vermi_R to detect only P. vermicola and not P. rettgeri ), annealing was carried out at 68∘ C for 1 min and polymerization was carried out at 72∘ C for 1.5 min. PCR reactions were performed using a PTC-225 DNA Engine TetradTM Cycler (MJ Research Inc., Waltham, MA, USA).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    TSC22D4 deficiency counteracts diabetic hyperglycaemia and insulin resistance. ( a ) Glucose tolerance test in control or TSC22D4 shRNA adenovirus–injected db/db mice 1 week after injection. Glucose was injected i.p. at a concentration of 1 g glucose kg −1 body weight (means±s.e.m., n ≥6). ( b ) Serum glucose levels in same mice as in a after 16 h fasting and refeeding. ( c ) Serum insulin levels in same mice as in a . ( d ) Serum C-Peptide levels in same mice as in a after 16 h fasting and refeeding. ( e ) Representative western blot of liver extracts from control (NC shRNA) or TSC22D4 (TSC shRNA) shRNA adenovirus–injected db/db mice; same mice as in a using indicated antibodies. Blot representative of 6 similar samples. ( f ) Glucose tolerance test in control or TSC22D4 shRNA adenovirus–injected NZO mice 1 week after injection. Glucose was i.p. injected at a concentration of 1 g glucose kg −1 body weight (means±s.e.m., n ≥6). ( g ) Serum glucose levels in same mice as in f after 16 h fasting and refeeding. ( h ) HOMA-IR index in control or TSC22D4 miRNA AAV–injected (at 5 weeks of age) db/db mice 4 weeks after miRNA injection (means±s.e.m., n ≥6). ( i ) Serum insulin levels in same mice as in h 10 weeks after miRNA injection. ( j ) Glucose tolerance test in the same mice as in h 4 weeks after miRNA injection. Glucose was injected i.p at a concentration of 1 g glucose kg −1 body weight. ( k ) Weekly serum glucose quantification of same mice as in h . ( l – n ) Quantitative PCR analysis of phosphoenolpyruvate carboxykinase ( Pepck1 ), peroxisome proliferator-activated receptor gamma coactivator 1-alpha ( Pgc-1alpha ), glucose-6 phosphatase ( G6Pase ), carnitine palmitoyltransferase I ( Cpt1 ), acyl-CoA synthetase long-chain family member 1 ( Acsl1 ), peroxisome proliferator-activated receptor alpha ( PPARalpha ), acetyl-CoA carboxylase alpha ( Acaca ), fatty acid synthase ( Fasn ), stearoyl-coA desaturase-1 ( Scd1 ), sterol regulatory element-binding protein 1 ( Srebp1 ) in livers of same mice as in h 10 weeks after miRNA injection. Error bars in l − n represent standard deviation (s.d.). Statistical analysis: Student's t -test, * P ≤0.05; ** P ≤0.01; *** P ≤0.001.

    Journal: Nature Communications

    Article Title: Control of diabetic hyperglycaemia and insulin resistance through TSC22D4

    doi: 10.1038/ncomms13267

    Figure Lengend Snippet: TSC22D4 deficiency counteracts diabetic hyperglycaemia and insulin resistance. ( a ) Glucose tolerance test in control or TSC22D4 shRNA adenovirus–injected db/db mice 1 week after injection. Glucose was injected i.p. at a concentration of 1 g glucose kg −1 body weight (means±s.e.m., n ≥6). ( b ) Serum glucose levels in same mice as in a after 16 h fasting and refeeding. ( c ) Serum insulin levels in same mice as in a . ( d ) Serum C-Peptide levels in same mice as in a after 16 h fasting and refeeding. ( e ) Representative western blot of liver extracts from control (NC shRNA) or TSC22D4 (TSC shRNA) shRNA adenovirus–injected db/db mice; same mice as in a using indicated antibodies. Blot representative of 6 similar samples. ( f ) Glucose tolerance test in control or TSC22D4 shRNA adenovirus–injected NZO mice 1 week after injection. Glucose was i.p. injected at a concentration of 1 g glucose kg −1 body weight (means±s.e.m., n ≥6). ( g ) Serum glucose levels in same mice as in f after 16 h fasting and refeeding. ( h ) HOMA-IR index in control or TSC22D4 miRNA AAV–injected (at 5 weeks of age) db/db mice 4 weeks after miRNA injection (means±s.e.m., n ≥6). ( i ) Serum insulin levels in same mice as in h 10 weeks after miRNA injection. ( j ) Glucose tolerance test in the same mice as in h 4 weeks after miRNA injection. Glucose was injected i.p at a concentration of 1 g glucose kg −1 body weight. ( k ) Weekly serum glucose quantification of same mice as in h . ( l – n ) Quantitative PCR analysis of phosphoenolpyruvate carboxykinase ( Pepck1 ), peroxisome proliferator-activated receptor gamma coactivator 1-alpha ( Pgc-1alpha ), glucose-6 phosphatase ( G6Pase ), carnitine palmitoyltransferase I ( Cpt1 ), acyl-CoA synthetase long-chain family member 1 ( Acsl1 ), peroxisome proliferator-activated receptor alpha ( PPARalpha ), acetyl-CoA carboxylase alpha ( Acaca ), fatty acid synthase ( Fasn ), stearoyl-coA desaturase-1 ( Scd1 ), sterol regulatory element-binding protein 1 ( Srebp1 ) in livers of same mice as in h 10 weeks after miRNA injection. Error bars in l − n represent standard deviation (s.d.). Statistical analysis: Student's t -test, * P ≤0.05; ** P ≤0.01; *** P ≤0.001.

