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  • 99
    Thermo Fisher high capacity cdna reverse transcription kit
    Monoamine oxidase-A (MAO-A) gene is upregulated by alcohol. After 24 hours treatment with 0.1% alcohol, the dendritic cells were harvested, cytoplasmic <t>RNA</t> from the cell pellet was extracted, reverse transcribed to <t>cDNA,</t> and the cDNA was used for the
    High Capacity Cdna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 119667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine rnaimax
    IRF3 is phosphorylated, translocates to the nucleus, and binds the CXCL10 promoter in HT29 cells in response to addition of poly(I:C). A , Western blots of HT29 cells were stimulated with poly(I:C) alone ( Poly(I:C) , 2.5 μg/ml) or transfected with poly(I:C) using <t>Lipofectamine</t> <t>RNAimax</t> ( LF + Poly(I:C) , 0–1200 min) and stained with antibodies against phospho-IRF3 Ser-396 , total IRF3, phosphor-p65 Ser-536 , total p65, or GAPDH. The results are representative of two independent experiments. MW , molecular weight. B and C , nuclear accumulation of IRF3 ( B ) and IRF1 ( C ) in HT29 cells left untreated ( 0 ), stimulated with poly(I:C) (5–2 μg/ml), or transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml) for 3 h or overnight ( o/n ). Stimulated cells were fixed and immunostained for IRF3 or IRF1, and cell nuclei were stained with Hoechst 3342. Cells were visualized by automated imaging, and analysis was done using ScanR. The results show the percentage of cells with positive staining of IRF3 and IRF1 in the nucleus. The results show mean ± S.D. of triplicate samples with a minimum of 1300 cells assayed and are representative of three independent experiments. D , CXCL10 promotor occupancy by IRF3 in HT29 cells after poly(I:C) (2 μg/ml) stimulation for 3 h. IRF3 binding to the CXCL10 promoter was investigated by ChIP followed by qPCR of the CXCL10 promoter region. RNA polymerase II occupancy was measured as a control. E and F , CXCL10 production ( left panels ) and IRF mRNA expression ( right panels ) in HT29 cells left untreated ( No add ), treated with siRNA against IRF3 ( E ) or IRF7 ( F ) (10 n m ), NS RNA (10 n m ), or transfection reagent alone ( LF ) for 24 h. Cells were subsequently stimulated with poly(I:C) (2.5 μg/ml) for 6 h. CXCL10 release was assessed by ELISA, whereas silencing of IRF3 and 7 was confirmed by assessing mRNA expression by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicate samples.
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    Thermo Fisher lipofectamine 2000
    Colony survival assay in A549 cells following transfection with <t>Lipofectamine</t> 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.
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    Thermo Fisher trizol reagent
    Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The <t>vRNA</t> of the cell-bound HCV was extracted with <t>Trizol</t> reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.
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    Thermo Fisher rna
    GS-5806 inhibits RSV fusion, but not attachment, to HEp-2 cells. (A) HEp-2 cells were infected with RSV A2 at 4°C for 3 h, washed to remove unbound virus, and then shifted to 37°C for 30 h to facilitate fusion; 50 μg/ml heparin, 1.25 nM GS-5806, 20 nM VP-14637, or 100 nM BMS-433771 was added to the cells either throughout the experiment, only during viral attachment, or only during viral fusion. Intercellular RSV <t>RNA</t> was quantified by quantitative <t>PCR</t> (qPCR) and compared to an uninhibited DMSO-treated infection. The error bars indicate standard deviations. (B) GS-5806 (10 nM) inhibits syncytium formation in 293T cells transiently expressing the RSV F protein.
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    Thermo Fisher taqman gene expression assays
    Real-time <t>PCR</t> showing the relative RNA expression level of Jam2 in mice with different genotypes. (A) <t>TaqMan</t> assay showed about 1.5-fold increase of Jam2 expression in Ts65Dn mice compared to WT. (B) TaqMan assay showed about 40% decrease of Jam2 expression
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    Thermo Fisher qubit fluorometer
    <t>DNA</t> degradation. DNA recovery after bisulfite treatment was determined with the <t>Qubit</t> fluorometer. DNA recovery with the Epigentek kit was 33.2% ± 3.4%, with the Promega kit was 52% ± 3%, with the Diagenode kit was 55% ± 2.6% and with the Qiagen kit was 50.2% ± 2%. The BisulFlash DNA Modification kit was shown to have a significantly decreased average yield with respect to all other kits (FWER
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    Thermo Fisher agarose gel electrophoresis
    <t>DNA</t> degradation. DNA recovery after bisulfite treatment was determined with the <t>Qubit</t> fluorometer. DNA recovery with the Epigentek kit was 33.2% ± 3.4%, with the Promega kit was 52% ± 3%, with the Diagenode kit was 55% ± 2.6% and with the Qiagen kit was 50.2% ± 2%. The BisulFlash DNA Modification kit was shown to have a significantly decreased average yield with respect to all other kits (FWER
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    Thermo Fisher superscript vilo cdna synthesis kit
    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p
    Superscript Vilo Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17594 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 7900ht fast real time pcr system
    mRNA levels of SPARC and SLUG in melanoma cells at various stages of tumor development. ( A ) Analysis of SPARC and SLUG mRNA levels: relative gene expression levels of SPARC and SLUG in cultures of melanoma cells derived from RGP, VGP or metastatic melanoma tumors were evaluated by relative <t>Q-PCR</t> using an ABI Biosystems <t>7900HT</t> Fast Real Time PCR System and the SYBR Green dye detection protocol. Data were analyzed using the 2 −ddCt method and human 18S transcript level was used to normalize for each sample. Values are the mean of independent triplicates. RGP, Radial Growth Phase; VGP, Vertical Growth Phase. ( B ) Positive correlation between SPARC and SLUG in melanoma samples: regression analysis to determine the correlation between SPARC and SLUG in human melanoma samples. R , Spearman’s Rank Correlation Coefficient; P
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    Thermo Fisher 293ft cells
    Effect of MAZ on global genome organization. ( A ) Scatter plot showing differential intra-TAD activity in WT vs MAZ KO MNs (FDR cut-off=0.05). ( B ) Scatter plot showing differential loop activity in WT vs MAZ KO MNs (all loops, n=95119, FDR cut-off=0.005, | log (Fold Change) | cut-off=1.5, Up-regulated=741, Down-regulated: 22904). ( C ) Aggregate Peak Analysis (APA) of loops in WT vs MAZ KO MNs showing ChIP-seq signals of CTCF, MAZ, or both at any region covered by them. The resolution of APA is 5 kb. Histograms showing the distribution of loop distance in MAZ KO compared to WT related to the binding level of ChIP-seq. ( D ) Western blot analysis of FLAG, RAD21, and CTCF upon FLAG-MAZ immunoprecipitation from nuclear extract of <t>293FT</t> cells. ( E ) Proposed model for the effect of MAZ on loops via its interaction with cohesin (middle) or binding adjacent to CTCF and interaction with cohesin (right), in addition to existing loop-extrusion model of CTCF and cohesin (left, ( 44 , 45 )).
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    Thermo Fisher fbs
    ShRNA-mediated silencing of TRPM7 impaired invasion in pancreatic adenocarcinoma cells. (A) <t>Non-transfected</t> BxPC-3 cells assayed for invasion using the trans-well assay in the presence of 3% <t>FBS</t> or no FBS. (B,C) BxPC-3 cells transfected with non-targeting control (NC) shRNA or anti- TRPM7 shRNA analyzed for cell invasion in the presence of either 3% FBS (B) or 10% FBS (C). A representative image of the invaded cells stained with crystal violet is shown for each experimental group. Cell invasion is expressed as % control, and each column represents the mean ± standard error.
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    Thermo Fisher sybr green pcr master mix
    SNC1-RLD encodes a truncated TNL protein. (A) Alignment of deduced amino acid sequences of SNC1-Col (top) and SNC1-RLD (bottom) using the EBI-ClustalW tool ( http://www.ebi.ac.uk/Tools/clustalw/ ) [54] . Identical amino acids are indicated by asterisks. Colons and semi-colons show conserved substitutions and semi-conserved substitutions, respectively. Characters in blue, red and <t>green</t> show the amino acids corresponding to exon 1, exon 2 and exon 3, respectively. (B) SNC1 gene model as experimentally verified by reverse transcription <t>PCR</t> and 3′-RACE from Col-0 (middle) and RLD (bottom) compared with the TAIR9 gene model (top). Exons are indicated by boxes, introns by lines, and stop codons by red asterisks.
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    Thermo Fisher generacer kit
    StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using <t>GeneRacer™</t> 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P
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    Thermo Fisher agarose gel
    StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using <t>GeneRacer™</t> 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P
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    Thermo Fisher taqman universal pcr master mix
    Acute MMP-9/2 inhibition attenuates CCI-induced allodynia and neuroinflammation. ( A ) Gelatin zymography of sciatic nerve extracts (70 μg total protein/lane) obtained at 3 h, 6 h, 1 day, 3 days, 5 days, and 7 days post-CCI. The arrows point to the MMP-9 and MMP-2 species. ( B ) <t>Taqman</t> <t>qRT-PCR</t> for MMP-9 in the sham (Sh) and CCI sciatic nerves (day 1). The mean relative mRNA ± SEM of n = 6/group normalized to GAPDH compared to naïve (N) nerve (**, P
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    Thermo Fisher rnase free dnase
    Analysis of viral protein and DNA in HAV5.EGFP capsids . (A) Proteins from lysates of <t>RNase/DNase</t> treated or untreated mature and empty/intermediate capsids were separated by 10% SDS-PAGE, and stained by SYPRO Ruby protein stains. A 100 kDa hexon protein band was used for determining the capsid protein concentration using Kodak IM Network software. (B) Total yields of DNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. ( C ) EGFP expression. 293 cells were transduced by 10 fold serially diluted mature and empty/intermediate capsids with or without RNase/DNase treatment. At 48 h post transduction, cells were analysed for EGFP expression by a fluorescent microscope (i) EGFP, (ii) Phase contrast. Presence (+). Absence (-). Mature (Ma); Empty/Intermediate (E-I).
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    Thermo Fisher dnase i
    PCR-based detection of ClC-3 in rat pancreatic tissue from early postnatal rats cDNA was produced from RNA preparations from pancreas and kidney of 5-day-old and adult rats. cDNA amplification was performed with primers specific for rat kidney ClC-3 (upper panel) and rat p24A (lower panel). RT-PCR amplification products were separated by standard agarose gel electrophoresis and stained with ethidium bromide. In controls, primers without cDNA (water control) as well as DNase I-treated RNA samples from 5-day-old and adult rats were used in the PCR reaction mixture. Using a two-step amplification protocol (‘nested PCR’) a 517 bp fragment of ClC-3 cDNA could be amplified from kidney and pancreas of 5-day-old rats, as well as from the kidney of adult rats (upper panel). The integrity of the pancreas cDNA preparation from adult animals could be shown by amplification of a 266 bp cDNA fragment of the ubiquitously expressed protein p24A (lower panel). Molecular weight (MW) markers were added to the first and last lane.
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    Image Search Results


