Journal: JCI Insight

Article Title: PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms

doi: 10.1172/jci.insight.91722

Figure Lengend Snippet: Sex-dependent PRL1/2 and CNNM expression in BAT. ( A ) Tissue distribution of PRL1 and PRL2 proteins is shown. Tissues were lysed and immunoblotted with PRL1/2 and β-actin antibodies. EDL, extensor digitorum longus muscle; SOL, soleus muscle. ( B and C ) Protein expression of PRL1/2 ( B ) and CNNM3 ( C ) in BAT is shown. Tissue lysates were immunoblotted with PRL1/2, CNNM3, and calnexin (CNX) antibodies. Band intensities were normalized to those of CNX and indicated below the blots. ( D ) Quantitative PCR analyses were performed on RNA extracted from BAT. Data were normalized to Actb and expressed as fold change vs. WT. Data is expressed as mean ± SEM. Number of animals analyzed is indicated in parentheses in the figures. P values were calculated by two-way ANOVA. M vs. F, male versus female; W vs. K: WT versus KO. P > 0.05.

Article Snippet: Quantitative PCR (qPCR) was performed on a LightCycler 480 (Roche Life Science) with SYBR Green Master Mix (Roche Life Science) according to manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: JCI Insight

Article Title: PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms

doi: 10.1172/jci.insight.91722

Figure Lengend Snippet: Altered cellular metabolism in PRL2-KO MEF and BAT. ( A ) Mg 2+ content of immortalized PRL2-KO mouse embryonic fibroblasts (MEF) rescued with empty control (KO), Flag-PRL2 (WT), or Flag-PRL2 C101S (C/S) are shown. Expression of Flag-PRL2 transgene was immunoblotted with a Flag antibody. Actin antibody was used to confirm equal loading of lysates. ( B ) Fraction of cellular respiration devoted to uncoupled respiration of PRL2 (KO), C/S, or PRL2 (WT) MEF are shown. ( C–G ) Relative band intensity of the immunoblotting is shown. Protein lysates were obtained from 3 independent BAT samples after a week of feeding control diets (Mg [+]) or Mg 2+ -deficient diets (Mg [–]). They were immunoblotted with indicated antibodies, quantified, and normalized against total protein using stain-free technology. Three individual animals were used for each condition. Data is expressed as fold change versus Mg (+) WT. ND, not detectable. ( H ) Quantitative PCR analyses were performed on RNA extracted from BAT. Data were normalized to β-actin and expressed as fold change versus WT. ( I ) Amounts of each metabolite in BAT were quantified by GC-MS. Data were normalized to internal standard myristic acid-D27 and sample weight and expressed as fold change versus WT. Data is expressed as mean ± SEM ( A , B , H , I ) and mean ± SD ( C–G ). P values were calculated by one-way ANOVA with Dunnett’s multiple comparison ( A and B ), two-way ANOVA with Tukey’s multiple comparison (WT vs. KO, indicated as KO*) (Mg (+) vs Mg (-), indicated as Mg*) ( C–F ), Mann-Whitney test ( G ) and Student’s t -test ( H and I ). Two data points (9.03 and 7.23) in KO-Cs are outside the axis. *P

Article Snippet: Quantitative PCR (qPCR) was performed on a LightCycler 480 (Roche Life Science) with SYBR Green Master Mix (Roche Life Science) according to manufacturer’s instructions.

Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Gas Chromatography-Mass Spectrometry, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