    Article Snippet: From each RT–PCR, 2 μl were amplified in a 26 μl PCR reaction using the Brilliant SYBR green QPCR Core reagent kit from Stratagene) according to the manufacturer's instructions.

    Techniques: shRNA, Injection, Mouse Assay, Concentration Assay, Western Blot, Real-time Polymerase Chain Reaction, Pyrolysis Gas Chromatography, Binding Assay, Standard Deviation

    TSC22D4 acts via the LCN13 endocrine system. ( a ) Chromatin immunoprecipitation of LCN13 promoter regions (1–3) by antibodies against TSC22D4 in livers of wild-type mice. Fold enrichment relative to negative isotype control IgG is determined by qPCR. Region 4 represents a negative PCR control ( n =2–3). Similar results were obtained by using a different TSC22D4 antibody. ( b ) Quantitative PCR analysis of LCN13 in livers of control or TSC22D4 shRNA adenovirus-injected wild-type C57Bl/6 (left), db/db (middle), and NZO mice (below) (means± s.e.m., n ≥ 6 for each experiment). ( c ) Serum from control (NC shRNA) or TSC22D4 (TSC shRNA) shRNA adenovirus–injected C57Bl/6 mice 7 days after injection was immunoblotted with LCN13 antibody. Albumin antibody was used as loading control. ( d ) Representative western blot from control (PBS) or LCN13 (200 nM)-treated C2C12 myotube extracts using indicated antibodies. LCN13 (3 h) and insulin treatment for 15 min (30 nM) indicated, respectively. Right: densitometric analysis shown. **Indicates effect of insulin; ## Indicates effect of LCN13. ( e ) Glucose tolerance test in control (control shRNA), LCN13 (LCN13 shRNA), TSC22D4 (TSC22D4 shRNA), TSC22D4 plus LCN13 (TSC22D4 +LCN13 shRNA) shRNA adenovirus–injected db/db mice 1 week after injection. Glucose was injected i.p. at a concentration of 1 g glucose kg −1 body weight. *Indicates significance between NC and TSC22D4 group; # Indicates significance between TSC22D4 and TSC22D4 +LCN13 group; $ Indicates significance between NC and TSC22D4 +LCN13 group, (means±s.e.m., n ≥6). ( f ) HOMA-IR index in same mice as in e . ( g ) Serum glucose levels in same mice as in e . ( h ) Body weight of mice as in e . ( i ) Abdominal white adipose tissue (aWAT) mass in same mice as in e . ( j ) Inguinal white adipose tissue (iWAT) in same mice as in e . Statistical analysis: Student's t -test, * P ≤0.05; ** P ≤0.01; *** P ≤0.001, # P ≤0.05; ## P ≤0.01; ### P ≤0.001, $ P ≤0.05; $$ P ≤0.01; $$$ P ≤0.001.

    Journal: Nature Communications

    Article Title: Control of diabetic hyperglycaemia and insulin resistance through TSC22D4