    Monoamine oxidase-A (MAO-A) gene is upregulated by alcohol. After 24 hours treatment with 0.1% alcohol, the dendritic cells were harvested, cytoplasmic RNA from the cell pellet was extracted, reverse transcribed to cDNA, and the cDNA was used for the

    Journal: Alcoholism, clinical and experimental research

    Article Title: Upregulation of Serotonin Transporter by Alcohol in Human Dendritic Cells: Possible Implication in Neuroimmune Deregulation

    doi: 10.1111/j.1530-0277.2009.01010.x

    Figure Lengend Snippet: Monoamine oxidase-A (MAO-A) gene is upregulated by alcohol. After 24 hours treatment with 0.1% alcohol, the dendritic cells were harvested, cytoplasmic RNA from the cell pellet was extracted, reverse transcribed to cDNA, and the cDNA was used for the

    Article Snippet: Equal quantities of RNA from all the samples were reverse transcribed using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA).

    Techniques:

    Expression of the Gstp, Gstm, and Gstt genes in the mice homozygous for the deleted loci. (A) RNA was prepared from the duodenum (duo) and colon of two GstpΔ/Δ mice and their controls and was analyzed by Northern blot using the a full-length cDNA probe. The Gstp1 cDNA does not discriminated between Gstp1 and Gstp2 expression because of the high level of homology between these genes (99.3% by Wilbur-Lipman DNA alignment). As expected, a strong signal was observed in the lanes corresponding to wild-type mice but not in the lanes loaded with RNA from GstpΔ/Δ animals. A band is also detected in wild-type RNA from these tissues when analyzed with a full-length cDNA probe corresponding to Gstp3 . Although GSTP3 has 71.4% (Lipman-Pearson protein alignment) homology at the protein level with GSTP1/2, this cDNA probe has only 65% similarity index (Wilbur-Lipman DNA alignment) with Gstp1 and Gstp2 and thus will not hybridize to these transcripts in the conditions used in this study. Consistent with a deletion that encompasses this telomeric Gstp gene, this band is absent in lanes corresponding to the GstpΔ/Δ mice. To verify the quality and quantity of the bound RNA, filters were probed with mPges2 , a gene that is expressed at moderate levels throughout the intestinal tract. (B) RNA was isolated from liver, kidney, and bone marrow derived macrophages of wild-type and GsttΔ/Δ mice. Northern blot analysis using cDNA probes specific for Gstt1 and Gstt2 under stringent hybridization conditions showed high expression of both genes in these tissues. Gstt3 expression, detected using a full-length cDNA, was also observed in both liver and kidney, although longer exposure of the film was required. No signal was observed using the three Gstt probes in RNA prepared from GsttΔ/Δ mice. Gstt4 is expressed at low levels in most tissues, including bone marrow-derived macrophages. However, we identified robust expression of this gene after treatment of macrophages for 16 hours with LPS using a full-length cDNA probe specific for this gene. No signal was observed in lanes corresponding to RNA isolated from LPS-treated cells derived from GsttΔ/Δ mice. (C) Total RNA was prepared from the indicated tissues of GstmΔ/Δ mice and cohoused sex- and age-matched controls. Expression of the Gstm gene indicated in each of the panels from tissues known to express each family member was determined by qPCR using gene-specific primers obtained from Applied Biosciences. Expression levels were normalized to 18S RNA. Data were analyzed using the comparative C T method (ΔC T ) as described by Applied Biosystems. No signal (ND: not detected) was observed in all cases in samples prepared from mice homozygous for the GstmΔ/Δ locus. Values represent mean of three animals ± S.E.M.

    Journal: Drug Metabolism and Disposition

    Article Title:

    doi: 10.1124/dmd.113.056481

    Figure Lengend Snippet: Expression of the Gstp, Gstm, and Gstt genes in the mice homozygous for the deleted loci. (A) RNA was prepared from the duodenum (duo) and colon of two GstpΔ/Δ mice and their controls and was analyzed by Northern blot using the a full-length cDNA probe. The Gstp1 cDNA does not discriminated between Gstp1 and Gstp2 expression because of the high level of homology between these genes (99.3% by Wilbur-Lipman DNA alignment). As expected, a strong signal was observed in the lanes corresponding to wild-type mice but not in the lanes loaded with RNA from GstpΔ/Δ animals. A band is also detected in wild-type RNA from these tissues when analyzed with a full-length cDNA probe corresponding to Gstp3 . Although GSTP3 has 71.4% (Lipman-Pearson protein alignment) homology at the protein level with GSTP1/2, this cDNA probe has only 65% similarity index (Wilbur-Lipman DNA alignment) with Gstp1 and Gstp2 and thus will not hybridize to these transcripts in the conditions used in this study. Consistent with a deletion that encompasses this telomeric Gstp gene, this band is absent in lanes corresponding to the GstpΔ/Δ mice. To verify the quality and quantity of the bound RNA, filters were probed with mPges2 , a gene that is expressed at moderate levels throughout the intestinal tract. (B) RNA was isolated from liver, kidney, and bone marrow derived macrophages of wild-type and GsttΔ/Δ mice. Northern blot analysis using cDNA probes specific for Gstt1 and Gstt2 under stringent hybridization conditions showed high expression of both genes in these tissues. Gstt3 expression, detected using a full-length cDNA, was also observed in both liver and kidney, although longer exposure of the film was required. No signal was observed using the three Gstt probes in RNA prepared from GsttΔ/Δ mice. Gstt4 is expressed at low levels in most tissues, including bone marrow-derived macrophages. However, we identified robust expression of this gene after treatment of macrophages for 16 hours with LPS using a full-length cDNA probe specific for this gene. No signal was observed in lanes corresponding to RNA isolated from LPS-treated cells derived from GsttΔ/Δ mice. (C) Total RNA was prepared from the indicated tissues of GstmΔ/Δ mice and cohoused sex- and age-matched controls. Expression of the Gstm gene indicated in each of the panels from tissues known to express each family member was determined by qPCR using gene-specific primers obtained from Applied Biosciences. Expression levels were normalized to 18S RNA. Data were analyzed using the comparative C T method (ΔC T ) as described by Applied Biosystems. No signal (ND: not detected) was observed in all cases in samples prepared from mice homozygous for the GstmΔ/Δ locus. Values represent mean of three animals ± S.E.M.

    Article Snippet: Reverse transcription of RNA to cDNA for quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.

    Techniques: Expressing, Mouse Assay, Northern Blot, Isolation, Derivative Assay, Hybridization, Real-time Polymerase Chain Reaction

    IRF3 is phosphorylated, translocates to the nucleus, and binds the CXCL10 promoter in HT29 cells in response to addition of poly(I:C). A , Western blots of HT29 cells were stimulated with poly(I:C) alone ( Poly(I:C) , 2.5 μg/ml) or transfected with poly(I:C) using Lipofectamine RNAimax ( LF + Poly(I:C) , 0–1200 min) and stained with antibodies against phospho-IRF3 Ser-396 , total IRF3, phosphor-p65 Ser-536 , total p65, or GAPDH. The results are representative of two independent experiments. MW , molecular weight. B and C , nuclear accumulation of IRF3 ( B ) and IRF1 ( C ) in HT29 cells left untreated ( 0 ), stimulated with poly(I:C) (5–2 μg/ml), or transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml) for 3 h or overnight ( o/n ). Stimulated cells were fixed and immunostained for IRF3 or IRF1, and cell nuclei were stained with Hoechst 3342. Cells were visualized by automated imaging, and analysis was done using ScanR. The results show the percentage of cells with positive staining of IRF3 and IRF1 in the nucleus. The results show mean ± S.D. of triplicate samples with a minimum of 1300 cells assayed and are representative of three independent experiments. D , CXCL10 promotor occupancy by IRF3 in HT29 cells after poly(I:C) (2 μg/ml) stimulation for 3 h. IRF3 binding to the CXCL10 promoter was investigated by ChIP followed by qPCR of the CXCL10 promoter region. RNA polymerase II occupancy was measured as a control. E and F , CXCL10 production ( left panels ) and IRF mRNA expression ( right panels ) in HT29 cells left untreated ( No add ), treated with siRNA against IRF3 ( E ) or IRF7 ( F ) (10 n m ), NS RNA (10 n m ), or transfection reagent alone ( LF ) for 24 h. Cells were subsequently stimulated with poly(I:C) (2.5 μg/ml) for 6 h. CXCL10 release was assessed by ELISA, whereas silencing of IRF3 and 7 was confirmed by assessing mRNA expression by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicate samples.

