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  • 99
    Thermo Fisher sybr green pcr master mix
    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative <t>RT–PCR</t> using <t>SYBR®</t> Green in triplicates and normalized to β-Actin. All UPR markers except
    Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 101787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dnase i
    Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and <t>DNase</t> I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rneasy mini kit
    Gene regulation confirmed by Q-PCR in mouse and human EOC cell lines. A-B. Mouse ID8 P0/P1 cells (A) or human EOC cells (B) were seeded into 6-well plates in attached or low-attached plates in suspension, <t>RNAs</t> were extracted with the <t>RNeasy</t> mini kit (Qiagen, Valencia, CA) and reverse transcribed by M-MLV reverse transcriptase. Quantitative real-time PCR was performed on a Light Cycler 480 (Roche, Indianapolis, IN) with a SYBR Green I Master Mix (Roche, Indianapolis, IN). mRNA abundance was normalized to GAPDH.
    Rneasy Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 343576 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript rt reagent kit
    Gene regulation confirmed by Q-PCR in mouse and human EOC cell lines. A-B. Mouse ID8 P0/P1 cells (A) or human EOC cells (B) were seeded into 6-well plates in attached or low-attached plates in suspension, <t>RNAs</t> were extracted with the <t>RNeasy</t> mini kit (Qiagen, Valencia, CA) and reverse transcribed by M-MLV reverse transcriptase. Quantitative real-time PCR was performed on a Light Cycler 480 (Roche, Indianapolis, IN) with a SYBR Green I Master Mix (Roche, Indianapolis, IN). mRNA abundance was normalized to GAPDH.
    Primescript Rt Reagent Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 72258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rneasy kit
    Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab <t>PeqGold</t> RNA pure kit (PL), Qiagen <t>RNeasy</t> kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P
    Rneasy Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 111781 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaquick pcr purification kit
    Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab <t>PeqGold</t> RNA pure kit (PL), Qiagen <t>RNeasy</t> kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P
    Qiaquick Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 137129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa sybr premix ex taq
    Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab <t>PeqGold</t> RNA pure kit (PL), Qiagen <t>RNeasy</t> kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P
    Sybr Premix Ex Taq, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 49731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen quantitect reverse transcription kit
    Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab <t>PeqGold</t> RNA pure kit (PL), Qiagen <t>RNeasy</t> kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P
    Quantitect Reverse Transcription Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 44197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mgcl2
    Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab <t>PeqGold</t> RNA pure kit (PL), Qiagen <t>RNeasy</t> kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P
    Mgcl2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 104037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaquick gel extraction kit
    Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab <t>PeqGold</t> RNA pure kit (PL), Qiagen <t>RNeasy</t> kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P
    Qiaquick Gel Extraction Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 113321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taq dna polymerase
    Use of exo+ and exo− <t>Taq</t> polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the <t>DNA</t> template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 44901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna
    MS2-driven <t>RNA</t> packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of <t>DNAse-treated</t> supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase inhibitor
    Validation of HuR target circRNAs. (A) RT-qPCR analysis of <t>HeLa</t> circRNAs enriched in HuR IP samples compared with IgG samples; RNA levels were normalized to the background levels of GAPDH mRNA in each IP sample. (B) Example of PCR product validation; hsa_circ_0031288(CircPABPN1 ) was visualized on ethidium bromide-stained agarose gels to confirm the specificity of the circRNA amplification band (RT, reverse transcription). (C) Validation of circRNA PCR product from (B) by DNA sequencing analysis. All of the circRNAs in (A) were verified on agarose gels and many were further verified by sequencing. (D) RT-qPCR analysis to measure GAPDH mRNA, PABPN1 mRNA, and hsa_circ_0031288 ( CircPABPN1 ) with or without <t>RNase</t> R treatment; RNA levels in RNase R-treated samples were compared with mock (untreated) samples (using identical input RNA amounts). (E) Western blot analysis of HuR levels in the samples pulled down using control biotinylated GAPDH (3′UTR) and linear biotinylated CircPABPN1 . ‘Input’, HuR levels in the samples used for pulldown analysis. (F) Western blot analysis of HuR levels in pulldown assays using a biotinylated antisense oligomer targeting the junction of CircPABPN1 or a control biotinylated oligomer. Data in (A,D) are the means and ± SEM from three independent experiments. Data in (B,E,F) are representative of three independent experiments.
    Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23259 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rneasy micro kit
    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for <t>RNA</t> isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN <t>RNeasy</t> Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.
    Rneasy Micro Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 35634 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman universal pcr master mix
    Open chromatin conformation and POL II plus GATA1 recruitment mark Gfi1b promoter and downstream elements in expressing cells. (A) Gfi1b RNA quantification of cell lines and primary cells by real-time <t>PCR,</t> <t>TaqMan®</t> Gene Expression Assay Mm01336944_m1
    Taqman Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rneasy plant mini kit
    Open chromatin conformation and POL II plus GATA1 recruitment mark Gfi1b promoter and downstream elements in expressing cells. (A) Gfi1b RNA quantification of cell lines and primary cells by real-time <t>PCR,</t> <t>TaqMan®</t> Gene Expression Assay Mm01336944_m1
    Rneasy Plant Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 39071 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen qiaamp dna mini kit
    Open chromatin conformation and POL II plus GATA1 recruitment mark Gfi1b promoter and downstream elements in expressing cells. (A) Gfi1b RNA quantification of cell lines and primary cells by real-time <t>PCR,</t> <t>TaqMan®</t> Gene Expression Assay Mm01336944_m1
    Qiaamp Dna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 48891 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen dneasy blood and tissue kit
    Open chromatin conformation and POL II plus GATA1 recruitment mark Gfi1b promoter and downstream elements in expressing cells. (A) Gfi1b RNA quantification of cell lines and primary cells by real-time <t>PCR,</t> <t>TaqMan®</t> Gene Expression Assay Mm01336944_m1
    Dneasy Blood And Tissue Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 51440 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript rt master mix
    Open chromatin conformation and POL II plus GATA1 recruitment mark Gfi1b promoter and downstream elements in expressing cells. (A) Gfi1b RNA quantification of cell lines and primary cells by real-time <t>PCR,</t> <t>TaqMan®</t> Gene Expression Assay Mm01336944_m1
    Primescript Rt Master Mix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 12317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Open chromatin conformation and POL II plus GATA1 recruitment mark Gfi1b promoter and downstream elements in expressing cells. (A) Gfi1b RNA quantification of cell lines and primary cells by real-time <t>PCR,</t> <t>TaqMan®</t> Gene Expression Assay Mm01336944_m1
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    Bland-Altman analysis of HIV-1 viral loads in patient samples ( n = 47) of plasma versus DPS after <t>RNA</t> extraction with a <t>QIAamp</t> viral RNA mini kit (Qiagen) (A), the Abbott sample preparation system (B), and a Nuclisens manual extraction kit (bioMérieux)
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    HEATR2 splice mutation results in alteration of the final conserved HEAT repeat and protein instability. (A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled <t>DNA</t> signified those that had their DNA included in SNP genotyping. (B) Schematic of HEATR2 transcript showing the transversion mutation ( ENST00000297440:c.2432-1G > C ) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the HEATR2 transcript, resulting in a frameshift in translation ( Figure S2B ). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See Figure S3A ). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of HEATR2 transcript by RT-qPCR, when normalized to the reference TBP gene. (D) The PCD transversion mutation ( ENST00000297440:c.2432-1G > C ) does not affect HEATR2 transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) <t>cDNA</t> from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients ( Figure S2B ). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation ( ENST00000297440:c.2432-1G > C ) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see Figure S3B ). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.
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    Characteristics and applications of the USER technique. ( a ) A comparison of the ability of <t>DNA</t> polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm <t>Taq:</t> HotMaster™ Taq DNA Polymerase, Taq: Platinum ® Taq DNA Polymerase, Pwo: Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo ® C x Hotstart DNA polymerase. ( b ) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus -specific USER compatible vector, pNB1u. H 2 O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. ( c ) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.
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    Image Search Results


    Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative RT–PCR using SYBR® Green in triplicates and normalized to β-Actin. All UPR markers except

    Journal: Human Molecular Genetics

    Article Title: Curcumin facilitates a transitory cellular stress response in Trembler-J mice

    doi: 10.1093/hmg/ddt318

    Figure Lengend Snippet: Activation of BiP ( A ), Atf3 ( B ), Ero1-lβ ( C ), Chop ( D ) in Tr-J . Expression levels in sciatic nerves were measured by quantitative RT–PCR using SYBR® Green in triplicates and normalized to β-Actin. All UPR markers except

    Article Snippet: The cDNA was then PCR-amplified in triplicates and confirmed in two independent experiments by SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) using 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) on different animal groups to avoid biases.

    Techniques: Activation Assay, Expressing, Quantitative RT-PCR, SYBR Green Assay

    Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p

    Journal: Oncotarget

    Article Title: Polarization of macrophages in the tumor microenvironment is influenced by EGFR signaling within colon cancer cells

    doi: 10.18632/oncotarget.12207

    Figure Lengend Snippet: Cetuximab modulates macrophage polarization in an AOM/DSS mouse model A. Establishment of the AOM/DSS mouse model. AOM was injected intraperitoneally at 12.5 mg/kg body weight. After one week, mice were given drinking water containing 2.5% DSS for 5 days followed by 16 days of regular drinking water. After two cycles of DSS treatment, cetuximab (1 mg/mouse, twice a week) was injected intraperitoneally for a month, and the mice were then sacrificed. B. Representative images of colon tumors in normal (right), AOM/DSS (2AD) (middle), and cetuximab-treated AOM/DSS mice (2AD+cetu) (left). C. Tumor quantification. Cetuximab treatment (2AD + cetu) reduced tumor numbers compared to 2AD mice. D. p-EGFR (Y1068), EGFR, Arg1, and iNOS protein levels in normal mice, 2AD and 2AD+cetu mouse tumors were detected by Western blot. All experiments were repeated three times. E. Representative photomicrographs of immunostaining for p-EGFR (Y1068), PCNA, F4/80, Arg1, and iNOS. Scale bars: 25 μm. F. M1 marker (IL-12, iNOS) and M2 marker (IL-4, IL-10, Arg1) mRNA levels in normal mouse colon tissues, 2AD and 2AD+cetu mouse tumor tissues were evaluated by q-PCR. G. Percentages of CD11b + /F4/80 + and F4/80 + /CD206 + cells in normal, 2AD, and 2AD+cetu mice colon tissues were detected by flow cytometry. Colon tissues were cut into small pieces (1-2 mm) and incubated with collagenase D (1- mg/mL), dispase II (1 mg/mL), and DNase I (100 μg/mL) for 30-45 min in a shaking incubator at 37°C, and single-cell suspensions were then incubated with antibodies. Bars represent means ± SD (n = 3) for each treatment. * p

    Article Snippet: Briefly, colon tissues were cut into small pieces (1-2 mm) and incubated in 10 mL PRMI 1640 medium with 10 mM HEPES and 5% fetal bovine serum containing 10 mg (1 mg/mL) collagenase D (Sigma-Aldrich), 10 mg (1 mg/mL) dispase II (Roche, Germany), and 100 μL 10 mg/ml DNase I (100 μg/mL) (Sigma-Aldrich) for 30-45 min in a shaking incubator at 37°C.

    Techniques: Injection, Mouse Assay, Western Blot, Immunostaining, Marker, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Incubation

    Gene regulation confirmed by Q-PCR in mouse and human EOC cell lines. A-B. Mouse ID8 P0/P1 cells (A) or human EOC cells (B) were seeded into 6-well plates in attached or low-attached plates in suspension, RNAs were extracted with the RNeasy mini kit (Qiagen, Valencia, CA) and reverse transcribed by M-MLV reverse transcriptase. Quantitative real-time PCR was performed on a Light Cycler 480 (Roche, Indianapolis, IN) with a SYBR Green I Master Mix (Roche, Indianapolis, IN). mRNA abundance was normalized to GAPDH.

    Journal: PLoS ONE

    Article Title: Changes in mRNA/protein expression and signaling pathways in in vivo passaged mouse ovarian cancer cells

    doi: 10.1371/journal.pone.0197404

    Figure Lengend Snippet: Gene regulation confirmed by Q-PCR in mouse and human EOC cell lines. A-B. Mouse ID8 P0/P1 cells (A) or human EOC cells (B) were seeded into 6-well plates in attached or low-attached plates in suspension, RNAs were extracted with the RNeasy mini kit (Qiagen, Valencia, CA) and reverse transcribed by M-MLV reverse transcriptase. Quantitative real-time PCR was performed on a Light Cycler 480 (Roche, Indianapolis, IN) with a SYBR Green I Master Mix (Roche, Indianapolis, IN). mRNA abundance was normalized to GAPDH.