    doi: 10.1038/ncomms13267

    Figure Lengend Snippet: TSC22D4 acts via the LCN13 endocrine system. ( a ) Chromatin immunoprecipitation of LCN13 promoter regions (1–3) by antibodies against TSC22D4 in livers of wild-type mice. Fold enrichment relative to negative isotype control IgG is determined by qPCR. Region 4 represents a negative PCR control ( n =2–3). Similar results were obtained by using a different TSC22D4 antibody. ( b ) Quantitative PCR analysis of LCN13 in livers of control or TSC22D4 shRNA adenovirus-injected wild-type C57Bl/6 (left), db/db (middle), and NZO mice (below) (means± s.e.m., n ≥ 6 for each experiment). ( c ) Serum from control (NC shRNA) or TSC22D4 (TSC shRNA) shRNA adenovirus–injected C57Bl/6 mice 7 days after injection was immunoblotted with LCN13 antibody. Albumin antibody was used as loading control. ( d ) Representative western blot from control (PBS) or LCN13 (200 nM)-treated C2C12 myotube extracts using indicated antibodies. LCN13 (3 h) and insulin treatment for 15 min (30 nM) indicated, respectively. Right: densitometric analysis shown. **Indicates effect of insulin; ## Indicates effect of LCN13. ( e ) Glucose tolerance test in control (control shRNA), LCN13 (LCN13 shRNA), TSC22D4 (TSC22D4 shRNA), TSC22D4 plus LCN13 (TSC22D4 +LCN13 shRNA) shRNA adenovirus–injected db/db mice 1 week after injection. Glucose was injected i.p. at a concentration of 1 g glucose kg −1 body weight. *Indicates significance between NC and TSC22D4 group; # Indicates significance between TSC22D4 and TSC22D4 +LCN13 group; $ Indicates significance between NC and TSC22D4 +LCN13 group, (means±s.e.m., n ≥6). ( f ) HOMA-IR index in same mice as in e . ( g ) Serum glucose levels in same mice as in e . ( h ) Body weight of mice as in e . ( i ) Abdominal white adipose tissue (aWAT) mass in same mice as in e . ( j ) Inguinal white adipose tissue (iWAT) in same mice as in e . Statistical analysis: Student's t -test, * P ≤0.05; ** P ≤0.01; *** P ≤0.001, # P ≤0.05; ## P ≤0.01; ### P ≤0.001, $ P ≤0.05; $$ P ≤0.01; $$$ P ≤0.001.

    Article Snippet: From each RT–PCR, 2 μl were amplified in a 26 μl PCR reaction using the Brilliant SYBR green QPCR Core reagent kit from Stratagene) according to the manufacturer's instructions.

    Techniques: Chromatin Immunoprecipitation, Mouse Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, shRNA, Injection, Western Blot, Concentration Assay

    TSC22D4 levels correlate with insulin sensitivity in humans. ( a ) Quantitative PCR analysis of Tsc22d4 mRNA expression in livers of patients with type 2 diabetes (T2D, n =26) or normal glucose tolerance (NGT, n =40) Error bars indicate standard error of the mean (s.e.m.). ( b ) Correlation of hepatic expression of Tsc22d4 mRNA and fasting plasma glucose in the same patients as in a . ( c ) Correlation of human liver expression of Tsc22d4 mRNA and glucose infusion rate (GIR) during hyperinsulemic-euglycaemic clamp study in the same patients as in a . Statistical analysis for a : Student's t -test, b and c : Pearson correlation coefficient, * P ≤0.05; ** P ≤0.01; *** P ≤0.001.

    Journal: Nature Communications

    Article Title: Control of diabetic hyperglycaemia and insulin resistance through TSC22D4

    doi: 10.1038/ncomms13267

    Figure Lengend Snippet: TSC22D4 levels correlate with insulin sensitivity in humans. ( a ) Quantitative PCR analysis of Tsc22d4 mRNA expression in livers of patients with type 2 diabetes (T2D, n =26) or normal glucose tolerance (NGT, n =40) Error bars indicate standard error of the mean (s.e.m.). ( b ) Correlation of hepatic expression of Tsc22d4 mRNA and fasting plasma glucose in the same patients as in a . ( c ) Correlation of human liver expression of Tsc22d4 mRNA and glucose infusion rate (GIR) during hyperinsulemic-euglycaemic clamp study in the same patients as in a . Statistical analysis for a : Student's t -test, b and c : Pearson correlation coefficient, * P ≤0.05; ** P ≤0.01; *** P ≤0.001.

    Article Snippet: From each RT–PCR, 2 μl were amplified in a 26 μl PCR reaction using the Brilliant SYBR green QPCR Core reagent kit from Stratagene) according to the manufacturer's instructions.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Linear correlation between percent T509 allele in Blumeria graminis f. sp. hordei genomic DNA samples quantified by digital PCR and the percentage of the triazole resistant isolate Per (T509) in a genomic DNA mixture of Per and the triazole sensitive isolate Wagga (S509). Each point represents the average of triplicate chips.

    Journal: Frontiers in Microbiology

    Article Title: Improved Detection and Monitoring of Fungicide Resistance in Blumeria graminis f. sp. hordei With High-Throughput Genotype Quantification by Digital PCR

    doi: 10.3389/fmicb.2018.00706

    Figure Lengend Snippet: Linear correlation between percent T509 allele in Blumeria graminis f. sp. hordei genomic DNA samples quantified by digital PCR and the percentage of the triazole resistant isolate Per (T509) in a genomic DNA mixture of Per and the triazole sensitive isolate Wagga (S509). Each point represents the average of triplicate chips.