doi: 10.1093/nar/gkw760

Figure Lengend Snippet: Determination of replication origin locations by initiation site sequencing (ini-seq). ( A ) Validation of immunoprecipitation by quantification of nascent DNA at established replication origins. Immunoprecipitated DNA was quantified by real-time PCR, using primer pairs located at the replication origins located near the promoters of the MYC, MCM4 and TOP1 gene loci (origin), and at corresponding non-origin background sites (bg). Data are expressed as proportions of 0.1% amount of input DNA, mean values of triplicate determinations are shown for one IP experiment. Sequences for the PCR primer pairs are in Supplementary Table S1, and their genomic locations are mapped in panel B. ( B ) Verification of origin positions by initiation site sequencing (ini-seq). Positions of sequencing reads are visualised on the integrated genome viewer (IGV) for total input control DNA (input) and two independent biological replicates of immunoprecipitated nascent DNA (IP-A and IP-B). Peaks of nascent DNA enrichment were called by SICER, either by integrating the input DNA (w/i; blue bars), or not doing so (green bars). The areas of peaks present in both IP-A and IP-B (i.e. the intersect) are shown for both peak-calling strategies as indicated. Chromosomal 25kb regions are shown for the MYC locus (chromosome 8: 128.735–128.76Mb), the MCM4 locus (chromosome 8: 48.86-48.885Mb), and the TOP1 locus (chromosome 20: 39.648–39.673 Mb). Positions of reference genes (blue), transcription directionalities (black arrows), and primer sites used for qPCR analyses (black, origin sites; grey, background sites) are indicated. ( C ) Venn diagrams of overlap between enrichment peaks called by two modes of SICER in the human genome for libraries A and B (IP A and IP B). Overall counts of peaks are indicated.

Article Snippet: Quantitative PCR (qPCR) Quantitative PCR (qPCR) was performed on the LightCycler 480 II using SYBR Green I master mix according to the manufacturer's protocol (Roche).

Techniques: Sequencing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Biology Open

Article Title: Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics

doi: 10.1242/bio.019588

Figure Lengend Snippet: Differential regulation of RefilinA and RefilinB mRNAs and proteins. (A) Comparison of RefilinA and RefilinB mRNA levels in three cell sorting experiments (see Fig. S1C ). (B) Comparison of RefilinA and RefilinB mRNA levels during mouse brain development. 1 µg of cDNA from embryonic and postnatal mouse brain [E8.5; E10.5; E12.5; E15.5; E18.5; postnatal day (P)0; P5; P10] were used to perform quantitative PCR. β-actin gene was selected as stable reference gene in embryo for internal standardization ( Willems et al., 2006 ). Error bars represent the standard deviation (s.d.) of three independent experiments. We assigned the enrichment value of 1 for stage E8.5. (C) Western blot analyses of total cellular extracts of U373MG cells (lane 1), U373MG cells transfected with RefilinB or RefilinA constructs (lane 2), long term rat OPC cultures (lanes 3,5) or rat neural progenitors (lanes 4,6). OPC and neural progenitors were grown in Neurobasal medium supplemented with bFGF and PDGF (lanes 3,4) or bFGF and EGF (lanes 5,6). Anti-FLNA, anti-RefilinB, anti-RefilinA and anti-β-Tubulin antibodies were used as indicated. (D) Rat OPC cultures were shifted to differentiation culture medium for different period of time as indicated and lysed in SDS-sampling buffer. Total cell extracts (20 µg) were analysed by western blot using anti-FLNA, anti-RefilinB, and anti-GAPDH antibodies. A fivefold greater concentration of loaded sample was required to detect RefilinA (100 µg) with anti-RefilinA antibody.

Article Snippet: Quantitative PCR (qPCR) qPCR was performed on the LightCycler2.0 machine (Roche) using LightCycler® FastStart DNA MasterPLUS SYBR Green I mix (Roche) with the following program: 10 min incubation at 95°C, followed by 45 cycles of 95°C for 10 s, 55°C for 10 s and 72°C for 15 s, and a final step of 65°C for 15 s. The β-actin or Gapdh gene was selected as reference gene for internal standardization.