    Journal: The Journal of Biological Chemistry

    Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

    doi: 10.1074/jbc.M117.784090

    Figure Lengend Snippet: IRF3 is phosphorylated, translocates to the nucleus, and binds the CXCL10 promoter in HT29 cells in response to addition of poly(I:C). A , Western blots of HT29 cells were stimulated with poly(I:C) alone ( Poly(I:C) , 2.5 μg/ml) or transfected with poly(I:C) using Lipofectamine RNAimax ( LF + Poly(I:C) , 0–1200 min) and stained with antibodies against phospho-IRF3 Ser-396 , total IRF3, phosphor-p65 Ser-536 , total p65, or GAPDH. The results are representative of two independent experiments. MW , molecular weight. B and C , nuclear accumulation of IRF3 ( B ) and IRF1 ( C ) in HT29 cells left untreated ( 0 ), stimulated with poly(I:C) (5–2 μg/ml), or transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml) for 3 h or overnight ( o/n ). Stimulated cells were fixed and immunostained for IRF3 or IRF1, and cell nuclei were stained with Hoechst 3342. Cells were visualized by automated imaging, and analysis was done using ScanR. The results show the percentage of cells with positive staining of IRF3 and IRF1 in the nucleus. The results show mean ± S.D. of triplicate samples with a minimum of 1300 cells assayed and are representative of three independent experiments. D , CXCL10 promotor occupancy by IRF3 in HT29 cells after poly(I:C) (2 μg/ml) stimulation for 3 h. IRF3 binding to the CXCL10 promoter was investigated by ChIP followed by qPCR of the CXCL10 promoter region. RNA polymerase II occupancy was measured as a control. E and F , CXCL10 production ( left panels ) and IRF mRNA expression ( right panels ) in HT29 cells left untreated ( No add ), treated with siRNA against IRF3 ( E ) or IRF7 ( F ) (10 n m ), NS RNA (10 n m ), or transfection reagent alone ( LF ) for 24 h. Cells were subsequently stimulated with poly(I:C) (2.5 μg/ml) for 6 h. CXCL10 release was assessed by ELISA, whereas silencing of IRF3 and 7 was confirmed by assessing mRNA expression by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicate samples.

    Article Snippet: Silencing of TLR3 and TRIF SW620 or HT29 cells were transfected for 24–48 h with 10–20 nm siRNA against TLR3 (TLR3_5, Qiagen, SI02630768; TLR3_8, Qiagen, SI02655156) or non-silencing control siRNA (Qiagen, SI03650325) using Lipofectamine RNAiMAX (Invitrogen) for TLR3 knockdown or with siRNA against TICAM-1/TRIF (Ambion, s45115) for TICAM/TRIF knockdown using Lipofectamine RNAiMAX. siRNA and transfection reagent (ratio, 1:2) were preincubated for 15 min in RPMI medium before being added to newly seeded cells.

    Techniques: Western Blot, Transfection, Staining, Molecular Weight, Imaging, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    IFNβ is induced in IECs in response to transfection with poly(I:C) but not in response to poly(I:C) addition. A , HT29 cells were left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before CXCL10 in the supernatant was assessed by ELISA. B , kinetics of CXCL10 release assessed by ELISA in supernatant from HT29 cells stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 5, 12, 20, and 24 h. The results are presented as mean ± S.D. of triplicates. C , IFNβ mRNA induction in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone ( Poly(I:C) ), transfected with poly(I:C) complexed with Lipofectamine RNAimax ( LF + Poly(I:C) , 2 μg/ml), or treated with only Lipofectamine RNAimax ( LF ) for 20 h. IFNβ mRNA induction was determined by qPCR. The results are presented as relative induction compared with medium-treated Caco-2 cells. GAPDH served as an internal control. Results show mean -fold induction ± S.D. of triplicates. D , IFNβ protein production in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone, transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 20 h. IFNβ in the supernatant was assessed by ELISA, and the results show mean ± S.D. of three samples. E , HT29 cells were stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 6, 12, 20, or 24 h before CXCL10 and IFNβ mRNA induction was determined by qPCR. The results show relative induction with a non-treated sample as reference. GAPDH served as an internal control. The results show mean -fold induction ± S.D. of triplicates. F , CXCL10 mRNA induction in HT29 cells pretreated with cycloheximide (0, 15, or 30 μg/ml) for 30 min prior to stimulation with poly(I:C) (2.5 μg/ml) for 8 h. CXCL10 mRNA was determined by qPCR (normalized to medium control and the endogenous control TBP). G , viability in HT29 cells left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before viability was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. H , viability in IECs left untreated ( 0 ), stimulated with poly(I:C) alone (2 μg/ml), transfected with poly(I:C) using Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 43 h before the viability of the cells was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. The results show mean ± S.D. of five samples. All results are representative of at least two independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

    doi: 10.1074/jbc.M117.784090

    Figure Lengend Snippet: IFNβ is induced in IECs in response to transfection with poly(I:C) but not in response to poly(I:C) addition. A , HT29 cells were left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before CXCL10 in the supernatant was assessed by ELISA. B , kinetics of CXCL10 release assessed by ELISA in supernatant from HT29 cells stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 5, 12, 20, and 24 h. The results are presented as mean ± S.D. of triplicates. C , IFNβ mRNA induction in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone ( Poly(I:C) ), transfected with poly(I:C) complexed with Lipofectamine RNAimax ( LF + Poly(I:C) , 2 μg/ml), or treated with only Lipofectamine RNAimax ( LF ) for 20 h. IFNβ mRNA induction was determined by qPCR. The results are presented as relative induction compared with medium-treated Caco-2 cells. GAPDH served as an internal control. Results show mean -fold induction ± S.D. of triplicates. D , IFNβ protein production in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone, transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 20 h. IFNβ in the supernatant was assessed by ELISA, and the results show mean ± S.D. of three samples. E , HT29 cells were stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 6, 12, 20, or 24 h before CXCL10 and IFNβ mRNA induction was determined by qPCR. The results show relative induction with a non-treated sample as reference. GAPDH served as an internal control. The results show mean -fold induction ± S.D. of triplicates. F , CXCL10 mRNA induction in HT29 cells pretreated with cycloheximide (0, 15, or 30 μg/ml) for 30 min prior to stimulation with poly(I:C) (2.5 μg/ml) for 8 h. CXCL10 mRNA was determined by qPCR (normalized to medium control and the endogenous control TBP). G , viability in HT29 cells left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before viability was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. H , viability in IECs left untreated ( 0 ), stimulated with poly(I:C) alone (2 μg/ml), transfected with poly(I:C) using Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 43 h before the viability of the cells was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. The results show mean ± S.D. of five samples. All results are representative of at least two independent experiments.

    Article Snippet: Silencing of TLR3 and TRIF SW620 or HT29 cells were transfected for 24–48 h with 10–20 nm siRNA against TLR3 (TLR3_5, Qiagen, SI02630768; TLR3_8, Qiagen, SI02655156) or non-silencing control siRNA (Qiagen, SI03650325) using Lipofectamine RNAiMAX (Invitrogen) for TLR3 knockdown or with siRNA against TICAM-1/TRIF (Ambion, s45115) for TICAM/TRIF knockdown using Lipofectamine RNAiMAX. siRNA and transfection reagent (ratio, 1:2) were preincubated for 15 min in RPMI medium before being added to newly seeded cells.

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, MTT Assay

    Functional characteristics of miR-548aq-3p as anti-angiogenic miRNAs. ( A ) Healthy ECFCs were transduced with mock lentivirus (M) or lentivirus overexpressing miR-548aq-3p (OE). 48 hours after transduction, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( B ) Representative images from the tube formation assays of non-transduced controls treated with or without 10 nM vinblastine (VB), mock infected (M) and miR-548aq-3p overexpressed (OE) healthy ECFCs (original magnification 10×, scale bar 300 μm). ( C ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( B ). ( D ) CAD ECFCs were transfected with mock (M) or miR-548aq-3p inhibitor (KD) using Lipofectamine RNAiMAX. 48 hours after transfection, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( E ) Representative images from the tube formation assay of CAD ECFCs treated with or without 10 nM vinblastine (VB), mock transfected (M) and miR-548aq-3p knockdown (KD) (original magnification 10×, scale bar 300 μm). ( F ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( E ).