    Article Snippet: Each 10 μl PCR reaction contained 0.5 U MyTaq DNA polymerase (Bioline) with 1 × MyTaq reaction buffer, 0.4 μM of each primer and 1 μL DNA template.

    Techniques: Digital PCR

    Scatter plots for Blumeria graminis f. sp. hordei ( Bgh ) S509T digital PCR assay using known ratios of genomic DNA for the triazole resistant Bgh isolate Per (contains T509 allele) and triazole sensitive Bgh isolate Wagga (contains S509 allele). Scatter plots were obtained by Quantstudio TM 3D AnalysisSuite TM by digital PCR. Wells with T509 alleles are represented by FAM signals (blue), S509 alleles are represented by VIC signals (red), detection of both alleles are represented by green signals, and wells without any alleles (passive reference) are represented by ROX signals (yellow). (A) 100% Wagga gDNA (S509 allele only). (B) 0.1% Per gDNA; T509 frequency calculated by dPCR = 0.127%. (C) 1% Per gDNA; T509 dPCR frequency = 1.06%. (D) 10% Per gDNA; T509 dPCR frequency = 12.59%.

    Journal: Frontiers in Microbiology

    Article Title: Improved Detection and Monitoring of Fungicide Resistance in Blumeria graminis f. sp. hordei With High-Throughput Genotype Quantification by Digital PCR

    doi: 10.3389/fmicb.2018.00706

    Figure Lengend Snippet: Scatter plots for Blumeria graminis f. sp. hordei ( Bgh ) S509T digital PCR assay using known ratios of genomic DNA for the triazole resistant Bgh isolate Per (contains T509 allele) and triazole sensitive Bgh isolate Wagga (contains S509 allele). Scatter plots were obtained by Quantstudio TM 3D AnalysisSuite TM by digital PCR. Wells with T509 alleles are represented by FAM signals (blue), S509 alleles are represented by VIC signals (red), detection of both alleles are represented by green signals, and wells without any alleles (passive reference) are represented by ROX signals (yellow). (A) 100% Wagga gDNA (S509 allele only). (B) 0.1% Per gDNA; T509 frequency calculated by dPCR = 0.127%. (C) 1% Per gDNA; T509 dPCR frequency = 1.06%. (D) 10% Per gDNA; T509 dPCR frequency = 12.59%.

    Article Snippet: Each 10 μl PCR reaction contained 0.5 U MyTaq DNA polymerase (Bioline) with 1 × MyTaq reaction buffer, 0.4 μM of each primer and 1 μL DNA template.

    Techniques: Digital PCR

    Overview of the Long-16S method. (A) 16S rRNA gene template molecules are tagged with unique tags via two single rounds of annealing and extension with tagged forward and reverse primers containing random tags (see Fig. 2 ), that also contain Illumina adapter sequences. After removal of tagged primers, tagged templates are amplified via PCR using primers complementary to the adapter sequences. Libraries from one or more samples can then be pooled and sequenced on the MiSeq. Blue triangles and yellow stars indicate random tags of 10 nt. (B) Full-length 16S rRNA gene amplicon Illumina libraries are tagmented using the standard Nextera method, and two pools of products are amplified which contain either the left end of the tagged amplicons and an internal region, or the right end of the amplicon and an internal region. This procedure adds Nextera adapters for sequencing at the internal end of the fragments. (C) Both full-length and tagmented libraries are paired end sequenced, and the unique molecular tags are used to computationally group sequences from the same progenitor 16S rRNA gene molecule for assembly of near full-length sequences.

    Journal: PeerJ

    Article Title: A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

    doi: 10.7717/peerj.2492

    Figure Lengend Snippet: Overview of the Long-16S method. (A) 16S rRNA gene template molecules are tagged with unique tags via two single rounds of annealing and extension with tagged forward and reverse primers containing random tags (see Fig. 2 ), that also contain Illumina adapter sequences. After removal of tagged primers, tagged templates are amplified via PCR using primers complementary to the adapter sequences. Libraries from one or more samples can then be pooled and sequenced on the MiSeq. Blue triangles and yellow stars indicate random tags of 10 nt. (B) Full-length 16S rRNA gene amplicon Illumina libraries are tagmented using the standard Nextera method, and two pools of products are amplified which contain either the left end of the tagged amplicons and an internal region, or the right end of the amplicon and an internal region. This procedure adds Nextera adapters for sequencing at the internal end of the fragments. (C) Both full-length and tagmented libraries are paired end sequenced, and the unique molecular tags are used to computationally group sequences from the same progenitor 16S rRNA gene molecule for assembly of near full-length sequences.