    Article Snippet: For quantitative real-time PCR (Q-PCR), mouse ID8 P0/P1 cells were seeded into 6-well plates in attached or low-attached plates in suspension, RNAs were extracted with the RNeasy mini kit (Qiagen, Valencia, CA) and reverse transcribed by M-MLV reverse transcriptase.

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

    Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab PeqGold RNA pure kit (PL), Qiagen RNeasy kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P

    Journal: ACS chemical biology

    Article Title: Quantification of cellular poly(ADP-ribosyl)ation by stable isotope dilution mass spectrometry reveals tissue- and drug-dependent stress response dynamics

    doi: 10.1021/cb400170b

    Figure Lengend Snippet: Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab PeqGold RNA pure kit (PL), Qiagen RNeasy kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P

    Article Snippet: PAR extractions via the High pure miRNA isolation kit (Roche), PeqGOLD RNA pure (Peqlab), RNeasy kit (Qiagen) were performed analogous to the manufacturers’ recommendations for the purification of RNA.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Affinity Chromatography, RNA Extraction, Isolation, Dot Blot, Chromatography

    Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.

    Journal: PLoS ONE

    Article Title: Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

    doi: 10.1371/journal.pone.0070942

    Figure Lengend Snippet: Use of exo+ and exo− Taq polymerase. Eprobe mediated real-time PCR experiments were performed by using an exo+ (Amplitaq) and exo− (Genotyping Master) Taq polymerase and Eprobes with different melting temperatures. Amplification curves (Random fluorescent units (RFU) plotted against PCR cycle number) using a 7 times serial dilution of the DNA template are shown on the left. The R-squared values of the PCR efficiency plots are indicated in the graphs. Differential melting curve analysis by plotting –dF/dT against temperature is shown on the right. Main peaks are indicated in the graph to show the different T M values for both Eprobes. EGFR wild-type plasmid DNA concentrations are indicated by colors: Red: 1.5×10 8 copies, Dark blue: 1.5×10 7 copies, Yellow: 1.5×10 6 copies, Green: 1.5×10 5 copies, Pink: 1.5×10 4 copies, Sky blue: 1.5×10 3 copies, Brown: 150 copies, Orange: TE negative control. A: Amplitaq and Eprobe 215-21 wt TO. B: Genotyping Mastermix and Eprobe 215-21 TO. C: Amplitaq and Eprobe 205-13 wt TO. D: Genotyping Master and Eprobe 205-13 wt TO.

    Article Snippet: To test this hypothesis, we compared Eprobe 215-21 wt TO along with the shorter Eprobe 205-13 wt TO using a Taq DNA polymerase with a 5′–3′ exonuclease activity (Applied Biosystems AmpliTaq Gold) and a Taq DNA polymerase lacking the 5′–3′ exonuclease activity (Roche Genotyping Master).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Serial Dilution, Plasmid Preparation, Negative Control

    MS2-driven RNA packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of DNAse-treated supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles

    doi: 10.1038/mtm.2015.39

    Figure Lengend Snippet: MS2-driven RNA packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of DNAse-treated supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.

    Article Snippet: In total, 4 µl RNA treated with TURBO DNAse (2 U/µl) was used for cDNA synthesis with the Superscript First-strand Synthesis System (Life Technologies).

    Techniques: Plasmid Preparation, Construct, Positive Control, Derivative Assay, Expressing, Luciferase, Sequencing, Transmission Assay, Electron Microscopy, Negative Staining, Microscopy, Quantitative RT-PCR, Transfection, Amplification, Concentration Assay