Techniques: FACS, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Transfection, Construct, Sampling, Concentration Assay

Journal: Molecular and Cellular Biology

Article Title: S-Phase Cyclin-Dependent Kinases Promote Sister Chromatid Cohesion in Budding Yeast ▿

doi: 10.1128/MCB.05323-11

Figure Lengend Snippet: Relative abundance of Mcd1/Scc1-Myc at CEN16 , TRP1 , and LYS4 during S phase in hydroxyurea or after mitotic arrest in nocodazole. (A) Western blotting of Mcd1/Scc1-Myc in wild-type and clb5Δ clb6Δ cells in whole-cell extracts (WCE) or after immunoprecipitation (IP). PSTAIRE is a loading control. (B) Real-time quantitative PCR of the CEN16 locus in chromatin immunoprecipitates from extracts prepared after cells were arrested in G 1 with mating pheromone or after mitotic arrest following G 1 synchrony and release in the presence of nocodazole and hydroxyurea. (C) Analysis of synchronous cell cycles of wild-type and clb5Δ clb6Δ cells following G 1 arrest and release into medium containing 75 mM hydroxyurea. (Left) Real-time quantitative PCR of the TRP1 and LYS4 loci in chromatin immunoprecipitates from samples at the indicated time points (see Materials and Methods for details). (Right) FACScan analysis of DNA content at each time point shown on the left. (D) A model describing how Pds1 may act redundantly with Clb5- and Clb6-Cdc28 to ensure cohesion is maintained during an unperturbed S phase. In the absence of replication stress, either efficient cohesin loading (Clb5 and Clb6 dependent) or robust protection of cohesin from cleavage (Pds1 dependent) is sufficient for cohesion. When replication is perturbed by reduced nucleotide pools, both mechanisms are required to ensure cohesion in S phase; otherwise, spindle tension results in loss of cohesion under these conditions.

Article Snippet: DNA was analyzed by real-time quantitative PCR (qPCR) using the Roche LightCycler 480 system, and the primer pairs were designed to detect Mcd1/Scc1-Myc occupancy at CEN16 , TRP1 , and LYS4 loci according to the genome-wide mapping of Mcd1/Scc1 ( ).

Techniques: Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Activated Clotting Time Assay

Journal: BMC Plant Biology

Article Title: Genome-wide Identification and Expression Analysis of the CDPK Gene Family in Grape, Vitis spp

doi: 10.1186/s12870-015-0552-z

Figure Lengend Snippet: Expression of CDPK genes in Chinese wild grape ( Vitis pseudoreticulata ) in response to treatment with plant hormones. Expression was measured by reverse transcription, followed by real-time, quantitative PCR, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps ( a ) and histogram ( b ). Grape Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. a Expression profile of VpCDPK gene family under ABA, SA, MeJA, and ethylene (ETH) treatments. The color scale represents log2 expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance. b Detailed expression levels of four VpCDPK genes showed unusual expression patterns under ABA, SA, MeJA, and ethylene (ETH) treatments. The data were showed as mean value ± SD. * and ** represent statistically significant (p

Article Snippet: Quantitative PCR (qPCR) was carried out using SYBR green (Takara, Dalian, China) on an IQ5 real time PCR machine (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Plant Biology

Article Title: Genome-wide Identification and Expression Analysis of the CDPK Gene Family in Grape, Vitis spp

doi: 10.1186/s12870-015-0552-z

Figure Lengend Snippet: Expression of CDPK genes in Chinese wild grape ( Vitis pseudoreticulata ) under salt and temperature stress treatments. Expression was measured by reverse transcription, followed by real-time, quantitative PCR, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps a and histogram b . Grape Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. a Expression profile of VpCDPK gene family under NaCl, 4 °C, and 42 °C treatments. The color scale represents log2 expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance. b Detailed expression levels of four VpCDPK genes showed unusual expression patterns under NaCl, 4 °C, and 42 °C treatments. The data were showed as mean value ± SD. * and ** represent statistically significant (p

Article Snippet: Quantitative PCR (qPCR) was carried out using SYBR green (Takara, Dalian, China) on an IQ5 real time PCR machine (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Plant Biology