    Article Snippet: Each PCR reaction contained a combination of one of the Illumina provided Nextera XT PCR primers and one of the primers from the enrichment PCR above, so as to amplify only those fragments of interest; specifically, we combined primers PE_1 and an Illumina Index 1 primer (N706) in one PCR reaction and PE_2 and an Illumina Index 2 primer (S504) in the second.

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing

    qRT-PCR analysis of JAK2 and LMO2 expression. ( a ) The mean mRNA values of JAK2 and LMO2 in T-LBLs harboring JAK2 genetic alterations were normalized to those of a pool of fetal thymuses. Data represent three independent replicates. Error bars represent the s.d.; * P

    Journal: Leukemia

    Article Title: Contribution of JAK2 mutations to T-cell lymphoblastic lymphoma development

    doi: 10.1038/leu.2015.202

    Figure Lengend Snippet: qRT-PCR analysis of JAK2 and LMO2 expression. ( a ) The mean mRNA values of JAK2 and LMO2 in T-LBLs harboring JAK2 genetic alterations were normalized to those of a pool of fetal thymuses. Data represent three independent replicates. Error bars represent the s.d.; * P

    Article Snippet: All JAK2 mutants were generated by PCR reactions with QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Quantitative RT-PCR, Expressing

    Methylation-specific PCR (MSP) for SHP1 , SOCS1 and SOCS3 genes and methylation density at the SOCS3 promoter, in T-LBLs with mutant JAK2. ( a ) Schematic depiction of the CpG-island around the transcription start site of the genes (long black arrow). Short vertical lines represent CpG dinucleotides. ( b ) MSP analysis. The presence of a PCR band under lanes M or U indicated methylated or unmethylated CpG islands. Normal lymphocytes (NL) and in vitro methylated DNA (IVD) were used as a positive control. ( c ) Methylation profile of SOCS3 in two fetal thymuses and T-LBLs harboring JAK2 genetic alterations. The presence of methylated (black squares) or unmethylated (white squares) CpG sites is indicated in 10 sequenced clones for every tumor. Methylation density is indicated as the percentage of methylated sites.

    Journal: Leukemia

    Article Title: Contribution of JAK2 mutations to T-cell lymphoblastic lymphoma development

    doi: 10.1038/leu.2015.202

    Figure Lengend Snippet: Methylation-specific PCR (MSP) for SHP1 , SOCS1 and SOCS3 genes and methylation density at the SOCS3 promoter, in T-LBLs with mutant JAK2. ( a ) Schematic depiction of the CpG-island around the transcription start site of the genes (long black arrow). Short vertical lines represent CpG dinucleotides. ( b ) MSP analysis. The presence of a PCR band under lanes M or U indicated methylated or unmethylated CpG islands. Normal lymphocytes (NL) and in vitro methylated DNA (IVD) were used as a positive control. ( c ) Methylation profile of SOCS3 in two fetal thymuses and T-LBLs harboring JAK2 genetic alterations. The presence of methylated (black squares) or unmethylated (white squares) CpG sites is indicated in 10 sequenced clones for every tumor. Methylation density is indicated as the percentage of methylated sites.

    Article Snippet: All JAK2 mutants were generated by PCR reactions with QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Methylation, Polymerase Chain Reaction, Mutagenesis, In Vitro, Positive Control, Clone Assay

    TEL-JAK2 translocation and mutational analysis of JAK2. ( a ) Schematic representation of the fusion protein resulting from the t(9;12)(p24;p13) translocation. ( b ) PCR amplification of cDNA from a representation of the samples and HEK293T transfected with TEL-JAK2 positive control, using primers covering the breakpoint of this translocation. ( c ) Western blot analysis of STAT5 and phospho-Y694-STAT5 in T-LBLs and fetal thymus as control (CT). ( d ) Schematic representation of JAK2 protein showing all validated mutations at pseudokinase domain. All mutations were found in two T-LBL samples, both of them exhibiting several transcript variants.

    Journal: Leukemia

    Article Title: Contribution of JAK2 mutations to T-cell lymphoblastic lymphoma development

    doi: 10.1038/leu.2015.202

    Figure Lengend Snippet: TEL-JAK2 translocation and mutational analysis of JAK2. ( a ) Schematic representation of the fusion protein resulting from the t(9;12)(p24;p13) translocation. ( b ) PCR amplification of cDNA from a representation of the samples and HEK293T transfected with TEL-JAK2 positive control, using primers covering the breakpoint of this translocation. ( c ) Western blot analysis of STAT5 and phospho-Y694-STAT5 in T-LBLs and fetal thymus as control (CT). ( d ) Schematic representation of JAK2 protein showing all validated mutations at pseudokinase domain. All mutations were found in two T-LBL samples, both of them exhibiting several transcript variants.