    Journal: Scientific Reports

    Article Title: miR-548aq-3p is a novel target of Far infrared radiation which predicts coronary artery disease endothelial colony forming cell responsiveness

    doi: 10.1038/s41598-020-63311-1

    Figure Lengend Snippet: Functional characteristics of miR-548aq-3p as anti-angiogenic miRNAs. ( A ) Healthy ECFCs were transduced with mock lentivirus (M) or lentivirus overexpressing miR-548aq-3p (OE). 48 hours after transduction, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( B ) Representative images from the tube formation assays of non-transduced controls treated with or without 10 nM vinblastine (VB), mock infected (M) and miR-548aq-3p overexpressed (OE) healthy ECFCs (original magnification 10×, scale bar 300 μm). ( C ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( B ). ( D ) CAD ECFCs were transfected with mock (M) or miR-548aq-3p inhibitor (KD) using Lipofectamine RNAiMAX. 48 hours after transfection, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( E ) Representative images from the tube formation assay of CAD ECFCs treated with or without 10 nM vinblastine (VB), mock transfected (M) and miR-548aq-3p knockdown (KD) (original magnification 10×, scale bar 300 μm). ( F ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( E ).

    Article Snippet: To knockdown miR-548aq-3p in ECFCs, a commercial synthetic miRIDIAN microRNA Hairpin Inhibitor (hsa-miR-548aq-3p, IH-302531-01-0005) (Dharmacon, Lafayette, CO, USA) was added to the culture medium at a final concentration of 20 nM at 70~80% cell confluence using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA).

    Techniques: Functional Assay, Transduction, Expressing, Quantitative RT-PCR, Infection, Transfection, Tube Formation Assay

    Intracellular amount of emodin in K562/ADM and K562 cells. Both cells were incubated with various concentrations of emodin (0.1–10 μM) at 37°C or 4°C for 2 h (A); K562/ADM cells were co-treated with 0, 1, 3, 10 μM of verapamil or 0, 2, 4, 10 μM of cyclosporine A and emodin (5 μM) for 2 h (B). RNAi of P-gp (C). A-: K562/ADM cells transfected with negative control siRNA; AL: K562/ADM cells transfected with Lipofectamine RNAiMAX Reagent; AP: K562/ADM cells transfected with P-gp siRNA; A: K562/ADM cells; S: K562 cells. After transfection with P-gp siRNA, intracellular amount of emodin (5 μM) was enhanced compared with other control groups (D). Data were represented as the mean ± S.D. of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein

    doi: 10.1371/journal.pone.0187971

    Figure Lengend Snippet: Intracellular amount of emodin in K562/ADM and K562 cells. Both cells were incubated with various concentrations of emodin (0.1–10 μM) at 37°C or 4°C for 2 h (A); K562/ADM cells were co-treated with 0, 1, 3, 10 μM of verapamil or 0, 2, 4, 10 μM of cyclosporine A and emodin (5 μM) for 2 h (B). RNAi of P-gp (C). A-: K562/ADM cells transfected with negative control siRNA; AL: K562/ADM cells transfected with Lipofectamine RNAiMAX Reagent; AP: K562/ADM cells transfected with P-gp siRNA; A: K562/ADM cells; S: K562 cells. After transfection with P-gp siRNA, intracellular amount of emodin (5 μM) was enhanced compared with other control groups (D). Data were represented as the mean ± S.D. of three independent experiments. * P

    Article Snippet: The Lipofectamine RNAiMAX Reagent was purchased from Invitrogen Trading Co., Ltd (Shanghai, China).

    Techniques: Incubation, Transfection, Negative Control

    Colony survival assay in A549 cells following transfection with Lipofectamine 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.

    Journal: Molecules

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy

    doi: 10.3390/molecules25081994

    Figure Lengend Snippet: Colony survival assay in A549 cells following transfection with Lipofectamine 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.

    Article Snippet: Negative controls consisted of untreated cells or cells transfected with control scrambled siRNA while positive control cells were transfected with Lipofectamine 2000 (Life Technologies) lipoplexes, which were prepared as described above.

    Techniques: Clonogenic Cell Survival Assay, Transfection, Negative Control, Clonogenic Assay, Software

    ERCC1 and XPF gene knockdown efficiency was validated in lung adenocarcinoma cells (A549) after 72 h following double transfection of Lipofectamine 2000 (LF) or micelleplexes (PPP) with 100 pmol ERCC1-XPF siRNA or negative control (NC) siRNA. ERCC1 and XPF expression was normalized with GAPDH expression and quantified by real time PCR. Data points indicate mean ± SD. (n = 6).

    Journal: Molecules

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy

    doi: 10.3390/molecules25081994

    Figure Lengend Snippet: ERCC1 and XPF gene knockdown efficiency was validated in lung adenocarcinoma cells (A549) after 72 h following double transfection of Lipofectamine 2000 (LF) or micelleplexes (PPP) with 100 pmol ERCC1-XPF siRNA or negative control (NC) siRNA. ERCC1 and XPF expression was normalized with GAPDH expression and quantified by real time PCR. Data points indicate mean ± SD. (n = 6).

    Article Snippet: Negative controls consisted of untreated cells or cells transfected with control scrambled siRNA while positive control cells were transfected with Lipofectamine 2000 (Life Technologies) lipoplexes, which were prepared as described above.

    Techniques: Transfection, Negative Control, Expressing, Real-time Polymerase Chain Reaction

    Western Blot analysis of excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum group F (XPF) protein levels within A549 cells following ( A ) single transfection of micelleplexes (PPP) loaded with 50 pmol small interfering RNA (siRNA) after 72 h and ( B ) double transfection of micelleplexes (PPP) loaded with 100 pmol siRNA after 72h. Lipofectamine 2000 (LF) lipoplexes were prepared according to manufacturer’s protocol with 50 pmol (A) and 100 pmol (B) siRNA (n = 2).

    Journal: Molecules

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy

    doi: 10.3390/molecules25081994

    Figure Lengend Snippet: Western Blot analysis of excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum group F (XPF) protein levels within A549 cells following ( A ) single transfection of micelleplexes (PPP) loaded with 50 pmol small interfering RNA (siRNA) after 72 h and ( B ) double transfection of micelleplexes (PPP) loaded with 100 pmol siRNA after 72h. Lipofectamine 2000 (LF) lipoplexes were prepared according to manufacturer’s protocol with 50 pmol (A) and 100 pmol (B) siRNA (n = 2).

    Article Snippet: Negative controls consisted of untreated cells or cells transfected with control scrambled siRNA while positive control cells were transfected with Lipofectamine 2000 (Life Technologies) lipoplexes, which were prepared as described above.

    Techniques: Western Blot, Transfection, Small Interfering RNA

    Effects of dominant negative form of monomeric GTP-binding proteins on the induction of the promoter activity of IL-6 by H. pylori infection. MKN28 cells were cotransfected with 0.5 μg of the p1168hu.IL6P-luc+ plasmid together with 0.3 μg of one of the four plasmids, pCMV- RhoN19 , pCMV- RacN17 , pCMV- RasN17 , or pCMV plus 10 ng of Renilla plasmid as an internal control using LipofectAMINE. The transfected cells were subsequently infected with H. pylori . MKN28 cells were also cotransfected with the p1168hu.IL6P-luc+ plasmid together with either 50 ng of pCMV or pCMV-RafS621A plus 10 ng of Renilla plasmid and subsequently infected with H. pylori . Untreated plates served as controls. Shown is the normalized luciferase activity expressed as fold increase of luciferase activity in H. pylori -infected cells relative to uninfected controls. Five independent transfections, each run in triplicate, were performed. Data are expressed as mean ± SE; ** p

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of Interleukin-6 Promoter Activation in Gastric Epithelial Cells Infected with Helicobacter pylori

    doi: 10.1091/mbc.E05-05-0426

    Figure Lengend Snippet: Effects of dominant negative form of monomeric GTP-binding proteins on the induction of the promoter activity of IL-6 by H. pylori infection. MKN28 cells were cotransfected with 0.5 μg of the p1168hu.IL6P-luc+ plasmid together with 0.3 μg of one of the four plasmids, pCMV- RhoN19 , pCMV- RacN17 , pCMV- RasN17 , or pCMV plus 10 ng of Renilla plasmid as an internal control using LipofectAMINE. The transfected cells were subsequently infected with H. pylori . MKN28 cells were also cotransfected with the p1168hu.IL6P-luc+ plasmid together with either 50 ng of pCMV or pCMV-RafS621A plus 10 ng of Renilla plasmid and subsequently infected with H. pylori . Untreated plates served as controls. Shown is the normalized luciferase activity expressed as fold increase of luciferase activity in H. pylori -infected cells relative to uninfected controls. Five independent transfections, each run in triplicate, were performed. Data are expressed as mean ± SE; ** p

    Article Snippet: MKN28 cells were transfected with TranSilent shRNA Vectors for NF-κB p50 and NF-κB p65 (0.75 μg for each) or 1.5 μg of empty vectors (Panomics, Redwood City, CA) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions.

    Techniques: Dominant Negative Mutation, Binding Assay, Activity Assay, Infection, Plasmid Preparation, Transfection, Luciferase

    Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.

    Article Snippet: The virion RNA (vRNA) of cell-bound HCV was isolated with a Trizol Reagent (Invitrogen) or RNAzol Reagent (Molecular Research Center).