Article Title: Genome-wide Identification and Expression Analysis of the CDPK Gene Family in Grape, Vitis spp

doi: 10.1186/s12870-015-0552-z

Figure Lengend Snippet: Expression of CDPK genes in Chinese wild grape ( Vitis pseudoreticulata ) during powdery mildew infection. Expression was measured by reverse transcription, followed by real-time, quantitative PCR, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps (A) and histogram (B). Grape Actin1 (GenBank Accession number AY680701) was used as an internal control. The experiments were repeated three times and gave consistent results. Mean values and SDs were obtained from three biological and three technical replicates. (A) Expression profile of VpCDPK gene family under powdery mildew infection. The color scale represents log2 expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance. (B) Detailed expression levels of four VpCDPK genes that significantly up-regulated during powdery mildew infection. The data were showed as mean value ± SD. * and ** represent statistically significant (p

Article Snippet: Quantitative PCR (qPCR) was carried out using SYBR green (Takara, Dalian, China) on an IQ5 real time PCR machine (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.

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

Journal: Carcinogenesis

Article Title: Flaxseed lignans enriched in secoisolariciresinol diglucoside prevent acute asbestos-induced peritoneal inflammation in mice

doi: 10.1093/carcin/bgv174

Figure Lengend Snippet: Flaxseed lignan diet blunts proinflammatory and profibrogenic cytokine levels induced by asbestos exposure and alters cytokine and cytokine receptor gene expression. PLF levels (pg/ml) of proinflammatory TNFα ( A ) and profibrogenic free, active TGFß1 ( C ) were evaluated 3 days post asbestos exposure from mouse cohorts fed CTL or FLC diets. PLF WBC mRNA expression of cytokines, TNFα ( B ) and TGFß1 ( D ) and cytokine receptors, TNFαR1 ( E ) and TGFßR1 ( F ) was determined using qPCR. Levels of target gene mRNA were normalized to 18S ribosomal RNA and values are expressed as fold change from CTL. # indicates a statistically significant difference (#### P

Article Snippet: Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the high capacity RNA to cDNA kit supplied by Applied Biosystems Quantitative polymerase chain reaction (qPCR) and performed using TaqMan® probe-based gene expression assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA).

Techniques: Expressing, CTL Assay, Real-time Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: Flaxseed lignans enriched in secoisolariciresinol diglucoside prevent acute asbestos-induced peritoneal inflammation in mice

doi: 10.1093/carcin/bgv174

Figure Lengend Snippet: Flaxseed lignan diet abrogates increased nitrosative and oxidative stress from exposure to asbestos fibers. Mouse cohorts (13 weeks old) were fed CTL ( n = 16) or FLC ( n = 17) diets for 7 days prior to exposure to 400 µg of crocidolite asbestos fibers. PLF was evaluated 3 days following asbestos exposure for nitrite ( A ) and MDA concentrations ( C ). PLF WBC mRNA expression of iNOS ( B ), HO-1 ( D ), Nqo1 ( E ) and Gstm1 ( F ) was determined using qPCR. Levels of target gene mRNA were normalized to 18S ribosomal RNA and values are expressed as fold change from CTL. # indicates a statistically significant difference (#### P

Article Snippet: Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the high capacity RNA to cDNA kit supplied by Applied Biosystems Quantitative polymerase chain reaction (qPCR) and performed using TaqMan® probe-based gene expression assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA).

Techniques: CTL Assay, Multiple Displacement Amplification, Expressing, Real-time Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: Flaxseed lignans enriched in secoisolariciresinol diglucoside prevent acute asbestos-induced peritoneal inflammation in mice

doi: 10.1093/carcin/bgv174

Figure Lengend Snippet: Flaxseed lignan diet ameliorates asbestos-induced WBC proinflammatory cytokine release and gene expression levels. PLF levels (pg/ml for IL-1ß and IL-6 and ng/ml for HMGB1) of proinflammatory cytokines IL-1ß ( A ), IL-6 ( C ) and HMGB1 ( E ) were evaluated 3 days post asbestos exposure. PLF WBC mRNA expression of IL-1ß ( B ), IL-6 ( D ) and HMGB1 ( F ) was determined using qPCR. Levels of target gene mRNA were normalized to 18S ribosomal RNA and values are expressed as fold change from CTL. # indicates a statistically significant difference (#### P