    Article Snippet: All JAK2 mutants were generated by PCR reactions with QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Translocation Assay, Polymerase Chain Reaction, Amplification, Transfection, Positive Control, Western Blot

    In vitro alcohol exposure of human monocytes does not inhibit TLR3-TRIF cytokine/chemokine expression Adherence isolated human monocytes from healthy donors were treated with 25 μg/ml polyI:C or 25 mM alcohol (EtOH) for 2 hours or pre-exposed to 25 mM alcohol (EtOH) for 1, 5 or 24 hours followed by polyI:C stimulation for 2 hours. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of polyI:C stimulation were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to untreated. (D) Secreted RANTES protein after overnight (18–22 hours) polyI:C stimulation was analyzed by ELISA. Data summarize mean ± SD of 6 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human binge alcohol intake inhibits TLR4-MyD88 and TLR4-TRIF responses but not TLR3-TRIF pathway: HspA1A and PP1 play selective regulatory roles

    doi: 10.4049/jimmunol.1600924

    Figure Lengend Snippet: In vitro alcohol exposure of human monocytes does not inhibit TLR3-TRIF cytokine/chemokine expression Adherence isolated human monocytes from healthy donors were treated with 25 μg/ml polyI:C or 25 mM alcohol (EtOH) for 2 hours or pre-exposed to 25 mM alcohol (EtOH) for 1, 5 or 24 hours followed by polyI:C stimulation for 2 hours. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of polyI:C stimulation were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to untreated. (D) Secreted RANTES protein after overnight (18–22 hours) polyI:C stimulation was analyzed by ELISA. Data summarize mean ± SD of 6 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Article Snippet: The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL.

    Techniques: In Vitro, Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    In vitro and in vivo alcohol induces HspA1A expression in human monocytes (A) Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption and total RNA was analyzed for HspA1A mRNA by qRT-PCR. Graph depicts mean ± SD of 9 independent experiments. (B) Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption and HspA1A protein was detected in whole cell lysates by western blotting. The densitometry graph represents quantitation of bands seen in the gel and depicts mean ± SD (n=2). Representative gels are shown with HspA1A (top) and loading control, β-actin (bottom). Gel pictures cropped from different lanes of a single gel for comparison. (C) Human monocytes isolated by adherence were exposed to 25 mM alcohol (EtOH) for 6–20 hours and HspA1A protein was detected in whole cell lysates by western blotting. The densitometry graph represents quantitation of bands seen in the gel and depicts mean ± SD (n=4) and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human binge alcohol intake inhibits TLR4-MyD88 and TLR4-TRIF responses but not TLR3-TRIF pathway: HspA1A and PP1 play selective regulatory roles

    doi: 10.4049/jimmunol.1600924

    Figure Lengend Snippet: In vitro and in vivo alcohol induces HspA1A expression in human monocytes (A) Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption and total RNA was analyzed for HspA1A mRNA by qRT-PCR. Graph depicts mean ± SD of 9 independent experiments. (B) Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption and HspA1A protein was detected in whole cell lysates by western blotting. The densitometry graph represents quantitation of bands seen in the gel and depicts mean ± SD (n=2). Representative gels are shown with HspA1A (top) and loading control, β-actin (bottom). Gel pictures cropped from different lanes of a single gel for comparison. (C) Human monocytes isolated by adherence were exposed to 25 mM alcohol (EtOH) for 6–20 hours and HspA1A protein was detected in whole cell lysates by western blotting. The densitometry graph represents quantitation of bands seen in the gel and depicts mean ± SD (n=4) and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Article Snippet: The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL.

    Techniques: In Vitro, In Vivo, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Quantitation Assay

    In vivo alcohol exposure significantly inhibits TLR4-TRIF cytokine/chemokine expression by human monocytes Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption, following which the cells were stimulated with 100 ng/ml LPS. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of LPS stimulation were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to baseline. (D) Secreted RANTES protein after overnight (18–22 hours) LPS stimulation was analyzed by ELISA. Data summarize mean ± SD of 6–9 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human binge alcohol intake inhibits TLR4-MyD88 and TLR4-TRIF responses but not TLR3-TRIF pathway: HspA1A and PP1 play selective regulatory roles

    doi: 10.4049/jimmunol.1600924

    Figure Lengend Snippet: In vivo alcohol exposure significantly inhibits TLR4-TRIF cytokine/chemokine expression by human monocytes Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption, following which the cells were stimulated with 100 ng/ml LPS. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of LPS stimulation were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to baseline. (D) Secreted RANTES protein after overnight (18–22 hours) LPS stimulation was analyzed by ELISA. Data summarize mean ± SD of 6–9 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Article Snippet: The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL.