    Techniques: Inhibition, Purification, Incubation, Cell Culture, Quantitative RT-PCR

    Blockade of HCV1b cell attachment by apoE monoclonal antibody mAb23. DHHs at day-11 were incubated with HCV1b in the absence (Control) or presence of 10 µg/ml of normal mouse IgG1 (mIgG1) or increasing amounts of apoE mAb23 (0.4, 2, and 10 µg/ml) at 37°C for 2 hrs. The unbound HCV was removed by washing cells with 1x PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV vRNA were quantified by a real-time RT-PCR method using SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen). Reactions were run in a StepOnePlus real-time PCR system (Applied Biosystems) using the conditions provided by the qPCR kit. A house-keeping gene GAPDH was used as an internal control, which was quantified using Hu-GAPDH primer/probe mix containing vic-TAMRA (Applied Biosystems). The levels of HCV vRNA were calculated from the average data of three experiments upon normalization with the level of GAPDH.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Blockade of HCV1b cell attachment by apoE monoclonal antibody mAb23. DHHs at day-11 were incubated with HCV1b in the absence (Control) or presence of 10 µg/ml of normal mouse IgG1 (mIgG1) or increasing amounts of apoE mAb23 (0.4, 2, and 10 µg/ml) at 37°C for 2 hrs. The unbound HCV was removed by washing cells with 1x PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV vRNA were quantified by a real-time RT-PCR method using SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen). Reactions were run in a StepOnePlus real-time PCR system (Applied Biosystems) using the conditions provided by the qPCR kit. A house-keeping gene GAPDH was used as an internal control, which was quantified using Hu-GAPDH primer/probe mix containing vic-TAMRA (Applied Biosystems). The levels of HCV vRNA were calculated from the average data of three experiments upon normalization with the level of GAPDH.

    Article Snippet: The virion RNA (vRNA) of cell-bound HCV was isolated with a Trizol Reagent (Invitrogen) or RNAzol Reagent (Molecular Research Center).

    Techniques: Cell Attachment Assay, Incubation, Quantitative RT-PCR, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Effect of heparinase treatment on HCV1b attachment to DHHs. The day-11 DHHs in 12-well cell culture plates were incubated with varying concentrations of heparinase I in a buffer containing 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 4 mM CaCl 2 and 0.01% bovine serum albumin at 37°C for 1 hr [18] . The heparinase-treated DHHs were then incubated with HCV1b on ice for 2 hrs. The unbound HCV was removed and the cells were washed with 1x PBS for three times. The HCV1b vRNA of the cell-bound HCV was extracted with Trizol reagent and quantified by RT-qPCR using the StepOnePlus real-time PCR system same as that in Fig. 1.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Effect of heparinase treatment on HCV1b attachment to DHHs. The day-11 DHHs in 12-well cell culture plates were incubated with varying concentrations of heparinase I in a buffer containing 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 4 mM CaCl 2 and 0.01% bovine serum albumin at 37°C for 1 hr [18] . The heparinase-treated DHHs were then incubated with HCV1b on ice for 2 hrs. The unbound HCV was removed and the cells were washed with 1x PBS for three times. The HCV1b vRNA of the cell-bound HCV was extracted with Trizol reagent and quantified by RT-qPCR using the StepOnePlus real-time PCR system same as that in Fig. 1.

    Article Snippet: The virion RNA (vRNA) of cell-bound HCV was isolated with a Trizol Reagent (Invitrogen) or RNAzol Reagent (Molecular Research Center).

    Techniques: Cell Culture, Incubation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).

    Article Snippet: The virion RNA (vRNA) of cell-bound HCV was isolated with a Trizol Reagent (Invitrogen) or RNAzol Reagent (Molecular Research Center).

    Techniques: Inhibition, Derivative Assay, Binding Assay, Cell Culture, Incubation, Quantitative RT-PCR, Cell Attachment Assay

    In vivo expression of GlMBP1 in G. lamblia trophozoites. (A) Quantitative measurement of GlMBP1 transcripts. Total RNA was isolated from G. lamblia using TRIzol. cDNA was synthesized from 5 µg of RNA using the ImProm-II TM RT system and then analyzed with the Light Cycler 480 II Real-Time PCR System using LightCycler 490 DNA SYBR Green I Master (Roche Applied Science). Conditions for real-time PCR were as follows: pre-incubation at 95˚C for 5 min followed by 45 amplification cycles of 95˚C for 10 sec, 56˚C for 20 sec, and 72˚C for 10 sec. Real-time PCR was carried out in triplicate in a 96-well plate using the specific primers listed in Table 1 . The tim gene encoding triose-1-phosphate isomerase of G. lamblia was used as an endogenous control for the reactions. (B) Western blot analysis. Ten µg of Giardia extracts was separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with anti-GlMBP1 antibodies (1:1,000 dilution), followed by secondary antibodies (1:1,000 dilution).

    Journal: The Korean Journal of Parasitology

    Article Title: Identification of a Novel Microtubule-Binding Protein in Giardia lamblia

    doi: 10.3347/kjp.2016.54.4.461

    Figure Lengend Snippet: In vivo expression of GlMBP1 in G. lamblia trophozoites. (A) Quantitative measurement of GlMBP1 transcripts. Total RNA was isolated from G. lamblia using TRIzol. cDNA was synthesized from 5 µg of RNA using the ImProm-II TM RT system and then analyzed with the Light Cycler 480 II Real-Time PCR System using LightCycler 490 DNA SYBR Green I Master (Roche Applied Science). Conditions for real-time PCR were as follows: pre-incubation at 95˚C for 5 min followed by 45 amplification cycles of 95˚C for 10 sec, 56˚C for 20 sec, and 72˚C for 10 sec. Real-time PCR was carried out in triplicate in a 96-well plate using the specific primers listed in Table 1 . The tim gene encoding triose-1-phosphate isomerase of G. lamblia was used as an endogenous control for the reactions. (B) Western blot analysis. Ten µg of Giardia extracts was separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with anti-GlMBP1 antibodies (1:1,000 dilution), followed by secondary antibodies (1:1,000 dilution).

    Article Snippet: Total RNA was isolated from G. lamblia , using TRIzol (Invitrogen, Carlsbad, California, USA). cDNA was synthesized from 5 µg of RNA using the ImProm-IITM RT system (Promega, Madison, Wisconsin, USA) following the manufacturer’s directions. cDNA was then analyzed in the Light Cycler 480 II Real-Time PCR System (Roche Applied Science, Mannheim, Germany) using LightCycler 490 DNA SYBR Green I Master (Roche Applied Science).

    Techniques: In Vivo, Expressing, Isolation, Synthesized, Real-time Polymerase Chain Reaction, SYBR Green Assay, Incubation, Amplification, Size-exclusion Chromatography, Western Blot, SDS Page

    GS-5806 inhibits RSV fusion, but not attachment, to HEp-2 cells. (A) HEp-2 cells were infected with RSV A2 at 4°C for 3 h, washed to remove unbound virus, and then shifted to 37°C for 30 h to facilitate fusion; 50 μg/ml heparin, 1.25 nM GS-5806, 20 nM VP-14637, or 100 nM BMS-433771 was added to the cells either throughout the experiment, only during viral attachment, or only during viral fusion. Intercellular RSV RNA was quantified by quantitative PCR (qPCR) and compared to an uninhibited DMSO-treated infection. The error bars indicate standard deviations. (B) GS-5806 (10 nM) inhibits syncytium formation in 293T cells transiently expressing the RSV F protein.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: GS-5806 Inhibits a Broad Range of Respiratory Syncytial Virus Clinical Isolates by Blocking the Virus-Cell Fusion Process

    doi: 10.1128/AAC.01497-15

    Figure Lengend Snippet: GS-5806 inhibits RSV fusion, but not attachment, to HEp-2 cells. (A) HEp-2 cells were infected with RSV A2 at 4°C for 3 h, washed to remove unbound virus, and then shifted to 37°C for 30 h to facilitate fusion; 50 μg/ml heparin, 1.25 nM GS-5806, 20 nM VP-14637, or 100 nM BMS-433771 was added to the cells either throughout the experiment, only during viral attachment, or only during viral fusion. Intercellular RSV RNA was quantified by quantitative PCR (qPCR) and compared to an uninhibited DMSO-treated infection. The error bars indicate standard deviations. (B) GS-5806 (10 nM) inhibits syncytium formation in 293T cells transiently expressing the RSV F protein.

    Article Snippet: Approximately 25 ng of purified RNA was added to a PCR mixture that contained 0.9 μM RSV N Forward (5′-ATCCAGCAAATACACCATCCA-3′) and RSV N Reverse (5′-TTCTGCACATCATAATTAGGAGTATCAA-3′) primers, 0.2 μM RSV N probe (6-carboxyfluorescein [FAM]-CGGAGCACAGGAGAT-black hole quencher 1 [BHQ1]) and 1× TaqMan RNA-to-Ct 1-Step kit (Applied Biosystems, Foster City, CA).

    Techniques: Infection, Real-time Polymerase Chain Reaction, Expressing

    Detecting the expression of 11 selected miRNA in cardiac tissue samples. Total RNA was extracted from tissue samples using Trizol reagent following the manufacturer’s instructions. The expression level of candidate miRNAs was detected by TaqMan miRNA RT Real-Time PCR. Single-stranded cDNA was synthesized by using the TaqMan MicroRNA Reverse Transcription kit and then amplified by using TaqMan Universal PCR Master Mix together with miRNA-specific TaqMan MGB probes. The U6 snRNA was used for normalization. Each sample in each group was measured in triplicate and the experiment was repeated at least 3 times for the detection of miRNAs. The t test was used to analyze the results and P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Down-Regulation of miR-101 Contributes to Rheumatic Heart Disease Through Up-Regulating TLR2

    doi: 10.12659/MSM.893540

    Figure Lengend Snippet: Detecting the expression of 11 selected miRNA in cardiac tissue samples. Total RNA was extracted from tissue samples using Trizol reagent following the manufacturer’s instructions. The expression level of candidate miRNAs was detected by TaqMan miRNA RT Real-Time PCR. Single-stranded cDNA was synthesized by using the TaqMan MicroRNA Reverse Transcription kit and then amplified by using TaqMan Universal PCR Master Mix together with miRNA-specific TaqMan MGB probes. The U6 snRNA was used for normalization. Each sample in each group was measured in triplicate and the experiment was repeated at least 3 times for the detection of miRNAs. The t test was used to analyze the results and P

    Article Snippet: Total RNA was extracted from tissue samples using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. miRNA expression level was determined by TaqMan miRNA RT Real-Time PCR.