Article Snippet: Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the high capacity RNA to cDNA kit supplied by Applied Biosystems Quantitative polymerase chain reaction (qPCR) and performed using TaqMan® probe-based gene expression assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, CTL Assay

Journal: PLoS ONE

Article Title: Matrix Metalloproteinase-9 Is Involved in Chronic Lymphocytic Leukemia Cell Response to Fludarabine and Arsenic Trioxide

doi: 10.1371/journal.pone.0099993

Figure Lengend Snippet: Fludarabine transcriptionally upregulates MMP-9 and induces its localization to the CLL cell membrane. (A,B) 10–15×10 6 CLL in RPMI/0.1% FBS cells from two different patients were treated with 3 or 5 µM fludarabine (Fluda) for 48 h and MMP-9 mRNA expression was analyzed by RT-PCR (A) and qPCR (B). Normalized average values (fold change) are shown. (C) 1.5×10 5 CLL cells from two different patients were incubated with or without 3 µM fludarabine for 48 h and MMP-9 surface expression was analyzed by flow cytometry. White areas, control/untreated cells; grey areas, fludarabine treated cells. Arrows indicate specific fluorescence (SF) values for each cell population. Normalized average values are also shown.

Article Snippet: Quantitative PCR (qPCR) Quantitative PCR (qPCR) was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), and the primers described above for MMP-9.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Incubation, Flow Cytometry, Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Matrix Metalloproteinase-9 Is Involved in Chronic Lymphocytic Leukemia Cell Response to Fludarabine and Arsenic Trioxide

doi: 10.1371/journal.pone.0099993

Figure Lengend Snippet: Upregulation and membrane localization of MMP-9 is an initial CLL cell response to the cytotoxic action of ATO. (A) Cell sorter biparametric diagrams of PI − CLL cells (1.5×10 5 ) treated or not with ATO for 24 h and analyzed for MMP-9 expression. Numbers indicate the percentage of cells expressing MMP-9 in the early apoptotic (Annexin V + , top) and live (Annexin V − , bottom) cell compartments. (B) Flow cytometric analysis of MMP-9 expression in control or ATO-treated CLL cells with or without previous incubation with 50 µM Z-VAD-FMK. Histograms from two representative cases are shown. White areas: control cells; grey areas: ATO-treated cells. Arrows indicate specific fluorescence (SF). Normalized average values for all five samples analyzed are shown. The average % of early apoptotic (Ann V + /PI − ) cells in these samples is also shown. (C) 10–15×10 6 CLL cells treated as in (B) were analyzed for MMP-9 mRNA expression by qPCR, using TBP as an internal control. Average normalized values (fold change) are shown. (D,E) 10–15×10 6 CLL cells were treated with 1 µM ATO for 24 h and MMP-9 mRNA expression analyzed by RT-PCR (D) and qPCR (E). Normalized average values (fold change) are shown. (F) CLL cells treated as in (D, E) were analyzed for MMP-9 surface expression by flow cytometry with an anti-MMP-9 pAb or a control pAb. Histograms for the same samples used in (D, E) are shown. Arrows, white and grey areas are as in (B). Normalized average SF values of all four samples studied are shown. *P≤0.05; ***P≤0.001.

Article Snippet: Quantitative PCR (qPCR) Quantitative PCR (qPCR) was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), and the primers described above for MMP-9.

Techniques: Expressing, Flow Cytometry, Incubation, Fluorescence, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Cytometry

Journal: PLoS ONE

Article Title: Matrix Metalloproteinase-9 Is Involved in Chronic Lymphocytic Leukemia Cell Response to Fludarabine and Arsenic Trioxide