    Validation of HuR target circRNAs. (A) RT-qPCR analysis of HeLa circRNAs enriched in HuR IP samples compared with IgG samples; RNA levels were normalized to the background levels of GAPDH mRNA in each IP sample. (B) Example of PCR product validation; hsa_circ_0031288(CircPABPN1 ) was visualized on ethidium bromide-stained agarose gels to confirm the specificity of the circRNA amplification band (RT, reverse transcription). (C) Validation of circRNA PCR product from (B) by DNA sequencing analysis. All of the circRNAs in (A) were verified on agarose gels and many were further verified by sequencing. (D) RT-qPCR analysis to measure GAPDH mRNA, PABPN1 mRNA, and hsa_circ_0031288 ( CircPABPN1 ) with or without RNase R treatment; RNA levels in RNase R-treated samples were compared with mock (untreated) samples (using identical input RNA amounts). (E) Western blot analysis of HuR levels in the samples pulled down using control biotinylated GAPDH (3′UTR) and linear biotinylated CircPABPN1 . ‘Input’, HuR levels in the samples used for pulldown analysis. (F) Western blot analysis of HuR levels in pulldown assays using a biotinylated antisense oligomer targeting the junction of CircPABPN1 or a control biotinylated oligomer. Data in (A,D) are the means and ± SEM from three independent experiments. Data in (B,E,F) are representative of three independent experiments.

    Journal: RNA Biology

    Article Title: Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1

    doi: 10.1080/15476286.2017.1279788

    Figure Lengend Snippet: Validation of HuR target circRNAs. (A) RT-qPCR analysis of HeLa circRNAs enriched in HuR IP samples compared with IgG samples; RNA levels were normalized to the background levels of GAPDH mRNA in each IP sample. (B) Example of PCR product validation; hsa_circ_0031288(CircPABPN1 ) was visualized on ethidium bromide-stained agarose gels to confirm the specificity of the circRNA amplification band (RT, reverse transcription). (C) Validation of circRNA PCR product from (B) by DNA sequencing analysis. All of the circRNAs in (A) were verified on agarose gels and many were further verified by sequencing. (D) RT-qPCR analysis to measure GAPDH mRNA, PABPN1 mRNA, and hsa_circ_0031288 ( CircPABPN1 ) with or without RNase R treatment; RNA levels in RNase R-treated samples were compared with mock (untreated) samples (using identical input RNA amounts). (E) Western blot analysis of HuR levels in the samples pulled down using control biotinylated GAPDH (3′UTR) and linear biotinylated CircPABPN1 . ‘Input’, HuR levels in the samples used for pulldown analysis. (F) Western blot analysis of HuR levels in pulldown assays using a biotinylated antisense oligomer targeting the junction of CircPABPN1 or a control biotinylated oligomer. Data in (A,D) are the means and ± SEM from three independent experiments. Data in (B,E,F) are representative of three independent experiments.

    Article Snippet: For antisense oligo pulldown of hsa_circ_0031288 / circPABPN1 , HeLa cells were lysed in PEB buffer containing protease inhibitors (Roche) and RNase inhibitor (Thermo Fisher).

    Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, Staining, Amplification, DNA Sequencing, Sequencing, Western Blot

    Transcriptome-wide identification of HuR-associated circRNAs. (A,B) Schematic of the strategy used to identify globally HuR-interacting circRNAs in HeLa cells (A). Following cell lysis, RIP analysis was carried out using anti-HuR or control IgG antibodies; the presence of HuR in the IP samples was assessed by Western blot analysis [(B); HC, heavy IgG chain]. Total RNA was isolated and digested with RNase R, and circRNAs present in the sample were identified using circRNA microarrays (Arraystar). (C) Partial list of circRNAs highly enriched in HuR IP relative to IgG IP as identified in microarrays (n = 3).

    Journal: RNA Biology

    Article Title: Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1

    doi: 10.1080/15476286.2017.1279788

    Figure Lengend Snippet: Transcriptome-wide identification of HuR-associated circRNAs. (A,B) Schematic of the strategy used to identify globally HuR-interacting circRNAs in HeLa cells (A). Following cell lysis, RIP analysis was carried out using anti-HuR or control IgG antibodies; the presence of HuR in the IP samples was assessed by Western blot analysis [(B); HC, heavy IgG chain]. Total RNA was isolated and digested with RNase R, and circRNAs present in the sample were identified using circRNA microarrays (Arraystar). (C) Partial list of circRNAs highly enriched in HuR IP relative to IgG IP as identified in microarrays (n = 3).

    Article Snippet: For antisense oligo pulldown of hsa_circ_0031288 / circPABPN1 , HeLa cells were lysed in PEB buffer containing protease inhibitors (Roche) and RNase inhibitor (Thermo Fisher).