    Techniques: Expressing, miRNA RT, Real-time Polymerase Chain Reaction, Synthesized, Amplification, Polymerase Chain Reaction

    Real-time PCR showing the relative RNA expression level of Jam2 in mice with different genotypes. (A) TaqMan assay showed about 1.5-fold increase of Jam2 expression in Ts65Dn mice compared to WT. (B) TaqMan assay showed about 40% decrease of Jam2 expression

    Journal: Genetics

    Article Title: Penetrance of Congenital Heart Disease in a Mouse Model of Down Syndrome Depends on a Trisomic Potentiator of a Disomic Modifier

    doi: 10.1534/genetics.116.188045

    Figure Lengend Snippet: Real-time PCR showing the relative RNA expression level of Jam2 in mice with different genotypes. (A) TaqMan assay showed about 1.5-fold increase of Jam2 expression in Ts65Dn mice compared to WT. (B) TaqMan assay showed about 40% decrease of Jam2 expression

    Article Snippet: PCR was carried out using Taqman Gene Expression Assays (Applied Biosystems, Foster City, CA).

    Techniques: Real-time Polymerase Chain Reaction, RNA Expression, Mouse Assay, TaqMan Assay, Expressing

    Q-RT-PCR using TaqMan® (Life Technologies) for (A) heat shock protein 70 (Hsp70; Hspa1a) and (B) heat shock protein 40 (Hsp40; Dnajb1) performed on whole liver samples from Sham (n = 6), trauma with hemorrhagic shock (T/HS, n = 4), and T/HS animals resuscitated with IL-6 (T/HS-IL6, n = 4). Transcript values reported as relative quantification (RQ) in comparison to a normal rat liver. Values expressed as mean RQ ± SEM. “*”, “**” indicate group comparisons which are statistically different (p

    Journal: Scientific Reports

    Article Title: Modulation of the Unfolded Protein Response During Hepatocyte and Cardiomyocyte Apoptosis In Trauma/Hemorrhagic Shock

    doi: 10.1038/srep01187

    Figure Lengend Snippet: Q-RT-PCR using TaqMan® (Life Technologies) for (A) heat shock protein 70 (Hsp70; Hspa1a) and (B) heat shock protein 40 (Hsp40; Dnajb1) performed on whole liver samples from Sham (n = 6), trauma with hemorrhagic shock (T/HS, n = 4), and T/HS animals resuscitated with IL-6 (T/HS-IL6, n = 4). Transcript values reported as relative quantification (RQ) in comparison to a normal rat liver. Values expressed as mean RQ ± SEM. “*”, “**” indicate group comparisons which are statistically different (p

    Article Snippet: Briefly, total RNA (1 μg) was reverse transcribed using reverse transcription reagents (BioRad catalog no. 170-8842; Hercules, CA); 20% of each RT reaction was used in duplicate PCR reactions using TaqMan® Universal Master Mix II, with uracil N-glycosylase (PN 4440038) and specific primer and probe sets designed by the manufacturer (TaqMan Gene Expression Assay, Applied Biosystems, Darmstadt, Germany)—Hsp70 (Hspa1a; catalog no. Rn04224718_u1), Hsp40 (Dnajb1; catalog no. Rn 01426952_g1), and 18S rRNA (catalog no.Rn03928990_g1).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    DNA degradation. DNA recovery after bisulfite treatment was determined with the Qubit fluorometer. DNA recovery with the Epigentek kit was 33.2% ± 3.4%, with the Promega kit was 52% ± 3%, with the Diagenode kit was 55% ± 2.6% and with the Qiagen kit was 50.2% ± 2%. The BisulFlash DNA Modification kit was shown to have a significantly decreased average yield with respect to all other kits (FWER

    Journal: PLoS ONE

    Article Title: Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

    doi: 10.1371/journal.pone.0135058

    Figure Lengend Snippet: DNA degradation. DNA recovery after bisulfite treatment was determined with the Qubit fluorometer. DNA recovery with the Epigentek kit was 33.2% ± 3.4%, with the Promega kit was 52% ± 3%, with the Diagenode kit was 55% ± 2.6% and with the Qiagen kit was 50.2% ± 2%. The BisulFlash DNA Modification kit was shown to have a significantly decreased average yield with respect to all other kits (FWER

    Article Snippet: DNA yield and degradation In order to compare the DNA yield and hence, the degradation effect of each of the four different kits we used the Qubit fluorometer (Invitrogen) to determine the bisulfite-treated DNA concentration.

    Techniques: Modification

    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and cDNA were prepared using the RNesay mini kit (Qiagen) and SuperScript VILO cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p

    Journal: PLoS ONE

    Article Title: Mitochondrial DAMPs Increase Endothelial Permeability through Neutrophil Dependent and Independent Pathways

    doi: 10.1371/journal.pone.0059989

    Figure Lengend Snippet: mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and cDNA were prepared using the RNesay mini kit (Qiagen) and SuperScript VILO cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p

    Article Snippet: RNA and cDNA Preparation and Quantitative PCR RNA and cDNA were prepared using the RNesay mini kit (Qiagen, Valencia, CA) and the SuperScript VILO cDNA Synthesis kit (Life Technologies, Carlsbad, CA), respectively.

    Techniques: Expressing, Size-exclusion Chromatography, Isolation, Incubation, Real-time Polymerase Chain Reaction

    mRNA levels of SPARC and SLUG in melanoma cells at various stages of tumor development. ( A ) Analysis of SPARC and SLUG mRNA levels: relative gene expression levels of SPARC and SLUG in cultures of melanoma cells derived from RGP, VGP or metastatic melanoma tumors were evaluated by relative Q-PCR using an ABI Biosystems 7900HT Fast Real Time PCR System and the SYBR Green dye detection protocol. Data were analyzed using the 2 −ddCt method and human 18S transcript level was used to normalize for each sample. Values are the mean of independent triplicates. RGP, Radial Growth Phase; VGP, Vertical Growth Phase. ( B ) Positive correlation between SPARC and SLUG in melanoma samples: regression analysis to determine the correlation between SPARC and SLUG in human melanoma samples. R , Spearman’s Rank Correlation Coefficient; P

    Journal: PLoS ONE

    Article Title: The Epithelial-Mesenchymal Transition (EMT) Regulatory Factor SLUG (SNAI2) Is a Downstream Target of SPARC and AKT in Promoting Melanoma Cell Invasion

    doi: 10.1371/journal.pone.0040378

    Figure Lengend Snippet: mRNA levels of SPARC and SLUG in melanoma cells at various stages of tumor development. ( A ) Analysis of SPARC and SLUG mRNA levels: relative gene expression levels of SPARC and SLUG in cultures of melanoma cells derived from RGP, VGP or metastatic melanoma tumors were evaluated by relative Q-PCR using an ABI Biosystems 7900HT Fast Real Time PCR System and the SYBR Green dye detection protocol. Data were analyzed using the 2 −ddCt method and human 18S transcript level was used to normalize for each sample. Values are the mean of independent triplicates. RGP, Radial Growth Phase; VGP, Vertical Growth Phase. ( B ) Positive correlation between SPARC and SLUG in melanoma samples: regression analysis to determine the correlation between SPARC and SLUG in human melanoma samples. R , Spearman’s Rank Correlation Coefficient; P

    Article Snippet: Quantitative PCR was performed on 25 ng cDNA samples, in sealed 384-well microtiter plates using the SYBR Green™ PCR Master Mix (Applied Biosystems) with the 7900HT Fast Real-Time PCR System (Applied Biosystems).

    Techniques: Expressing, Derivative Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, SYBR Green Assay

    Effect of MAZ on global genome organization. ( A ) Scatter plot showing differential intra-TAD activity in WT vs MAZ KO MNs (FDR cut-off=0.05). ( B ) Scatter plot showing differential loop activity in WT vs MAZ KO MNs (all loops, n=95119, FDR cut-off=0.005, | log (Fold Change) | cut-off=1.5, Up-regulated=741, Down-regulated: 22904). ( C ) Aggregate Peak Analysis (APA) of loops in WT vs MAZ KO MNs showing ChIP-seq signals of CTCF, MAZ, or both at any region covered by them. The resolution of APA is 5 kb. Histograms showing the distribution of loop distance in MAZ KO compared to WT related to the binding level of ChIP-seq. ( D ) Western blot analysis of FLAG, RAD21, and CTCF upon FLAG-MAZ immunoprecipitation from nuclear extract of 293FT cells. ( E ) Proposed model for the effect of MAZ on loops via its interaction with cohesin (middle) or binding adjacent to CTCF and interaction with cohesin (right), in addition to existing loop-extrusion model of CTCF and cohesin (left, ( 44 , 45 )).