doi: 10.1371/journal.pone.0099993

Figure Lengend Snippet: Effect of ATO and fludarabine on MEC-1 cells. (A, B) 7.5×10 4 MEC-1 cells in IMDM/0.1% FBS were treated or not with the indicated concentrations of ATO (A) or fludarabine (Fluda) (B). After 24 h (ATO) or 48 h (Fluda), cell viability was determined by the MTT assay. Control cell viability was normalized to 100 and average values are shown. (C,D) 5×10 6 MEC-1 cells were treated with 3 µM ATO for the indicated times and MMP-9 mRNA expression was analyzed by RT-PCR, using GAPDH as internal control (C) and qPCR, using TBP as internal control (D). Normalized average values (fold change) are shown. (E) 1.5×10 5 MEC-1 cells were treated or not with 3 µM ATO for 24 h and MMP-9 surface expression was analyzed by flow cytometry, using an anti-MMP-9 pAb or a control pAb. Histograms from a representative experiment and normalized average values for the three experiments performed are shown. White areas, control/untreated cells; grey areas, ATO-treated cells. Arrows indicate the specific fluorescence. *P≤0.05 ; **P≤0.01; ***P≤0.001.

Article Snippet: Quantitative PCR (qPCR) Quantitative PCR (qPCR) was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), and the primers described above for MMP-9.

Techniques: MTT Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Matrix Metalloproteinase-9 Is Involved in Chronic Lymphocytic Leukemia Cell Response to Fludarabine and Arsenic Trioxide

doi: 10.1371/journal.pone.0099993

Figure Lengend Snippet: ATO transcriptionally upregulates MMP-9 in CLL cells. (A) 1.5×10 5 CLL cells in RPMI/0.1%FBS were incubated with or without the indicated concentrations of ATO. After 24 h, cells were analyzed by flow cytometry using FITC-Annexin V and PI. (B) 10–15×10 6 CLL cells were treated with 3 µM ATO for the indicated times and MMP-9 mRNA expression was analyzed by RT-PCR, using GAPDH mRNA as internal control. Normalized average values (fold change) are shown. (C) The same mRNA samples were also analyzed by qPCR using TBP as internal control and normalized average values (fold change) are shown. (D) 10–15×10 6 CLL cells were cultured with or without 3 µM ATO for 20 h. Cells were then treated or not (Control, Ctl) with 5 µM actinomycin D and mRNA expression was analyzed at the indicated times. Values represent the average MMP-9/GAPDH ratio from the two samples after normalizing control values to 100. Values for GAPDH mRNA are also shown. *P≤0.05; **P≤0.01; ***P≤0.001.

Article Snippet: Quantitative PCR (qPCR) Quantitative PCR (qPCR) was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), and the primers described above for MMP-9.

Techniques: Incubation, Flow Cytometry, Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Cell Culture, CTL Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Inhibitors of Signaling Pathways That Block Reversal of HIV-1 Latency

doi: 10.1128/AAC.01744-18

Figure Lengend Snippet: Quantitative PCR analysis of singly (SS) and multiply (MS) cellular HIV-1 RNA in the absence and presence of 1 μM prostratin and/or different concentrations of PF-3758309, danusertib, AZ 628, and P276-00. A total of 1 × 10 6 24ST1NLESG cells/well were exposed to prostratin (LRA) ± kinase inhibitor for 48 h, following which the total intracellular RNA from each condition was subjected to reverse transcription-quantitative PCR (RT-qPCR) analysis, as described in the Methods and Materials. Multiply spliced (MS) and singly spliced (SS) HIV-1 RNA were quantified relative to cellular IPO8 expression. Data are the average of 2 independent experiments.

Article Snippet: RNA was reverse transcribed using random primers with the AffinityScript multiple temperature reverse transcriptase (Agilent, Wilmington, DE), and quantitative PCR (qPCR) was performed using a Bio-Rad CFX96 real-time PCR detection system, using 2× Lightcycler 480 probes master (Roche, Indianapolis, IN).