    Techniques: In Vivo, Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    PP1 inhibition reverses alcohol-mediated inhibition of TLR4-TRIF chemokine RANTES but not TNFα RAW macrophages were stimulated with 100 ng/ml LPS for 2 hours, 50 mM alcohol (EtOH) and 0.25 μM tautomycetin alone or in combination for 24 hours or pre-exposed to alcohol and tautomycetin followed by LPS stimulation for indicated times. (A) TNFα and (B) RANTES mRNA was analyzed by qRT-PCR and normalized to LPS stimulation alone. Graph depicts mean ± SD of 3 independent experiments and statistical analysis was performed by one way ANOVA with Sidak’s test for multiple comparisons. (ns p > 0.05, * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human binge alcohol intake inhibits TLR4-MyD88 and TLR4-TRIF responses but not TLR3-TRIF pathway: HspA1A and PP1 play selective regulatory roles

    doi: 10.4049/jimmunol.1600924

    Figure Lengend Snippet: PP1 inhibition reverses alcohol-mediated inhibition of TLR4-TRIF chemokine RANTES but not TNFα RAW macrophages were stimulated with 100 ng/ml LPS for 2 hours, 50 mM alcohol (EtOH) and 0.25 μM tautomycetin alone or in combination for 24 hours or pre-exposed to alcohol and tautomycetin followed by LPS stimulation for indicated times. (A) TNFα and (B) RANTES mRNA was analyzed by qRT-PCR and normalized to LPS stimulation alone. Graph depicts mean ± SD of 3 independent experiments and statistical analysis was performed by one way ANOVA with Sidak’s test for multiple comparisons. (ns p > 0.05, * p

    Article Snippet: The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL.

    Techniques: Inhibition, Quantitative RT-PCR

    Exposure of human monocytes to in vitro alcohol significantly suppresses TLR4-TRIF cytokine/chemokine expression Adherence isolated human monocytes from healthy donors were treated with 100 ng/ml LPS or 25 mM alcohol (EtOH) for 2 hours or pre-exposed to 25 mM alcohol (EtOH) for 1, 3, 5, 7 or 24 hours followed by LPS stimulation for 2 hours. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of LPS stimulation were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to untreated. (D) Secreted RANTES protein after overnight (18–22 hours) LPS stimulation was analyzed by ELISA. Data summarize mean ± SD of 6 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human binge alcohol intake inhibits TLR4-MyD88 and TLR4-TRIF responses but not TLR3-TRIF pathway: HspA1A and PP1 play selective regulatory roles

    doi: 10.4049/jimmunol.1600924

    Figure Lengend Snippet: Exposure of human monocytes to in vitro alcohol significantly suppresses TLR4-TRIF cytokine/chemokine expression Adherence isolated human monocytes from healthy donors were treated with 100 ng/ml LPS or 25 mM alcohol (EtOH) for 2 hours or pre-exposed to 25 mM alcohol (EtOH) for 1, 3, 5, 7 or 24 hours followed by LPS stimulation for 2 hours. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of LPS stimulation were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to untreated. (D) Secreted RANTES protein after overnight (18–22 hours) LPS stimulation was analyzed by ELISA. Data summarize mean ± SD of 6 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Article Snippet: The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL.

    Techniques: In Vitro, Expressing, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    TLR3-TRIF cytokine/chemokine expression by human monocytes are not suppressed by in vivo binge alcohol consumption Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption, following which the cells were stimulated with 25 μg/ml polyI:C. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of polyI:C stimulation were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to baseline. (D) Secreted RANTES protein after overnight (18–22 hours) polyI:C stimulation was analyzed by ELISA. Data summarize mean ± SD of 6–9 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human binge alcohol intake inhibits TLR4-MyD88 and TLR4-TRIF responses but not TLR3-TRIF pathway: HspA1A and PP1 play selective regulatory roles

    doi: 10.4049/jimmunol.1600924

    Figure Lengend Snippet: TLR3-TRIF cytokine/chemokine expression by human monocytes are not suppressed by in vivo binge alcohol consumption Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption, following which the cells were stimulated with 25 μg/ml polyI:C. Levels of (A) IFNβ, (B) RANTES and (C) IP-10 mRNA after 2 hours of polyI:C stimulation were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to baseline. (D) Secreted RANTES protein after overnight (18–22 hours) polyI:C stimulation was analyzed by ELISA. Data summarize mean ± SD of 6–9 independent experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Article Snippet: The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL.