    Journal: bioRxiv

    Article Title: A CRISPR Screen Identifies Myc-associated Zinc Finger Protein (MAZ) as an Insulator Functioning at CTCF boundaries in Hox Clusters

    doi: 10.1101/2020.08.25.267237

    Figure Lengend Snippet: Effect of MAZ on global genome organization. ( A ) Scatter plot showing differential intra-TAD activity in WT vs MAZ KO MNs (FDR cut-off=0.05). ( B ) Scatter plot showing differential loop activity in WT vs MAZ KO MNs (all loops, n=95119, FDR cut-off=0.005, | log (Fold Change) | cut-off=1.5, Up-regulated=741, Down-regulated: 22904). ( C ) Aggregate Peak Analysis (APA) of loops in WT vs MAZ KO MNs showing ChIP-seq signals of CTCF, MAZ, or both at any region covered by them. The resolution of APA is 5 kb. Histograms showing the distribution of loop distance in MAZ KO compared to WT related to the binding level of ChIP-seq. ( D ) Western blot analysis of FLAG, RAD21, and CTCF upon FLAG-MAZ immunoprecipitation from nuclear extract of 293FT cells. ( E ) Proposed model for the effect of MAZ on loops via its interaction with cohesin (middle) or binding adjacent to CTCF and interaction with cohesin (right), in addition to existing loop-extrusion model of CTCF and cohesin (left, ( 44 , 45 )).

    Article Snippet: 293FT cells were cultured in standard medium as described in the manufacturer’s protocol (Thermo Fisher Scientific).

    Techniques: Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Western Blot, Immunoprecipitation

    Identification of proteins co-localizing with CTCF via native ChIP-MS in ESCs and MNs A. FLAG-tag integrated at the C-terminus of CTCF via CRISPR genome editing. AH1, 2: arm of homology 1, 2. B . FLAG pull-down followed by CTCF western blot in benzonase solubilized nuclear pellet (NP) of ESCs. C . Native FLAG-CTCF IP in ESCs and MNs results in identification of known CTCF interactors, and novel proteins. The mean peptide counts from two biological replicates of FLAG-CTCF IPs were normalized to the control FLAG IP from untagged cells. Candidates filtered from the top of the list are shown (see Data S2 for all). D. Western blot analysis of CTCF and MAZ in different cellular fractions in mESCs and MNs. CE: cytoplasmic extract, NE: nuclear extract, NP: nuclear pellet. E. Western blot analysis of CTCF, SMC1, and MAZ upon FLAG-CTCF immunoprecipitation from nuclear pellet of mESCs. F. Western blot analysis of FLAG, RAD21, and MAZ upon FLAG-CTCF immunoprecipitation from nuclear extract of 293FT cells.

    Journal: bioRxiv

    Article Title: A CRISPR Screen Identifies Myc-associated Zinc Finger Protein (MAZ) as an Insulator Functioning at CTCF boundaries in Hox Clusters

    doi: 10.1101/2020.08.25.267237

    Figure Lengend Snippet: Identification of proteins co-localizing with CTCF via native ChIP-MS in ESCs and MNs A. FLAG-tag integrated at the C-terminus of CTCF via CRISPR genome editing. AH1, 2: arm of homology 1, 2. B . FLAG pull-down followed by CTCF western blot in benzonase solubilized nuclear pellet (NP) of ESCs. C . Native FLAG-CTCF IP in ESCs and MNs results in identification of known CTCF interactors, and novel proteins. The mean peptide counts from two biological replicates of FLAG-CTCF IPs were normalized to the control FLAG IP from untagged cells. Candidates filtered from the top of the list are shown (see Data S2 for all). D. Western blot analysis of CTCF and MAZ in different cellular fractions in mESCs and MNs. CE: cytoplasmic extract, NE: nuclear extract, NP: nuclear pellet. E. Western blot analysis of CTCF, SMC1, and MAZ upon FLAG-CTCF immunoprecipitation from nuclear pellet of mESCs. F. Western blot analysis of FLAG, RAD21, and MAZ upon FLAG-CTCF immunoprecipitation from nuclear extract of 293FT cells.

    Article Snippet: 293FT cells were cultured in standard medium as described in the manufacturer’s protocol (Thermo Fisher Scientific).

    Techniques: Chromatin Immunoprecipitation, FLAG-tag, CRISPR, Western Blot, Immunoprecipitation

    ShRNA-mediated silencing of TRPM7 impaired invasion in pancreatic adenocarcinoma cells. (A) Non-transfected BxPC-3 cells assayed for invasion using the trans-well assay in the presence of 3% FBS or no FBS. (B,C) BxPC-3 cells transfected with non-targeting control (NC) shRNA or anti- TRPM7 shRNA analyzed for cell invasion in the presence of either 3% FBS (B) or 10% FBS (C). A representative image of the invaded cells stained with crystal violet is shown for each experimental group. Cell invasion is expressed as % control, and each column represents the mean ± standard error.

    Journal: Biology Open

    Article Title: Aberrant over-expression of TRPM7 ion channels in pancreatic cancer: required for cancer cell invasion and implicated in tumor growth and metastasis

    doi: 10.1242/bio.20137088

    Figure Lengend Snippet: ShRNA-mediated silencing of TRPM7 impaired invasion in pancreatic adenocarcinoma cells. (A) Non-transfected BxPC-3 cells assayed for invasion using the trans-well assay in the presence of 3% FBS or no FBS. (B,C) BxPC-3 cells transfected with non-targeting control (NC) shRNA or anti- TRPM7 shRNA analyzed for cell invasion in the presence of either 3% FBS (B) or 10% FBS (C). A representative image of the invaded cells stained with crystal violet is shown for each experimental group. Cell invasion is expressed as % control, and each column represents the mean ± standard error.

    Article Snippet: BxPC-3 cells at about 90% confluency in a 10-cm dish containing 10 ml OptiMem® medium were transfected with either 24 µg anti-TRPM7 shRNA or non-targeting shRNA using 60 µl Lipofectamine™ 2000 (Invitrogen™, Life Technologies, Grand Island, New York, USA) and then incubated at 37°C for 5 h. The transfected cells were incubated in RPMI 1640 medium containing 10% FBS (Life Technologies) at 37°C for 24 h, sorted, and collected by flow cytometry for GFP with emission light at a 488-nm wavelength using BD FACSCalibur® (BD Biosciences, San Jose, California, USA).

    Techniques: shRNA, Transfection, Staining

    SNC1-RLD encodes a truncated TNL protein. (A) Alignment of deduced amino acid sequences of SNC1-Col (top) and SNC1-RLD (bottom) using the EBI-ClustalW tool ( http://www.ebi.ac.uk/Tools/clustalw/ ) [54] . Identical amino acids are indicated by asterisks. Colons and semi-colons show conserved substitutions and semi-conserved substitutions, respectively. Characters in blue, red and green show the amino acids corresponding to exon 1, exon 2 and exon 3, respectively. (B) SNC1 gene model as experimentally verified by reverse transcription PCR and 3′-RACE from Col-0 (middle) and RLD (bottom) compared with the TAIR9 gene model (top). Exons are indicated by boxes, introns by lines, and stop codons by red asterisks.

    Journal: PLoS Pathogens

    Article Title: The Arabidopsis Resistance-Like Gene SNC1 Is Activated by Mutations in SRFR1 and Contributes to Resistance to the Bacterial Effector AvrRps4SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity

    doi: 10.1371/journal.ppat.1001172

    Figure Lengend Snippet: SNC1-RLD encodes a truncated TNL protein. (A) Alignment of deduced amino acid sequences of SNC1-Col (top) and SNC1-RLD (bottom) using the EBI-ClustalW tool ( http://www.ebi.ac.uk/Tools/clustalw/ ) [54] . Identical amino acids are indicated by asterisks. Colons and semi-colons show conserved substitutions and semi-conserved substitutions, respectively. Characters in blue, red and green show the amino acids corresponding to exon 1, exon 2 and exon 3, respectively. (B) SNC1 gene model as experimentally verified by reverse transcription PCR and 3′-RACE from Col-0 (middle) and RLD (bottom) compared with the TAIR9 gene model (top). Exons are indicated by boxes, introns by lines, and stop codons by red asterisks.

    Article Snippet: Quantitative real-time reverse transcription PCR (qPCR) was performed with SYBR GREEN PCR Master Mix and an ABI 7500 system (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P

    Journal: bioRxiv

    Article Title: Phytophthora infestans Ago1-bound miRNA promotes potato late blight disease

    doi: 10.1101/2020.01.28.924175

    Figure Lengend Snippet: StLL1 down-regulation is controlled by miR8788. a, Exons (filled box) and introns (black line) illustrating the lipase-like gene in potato ( StLL1 ). Alignment of miR8788-3p with StLL1 at the predicted binding site. The arrow with fraction above (8/10) indicates the cleavage site with the number of identical clones detected by 5’ RACE. b, 5’ RACE products of StLL1 in P. infestans -infected potato (cv. Bintje). R1 (lane 1-3) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 4-9), miR8788-specific product generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. c, 5’ RACE products of StLL1 in water-inoculated potato (cv. Bintje). R1 (lane 1) achieved using GeneRacer™ 5’ primer and target mRNA 3’ primer. R2 (lane 2-4), products generated with GeneRacer™ 5’ nested primer and target mRNA 3’ primer. M = 1 kb Plus DNA. d , Northern blot analysis using a γ- 32 P labelled RNA probe for miR8788-5p. M = γ- 32 P labeled GeneRuler Ultra Low Range DNA Ladder. H20 = water inoculation. P. infestans infected potato leaves (with strains 88069, pHAM34 : PiAgo1-GFP and pHAM34:eGFP ( GFP ). Samples (cv. Bintje) collected 5 dpi. miR8788-5p is indicated with black arrows. Lower panel = U6 snRNA from potato (loading control, probe cross-reacts with P. infestans U6). e, T-DNA constructs used in the luciferase reporter assay. 35S promoter (35S), REN luciferase (REN), U6 snRNA promoter (U6-26p), firefly luciferase (LUC), miR8788 target sequence in StLL1 (TS), non-specific sequence (NS) and Nos 3’ terminator (NosT). f, Luciferase reporter assay in N. benthamiana samples, 3 days post Agro-infiltration. Agro-infiltration with GV3101 (Control) or silencing construct (SC). Reporters: p35S:REN:LUC (TS), blue, p35S:REN:LUC (NS), red. The quantified LUC normalized to REN activities are shown (LUC/REN). Error bars indicate mean ± standard error of the mean ( n = 5, df = 19). *** = significant difference between the reporters during Agro-infiltration with SC (Student’s t- test: P

    Article Snippet: First, a touch down PCR with GeneRacer™ 5′ primer and a gene-specific reverse primer was run.