    Techniques: Lysis, Western Blot, Isolation

    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Journal: EMBO Molecular Medicine

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate

    doi: 10.15252/emmm.201606382

    Figure Lengend Snippet: Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Article Snippet: Total RNA was isolated using the RNeasy Micro Kit (Qiagen), and isoform‐specific expression analysis was performed as explained above.

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Serial Dilution, Incubation, Purification

    Open chromatin conformation and POL II plus GATA1 recruitment mark Gfi1b promoter and downstream elements in expressing cells. (A) Gfi1b RNA quantification of cell lines and primary cells by real-time PCR, TaqMan® Gene Expression Assay Mm01336944_m1

    Journal:

    Article Title: GFI1B controls its own expression binding to multiple sites

    doi: 10.3324/haematol.2009.012351

    Figure Lengend Snippet: Open chromatin conformation and POL II plus GATA1 recruitment mark Gfi1b promoter and downstream elements in expressing cells. (A) Gfi1b RNA quantification of cell lines and primary cells by real-time PCR, TaqMan® Gene Expression Assay Mm01336944_m1

    Article Snippet: Gfi1b cDNA was analyzed by real-time polymerase chain reaction (PCR) in a 25μL reaction containing Taqman Universal PCR Master Mix (Applied Biosystems) and TaqMan(R) Gene Expression Assay Mm01336944_m1 mix with a FAM-NFQ probe between Gfi1b exons 1 and 2 (Gene Expression Assays, Applied Biosystems) following the manufacturer’s recommendations.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Bland-Altman analysis of HIV-1 viral loads in patient samples ( n = 47) of plasma versus DPS after RNA extraction with a QIAamp viral RNA mini kit (Qiagen) (A), the Abbott sample preparation system (B), and a Nuclisens manual extraction kit (bioMérieux)

    Journal: Journal of Clinical Microbiology

    Article Title: Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing ▿

    doi: 10.1128/JCM.02255-08

    Figure Lengend Snippet: Bland-Altman analysis of HIV-1 viral loads in patient samples ( n = 47) of plasma versus DPS after RNA extraction with a QIAamp viral RNA mini kit (Qiagen) (A), the Abbott sample preparation system (B), and a Nuclisens manual extraction kit (bioMérieux)

    Article Snippet: However, we kept the QIAamp viral RNA mini kit (Qiagen) because the Biocentric viral load kit recommends this extraction method.

    Techniques: RNA Extraction, Sample Prep

    HIV-1 viral loads in patient samples ( n = 47) of plasma and DPS after RNA extractions with a QIAamp viral RNA mini kit (Qiagen), the Abbott sample preparation system, or a Nuclisens manual extraction kit (bioMérieux). (A) Comparison of

    Journal: Journal of Clinical Microbiology

    Article Title: Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing ▿

    doi: 10.1128/JCM.02255-08

    Figure Lengend Snippet: HIV-1 viral loads in patient samples ( n = 47) of plasma and DPS after RNA extractions with a QIAamp viral RNA mini kit (Qiagen), the Abbott sample preparation system, or a Nuclisens manual extraction kit (bioMérieux). (A) Comparison of

    Article Snippet: However, we kept the QIAamp viral RNA mini kit (Qiagen) because the Biocentric viral load kit recommends this extraction method.

    Techniques: Sample Prep

    HEATR2 splice mutation results in alteration of the final conserved HEAT repeat and protein instability. (A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled DNA signified those that had their DNA included in SNP genotyping. (B) Schematic of HEATR2 transcript showing the transversion mutation ( ENST00000297440:c.2432-1G > C ) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the HEATR2 transcript, resulting in a frameshift in translation ( Figure S2B ). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See Figure S3A ). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of HEATR2 transcript by RT-qPCR, when normalized to the reference TBP gene. (D) The PCD transversion mutation ( ENST00000297440:c.2432-1G > C ) does not affect HEATR2 transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) cDNA from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients ( Figure S2B ). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation ( ENST00000297440:c.2432-1G > C ) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see Figure S3B ). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.