Techniques: Real-time Polymerase Chain Reaction, Mass Spectrometry, Quantitative RT-PCR, Expressing

Journal: Toxicological Research

Article Title: CYP1B1 Activates Wnt/β-Catenin Signaling through Suppression of Herc5-Mediated ISGylation for Protein Degradation on β-Catenin in HeLa Cells

doi: 10.5487/TR.2017.33.3.211

Figure Lengend Snippet: CYP1B1 promotes the protein expression levels of β-catenin and cyclin D1 in HeLa cells. (A) RT-PCR analyses. HeLa cells were transfected with pcDNA3.1(zeo)-CYP1B1 (5 μg) or CYP1B1 specific siRNA (40 nM) for 48 hr. After RNA isolation, gene amplification of CYP1B1 was conducted. GAPDH mRNA was determined as a control. (B) Quantitative PCR analyses. Total RNA was isolated and qPCR for CYP1B1 was performed in triplicate. Data were normalized to the GAPDH value. The data represent mean ± SD. * p ≤0.05. (C) Western blot analyses. The cells were transfected with pcDNA 3.1(zeo)-CYP1B1 (5 μg) or CYP1B1 specific siRNA (40 nM). After stabilization for 48 hr, total protein was isolated from cell lysates, and the protein expression levels of CYP1B1, β-catenin, and cyclin D1 were measured. β-Actin was used as an internal loading control. Empty vector (pcDNA3.1(zeo))-transfected group was represented as control and CYP1B1-overexpressed group was represented as CYP1B1 in figures. Scrambled siRNA-transfected group was represented as siCON while CYP1B1 specific siRNA-transfected group was represented as si1B1 in figures.

Article Snippet: Quantitative PCR (qPCR) qPCR was performed with CFX Connect™ Real-Time PCR Detection System (Bio-Rad).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Isolation, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation

Journal: Journal of Animal Science and Biotechnology

Article Title: Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cell culture

doi: 10.1186/s40104-016-0068-x

Figure Lengend Snippet: Quantitative PCR results of miRNAs in differentiated and undifferentiated BMECs. a : Cellular miRNAs of BMECs. b : miRNAs in BMEC culture media. c : The miRNA expression ratio of medium/cells. + P

Article Snippet: Quantitative PCR (qPCR) analysis The miRNA qPCR was performed using a CFX96 thermal cycler (Bio-Rad) under the following program: first for 15 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C, with the Thunderbird SYBR qPCR kit (Toyobo, Tokyo) in combination with the miScript Primer Assay for let-7b, miR-21-5p, miR-23b-3p, miR-25, miR-26a, miR-30a, miR-103, miR-107, miR-148a, miR-155, miR-182, miR-191, miR-200c, miR-221, miR-223, miR-320a, miR-339a, and miR-375 (Qiagen).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Animal Science and Biotechnology

Article Title: Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cell culture

doi: 10.1186/s40104-016-0068-x

Figure Lengend Snippet: Quantitative PCR results of GAPDH and lactogenic markers (β- and κ-caseins) in differentiated and undifferentiated BMECs. * P

Article Snippet: Quantitative PCR (qPCR) analysis The miRNA qPCR was performed using a CFX96 thermal cycler (Bio-Rad) under the following program: first for 15 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C, with the Thunderbird SYBR qPCR kit (Toyobo, Tokyo) in combination with the miScript Primer Assay for let-7b, miR-21-5p, miR-23b-3p, miR-25, miR-26a, miR-30a, miR-103, miR-107, miR-148a, miR-155, miR-182, miR-191, miR-200c, miR-221, miR-223, miR-320a, miR-339a, and miR-375 (Qiagen).

Techniques: Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production

doi: 10.1038/s41467-018-05387-y

Figure Lengend Snippet: Lack of Opa1 alters mitochondrial morphology and cristae structure. a Transmission electron microscopy (TEM). Primary mature neutrophils from Opa1 N∆ and control mice were fixed and analyzed. Representative images are shown. Bars, 5 µm. Insets to the sides, bars, 1 µm. Mitochondria were further analyzed at higher magnification and statistical analyses are provided below in ( b ), ( e ), and ( f ). b The average numbers of mitochondria per neutrophil was quantified in at least 100 cells. Values are means ± SEM ( n = 3); n.s., not significant. c Quantitative PCR. Freshly purified mature neutrophils from Opa1 N∆ and control mice were analyzed for the number of DNA copies of mitochondrial mouse cytochrome c oxidase subunit 1 ( mt-Co1 ) relative to mouse β2 microglobulin ( B2M) which was used as a single copy nuclear-encoded reference gene. Values are means ± SEM ( n = 3); n.s., not significant. d Confocal microscopy. Primary mature mouse neutrophils from Opa1 N∆ and control mice were stained for TFAM expression (green) and DNA (red) using anti-TFAM antibody and PI. Bars, 10 µm; n = 3. e Average mitochondrial major axis length in freshly purified mature neutrophils from Opa1 N∆ and control mice. Images were acquired by TEM and subsequently analyzed using the measurement points module of Imaris software. Data were collected from at least five mitochondria per cell and more than 50 neutrophils per experiment. Values are means ± SEM. *** p