    Techniques: Binding Assay, Clone Assay, Infection, Generated, Northern Blot, Labeling, Construct, Luciferase, Reporter Assay, Sequencing

    Acute MMP-9/2 inhibition attenuates CCI-induced allodynia and neuroinflammation. ( A ) Gelatin zymography of sciatic nerve extracts (70 μg total protein/lane) obtained at 3 h, 6 h, 1 day, 3 days, 5 days, and 7 days post-CCI. The arrows point to the MMP-9 and MMP-2 species. ( B ) Taqman qRT-PCR for MMP-9 in the sham (Sh) and CCI sciatic nerves (day 1). The mean relative mRNA ± SEM of n = 6/group normalized to GAPDH compared to naïve (N) nerve (**, P

    Journal: Journal of Neuroinflammation

    Article Title: Immunodominant fragments of myelin basic protein initiate T cell-dependent pain

    doi: 10.1186/1742-2094-9-119

    Figure Lengend Snippet: Acute MMP-9/2 inhibition attenuates CCI-induced allodynia and neuroinflammation. ( A ) Gelatin zymography of sciatic nerve extracts (70 μg total protein/lane) obtained at 3 h, 6 h, 1 day, 3 days, 5 days, and 7 days post-CCI. The arrows point to the MMP-9 and MMP-2 species. ( B ) Taqman qRT-PCR for MMP-9 in the sham (Sh) and CCI sciatic nerves (day 1). The mean relative mRNA ± SEM of n = 6/group normalized to GAPDH compared to naïve (N) nerve (**, P

    Article Snippet: Gene expression levels were measured in a Mx4000™ Multiplex Quantitative PCR System (Agilent Technologies) using 50 ng of cDNA and 2x Taqman Universal PCR Master Mix (Ambion) with a one-step program: 95 °C, 10 min; 95 °C, 30 s; 60 °C, 1 min for 50 cycles.

    Techniques: Inhibition, Zymography, Quantitative RT-PCR

    Analysis of viral protein and DNA in HAV5.EGFP capsids . (A) Proteins from lysates of RNase/DNase treated or untreated mature and empty/intermediate capsids were separated by 10% SDS-PAGE, and stained by SYPRO Ruby protein stains. A 100 kDa hexon protein band was used for determining the capsid protein concentration using Kodak IM Network software. (B) Total yields of DNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. ( C ) EGFP expression. 293 cells were transduced by 10 fold serially diluted mature and empty/intermediate capsids with or without RNase/DNase treatment. At 48 h post transduction, cells were analysed for EGFP expression by a fluorescent microscope (i) EGFP, (ii) Phase contrast. Presence (+). Absence (-). Mature (Ma); Empty/Intermediate (E-I).

    Journal: Virology Journal

    Article Title: Packaging of viral RNAs in virions of adenoviruses

    doi: 10.1186/1743-422X-6-16

    Figure Lengend Snippet: Analysis of viral protein and DNA in HAV5.EGFP capsids . (A) Proteins from lysates of RNase/DNase treated or untreated mature and empty/intermediate capsids were separated by 10% SDS-PAGE, and stained by SYPRO Ruby protein stains. A 100 kDa hexon protein band was used for determining the capsid protein concentration using Kodak IM Network software. (B) Total yields of DNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. ( C ) EGFP expression. 293 cells were transduced by 10 fold serially diluted mature and empty/intermediate capsids with or without RNase/DNase treatment. At 48 h post transduction, cells were analysed for EGFP expression by a fluorescent microscope (i) EGFP, (ii) Phase contrast. Presence (+). Absence (-). Mature (Ma); Empty/Intermediate (E-I).

    Article Snippet: The isolated virion RNAs were treated with RNase-free DNase (Ambion) to eliminate the contaminated viral genomic DNA, followed by addition of 0.1 volume DNase inactivation reagent (DNA-free kit, Ambion).

    Techniques: SDS Page, Staining, Protein Concentration, Software, Isolation, Expressing, Transduction, Microscopy

    Analysis of RNA in HAV5.EGFP capsids with or without RNase/DNase treatment . (A) Total yields of RNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. (B) The [ 32 P]-labeled cDNAs were made by reverse transcription of 2 μg, RNase-free DNase treated RNAs from mature and empty/intermediate capsids with or without RNase/DNase treatment and hybridized to Hind III-digested pFHAV5, which contains E1A-deleted HAdV-5 genome. RT was primed by oligo-dT/hexamers. (C) Viral RNAs detected in RNAs from mature (Ma) and empty/intermediate (E-I) capsids after RNase/DNase treatment in Southern hybridization in panel B were quantitated by using PhosphorImager software.

    Journal: Virology Journal

    Article Title: Packaging of viral RNAs in virions of adenoviruses

    doi: 10.1186/1743-422X-6-16

    Figure Lengend Snippet: Analysis of RNA in HAV5.EGFP capsids with or without RNase/DNase treatment . (A) Total yields of RNAs isolated from mature and empty/intermediate capsids with or without RNase/DNase treatment. (B) The [ 32 P]-labeled cDNAs were made by reverse transcription of 2 μg, RNase-free DNase treated RNAs from mature and empty/intermediate capsids with or without RNase/DNase treatment and hybridized to Hind III-digested pFHAV5, which contains E1A-deleted HAdV-5 genome. RT was primed by oligo-dT/hexamers. (C) Viral RNAs detected in RNAs from mature (Ma) and empty/intermediate (E-I) capsids after RNase/DNase treatment in Southern hybridization in panel B were quantitated by using PhosphorImager software.

    Article Snippet: The isolated virion RNAs were treated with RNase-free DNase (Ambion) to eliminate the contaminated viral genomic DNA, followed by addition of 0.1 volume DNase inactivation reagent (DNA-free kit, Ambion).

    Techniques: Isolation, Labeling, Hybridization, Software

    PCR-based detection of ClC-3 in rat pancreatic tissue from early postnatal rats cDNA was produced from RNA preparations from pancreas and kidney of 5-day-old and adult rats. cDNA amplification was performed with primers specific for rat kidney ClC-3 (upper panel) and rat p24A (lower panel). RT-PCR amplification products were separated by standard agarose gel electrophoresis and stained with ethidium bromide. In controls, primers without cDNA (water control) as well as DNase I-treated RNA samples from 5-day-old and adult rats were used in the PCR reaction mixture. Using a two-step amplification protocol (‘nested PCR’) a 517 bp fragment of ClC-3 cDNA could be amplified from kidney and pancreas of 5-day-old rats, as well as from the kidney of adult rats (upper panel). The integrity of the pancreas cDNA preparation from adult animals could be shown by amplification of a 266 bp cDNA fragment of the ubiquitously expressed protein p24A (lower panel). Molecular weight (MW) markers were added to the first and last lane.

    Journal: The Journal of Physiology

    Article Title: Characterization of cell volume-sensitive chloride currents in freshly prepared and cultured pancreatic acinar cells from early postnatal rats

    doi: 10.1111/j.1469-7793.1998.453bb.x

    Figure Lengend Snippet: PCR-based detection of ClC-3 in rat pancreatic tissue from early postnatal rats cDNA was produced from RNA preparations from pancreas and kidney of 5-day-old and adult rats. cDNA amplification was performed with primers specific for rat kidney ClC-3 (upper panel) and rat p24A (lower panel). RT-PCR amplification products were separated by standard agarose gel electrophoresis and stained with ethidium bromide. In controls, primers without cDNA (water control) as well as DNase I-treated RNA samples from 5-day-old and adult rats were used in the PCR reaction mixture. Using a two-step amplification protocol (‘nested PCR’) a 517 bp fragment of ClC-3 cDNA could be amplified from kidney and pancreas of 5-day-old rats, as well as from the kidney of adult rats (upper panel). The integrity of the pancreas cDNA preparation from adult animals could be shown by amplification of a 266 bp cDNA fragment of the ubiquitously expressed protein p24A (lower panel). Molecular weight (MW) markers were added to the first and last lane.

    Article Snippet: RNA samples were pre-treated with DNase I (amplification grade, Gibco) prior to the RT reaction to ensure that the polymerase chain reaction (PCR) products were not amplified from genomic DNA contaminations.

    Techniques: Polymerase Chain Reaction, Produced, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Nested PCR, Molecular Weight