    Techniques: Expressing, In Vivo, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    In vivo alcohol exposure significantly inhibits TLR4-MyD88 dependent pro-inflammatory cytokine mRNA expression by human monocytes (A) Healthy human donors consumed alcohol (2 ml vodka/kg body weight in a total volume of 300 ml orange juice) over the period of an hour and their BAC was measured at baseline and 1 hour after alcohol consumption. Graph depicts mean ± SD of 10 independent experiments. (B-C) Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption, following which the cells were stimulated with 100 ng/ml LPS for 2 hours. Levels of (B) TNFα and (C) IL-6 mRNA were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to baseline. Data summarize mean ± SD of 6–8 experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human binge alcohol intake inhibits TLR4-MyD88 and TLR4-TRIF responses but not TLR3-TRIF pathway: HspA1A and PP1 play selective regulatory roles

    doi: 10.4049/jimmunol.1600924

    Figure Lengend Snippet: In vivo alcohol exposure significantly inhibits TLR4-MyD88 dependent pro-inflammatory cytokine mRNA expression by human monocytes (A) Healthy human donors consumed alcohol (2 ml vodka/kg body weight in a total volume of 300 ml orange juice) over the period of an hour and their BAC was measured at baseline and 1 hour after alcohol consumption. Graph depicts mean ± SD of 10 independent experiments. (B-C) Primary human monocytes were isolated from blood of healthy human donors at baseline and 1 hour and 24 hours after alcohol consumption, following which the cells were stimulated with 100 ng/ml LPS for 2 hours. Levels of (B) TNFα and (C) IL-6 mRNA were analyzed by qRT-PCR. Fold change in expression of genes was calculated with respect to baseline. Data summarize mean ± SD of 6–8 experiments and statistical analysis was performed by one way ANOVA with Dunnett’s test for multiple comparisons. (ns p > 0.05, * p

    Article Snippet: The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL.

    Techniques: In Vivo, Expressing, BAC Assay, Isolation, Quantitative RT-PCR

    HspA1A is required for alcohol-mediated inhibition of TLR4-MyD88 dependent TNFα but not TLR4-MyD88 independent RANTES (A) RAW macrophages were transfected with 10 nM siRNA targeting HspA1A or negative control (Neg ctrl) siRNA. Cells were subjected to heatshock (HS) (42°C for 45 min) and total RNA was used for HspA1A mRNA determination by qRT-PCR. Percent knockdown in HS cells was calculated with respect to untransfected. Graph depicts mean ± SD (n=3). Representative gels are shown to illustrate knockdown at protein level with HspA1A (top) and loading control, β-actin (bottom). (B,C) RAW macrophages were transfected with 10 nM siRNA targeting HspA1A or negative control (Neg ctrl) siRNA. 24 hours after transfection, macrophages were stimulated with 100 ng/ml LPS for 2 hours, 50 mM alcohol (EtOH) alone for 24 hours or pre-exposed to alcohol followed by LPS stimulation for indicated times. (B) TNFα and (C) RANTES mRNA was analyzed by qRT-PCR. Graph depicts mean ± SD of 3 independent experiments and statistical analysis was performed by one way ANOVA with Sidak’s test for multiple comparisons. (ns p > 0.05, * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Human binge alcohol intake inhibits TLR4-MyD88 and TLR4-TRIF responses but not TLR3-TRIF pathway: HspA1A and PP1 play selective regulatory roles

    doi: 10.4049/jimmunol.1600924

    Figure Lengend Snippet: HspA1A is required for alcohol-mediated inhibition of TLR4-MyD88 dependent TNFα but not TLR4-MyD88 independent RANTES (A) RAW macrophages were transfected with 10 nM siRNA targeting HspA1A or negative control (Neg ctrl) siRNA. Cells were subjected to heatshock (HS) (42°C for 45 min) and total RNA was used for HspA1A mRNA determination by qRT-PCR. Percent knockdown in HS cells was calculated with respect to untransfected. Graph depicts mean ± SD (n=3). Representative gels are shown to illustrate knockdown at protein level with HspA1A (top) and loading control, β-actin (bottom). (B,C) RAW macrophages were transfected with 10 nM siRNA targeting HspA1A or negative control (Neg ctrl) siRNA. 24 hours after transfection, macrophages were stimulated with 100 ng/ml LPS for 2 hours, 50 mM alcohol (EtOH) alone for 24 hours or pre-exposed to alcohol followed by LPS stimulation for indicated times. (B) TNFα and (C) RANTES mRNA was analyzed by qRT-PCR. Graph depicts mean ± SD of 3 independent experiments and statistical analysis was performed by one way ANOVA with Sidak’s test for multiple comparisons. (ns p > 0.05, * p

    Article Snippet: The qRT-PCR reaction was set up using 6.25 μL iTAQ universal SYBR Green PCR master mix (Bio-Rad Laboratories), 0.25 μM each of forward and reverse primers, and 2.5 μL cDNA (corresponding to 25 ng RNA) for a total reaction volume of 12.5 μL.

    Techniques: Inhibition, Transfection, Negative Control, Quantitative RT-PCR