    Journal: PLoS Genetics

    Article Title: HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus

    doi: 10.1371/journal.pgen.1004577

    Figure Lengend Snippet: HEATR2 splice mutation results in alteration of the final conserved HEAT repeat and protein instability. (A) Pedigree of related families of UK-Pakistani descent. IV∶4 identifies the proband, also designated by the arrow. Solid symbols (individuals IV∶1, IV∶4 and IV∶10) indicate those affected with PCD. Double lines indicate consanguineous marriages. The individuals labeled DNA signified those that had their DNA included in SNP genotyping. (B) Schematic of HEATR2 transcript showing the transversion mutation ( ENST00000297440:c.2432-1G > C ) affecting the splice acceptor site of the final exon. The mutation results in inactivation of this splice site and utilization of an adjacent downstream cryptic splice acceptor site in exon 13, causing a 2-nucleotide AG deletion in the HEATR2 transcript, resulting in a frameshift in translation ( Figure S2B ). This is predicted to alter the final 44 amino acids of the protein and add an additional 33 amino acids with creation of a novel termination signal at codon 888 in the 3′UTR (See Figure S3A ). This mutation disrupts the final highly conserved HEAT repeat and alters the C-terminus of the ARM-type fold superfamily domain (red). (C) Relative expression levels of HEATR2 transcript by RT-qPCR, when normalized to the reference TBP gene. (D) The PCD transversion mutation ( ENST00000297440:c.2432-1G > C ) does not affect HEATR2 transcript stability or gross splicing as shown by RT-PCR on parental control (C) and patient (P*) cDNA from LCLs. PCR products spanning the gene including the splice acceptor mutation at Exon 11–13 and Exon 12-3′UTR show no obvious alterations in size. Direct sequencing confirmed a 2 base pair deletion consistent with efficient splicing to the cryptic splice acceptor at the start of exon 13 in PCD patients ( Figure S2B ). (E) Western blot analysis on total protein extracts from unrelated control, heterozygous parental and homozygous patient LCLs demonstrates the PCD mutation ( ENST00000297440:c.2432-1G > C ) results in an elongated HEATR2 protein present at reduced levels implying instability. The slight shift in mobility of the protein in the patient is consistent with the predicted 3 kDa size shift due to the amino acid alterations described. β-actin is used as a loading control. (For longer exposure see Figure S3B ). (F) Levels of HEATR2 protein normalized relative to β-actin reveal that parental samples which are heterozygous for the mutation shows a reduction to ≈50% of that of unrelated controls whilst the homozygous patient sample shows a reduction to ≈3% of control levels.

    Article Snippet: Total RNA was isolated according to manufacturer's protocol using RNAeasy minicolumns (Qiagen), followed by DNase treatment with Turbo DNA-free kit (Ambion). cDNA was made using First Strand Synthesis of cDNA for RT-PCR (AMV) kit (Roche).

    Techniques: Mutagenesis, Labeling, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Western Blot

    Characteristics and applications of the USER technique. ( a ) A comparison of the ability of DNA polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm Taq: HotMaster™ Taq DNA Polymerase, Taq: Platinum ® Taq DNA Polymerase, Pwo: Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo ® C x Hotstart DNA polymerase. ( b ) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus -specific USER compatible vector, pNB1u. H 2 O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. ( c ) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.

    Journal: Nucleic Acids Research

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    doi: 10.1093/nar/gkl635

    Figure Lengend Snippet: Characteristics and applications of the USER technique. ( a ) A comparison of the ability of DNA polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm Taq: HotMaster™ Taq DNA Polymerase, Taq: Platinum ® Taq DNA Polymerase, Pwo: Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo ® C x Hotstart DNA polymerase. ( b ) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus -specific USER compatible vector, pNB1u. H 2 O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. ( c ) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.

    Article Snippet: PCR conditions PCR with the following DNA polymerases: HotMaster™ Taq DNA Polymerase (Eppendorf), Platinum® Taq DNA Polymerase (Invitrogen), Pwo DNA Polymerase (Roche), Phusion™ DNA Polymerase (Finnzymes), and PfuTurbo® Cx Hotstart DNA polymerase (PfuCx) (Stratagene) was performed according to manufacturers' instructions on pBAD-TOPO® (Invitrogen) containing the gene, At5g43440 (Accession no. AY143873).

    Techniques: Functional Assay, Expressing, Plasmid Preparation, Injection, Fluorescence