Article Snippet: Quantitative PCR (qPCR) assays were performed using iQ SYBR Green Supermix (Bio-Rad Laboratories).

Techniques: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Mouse Assay, Real-time Polymerase Chain Reaction, Purification, Confocal Microscopy, Staining, Expressing, Software

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Zinc finger protein 407 overexpression upregulates PPAR target gene expression and improves glucose homeostasis in mice

doi: 10.1152/ajpendo.00234.2016

Figure Lengend Snippet: Zinc finger protein 407 (Zfp407) mRNA overexpression in ZFP-TG mice. A : transgene construct with mouse Zfp407 cDNA tagged with Myc and DDK. B – D : endogenous ( B ), transgenic ( C ), and total Zfp407 mRNA expression ( D ) were measured by quantitative PCR and normalized to levels of Arbp in wild-type (WT) and ZFP-TG (TG) mice ( n = 4–10 mice/group). * P

Article Snippet: Quantitative PCR (qPCR) reactions were performed with the power SYBR green PCR Master Mix and run on a Bio-Rad (Hercules, CA) CFX Connect Real Time System.

Techniques: Over Expression, Mouse Assay, Construct, Transgenic Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: FEMS Microbiology Ecology

Article Title: Cold adaptation and replicable microbial community development during long-term low-temperature anaerobic digestion treatment of synthetic sewage

doi: 10.1093/femsec/fiy095

Figure Lengend Snippet: ( A ) qPCR data of Bacterial and Archaeal 16S copy numbers (per g biomass) on the right y-axis from biomass samples (x-axis) throughout the trial corresponding to the ratio of bacteria to archaea (expressed as a percentage) on the left y-axis. ( B ) qPCR data of Bacterial and Archaeal 16S rRNA transcripts copy numbers (per g biomass) on the right y-axis from biomass samples (x-axis) throughout the trial corresponding to the ratio of bacteria to archaea (expressed as a percentage) on the left y-axis.

Article Snippet: Quantitative-polymerase chain reaction Quantitative polymerase chain reaction (qPCR) was carried out for Archaeal and Bacterial domains using DNA and cDNA generated from granular biomass sampled from R1 and R2 and the fixed-film filter as described above. qPCR was performed using a LightCycler 480 (Roche, Manheim, Germany).

Techniques: Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Effect on the liver cancer cell invasion ability by studying the associations between autophagy and TRAP1 expression

doi: 10.3892/ol.2018.8774

Figure Lengend Snippet: (A) Reverse transcription-quantitative polymerase chain reaction was used to detect the TRAP1 mRNA expression levels in Hep3b2.1–7, HepG2, HepG2.2.15 and Sk-hep1 cells. (B) The protein levels of TRAP1 in the 4 liver cancer cell lines were determined by western blot analysis. β-actin was used as an internal control. (C) WES™ automated capillary-based size sorting system was used to measure the protein level of TRAP1 and β-actin in 4 liver cancer cell lines. (D) The protein levels of TRAP1 were examined in 4 liver cancer cells treated with 0, 5, 10 and 15 µmol/l rapamycin by western blot analysis. β-actin was used as an internal control. *P

Article Snippet: The mRNA and protein expression levels of tumor necrosis factor receptor-associated protein-1 (TRAP-1), Beclin1 and LC3-II/LC3-I were measured by reverse transcription-quantitative polymerase chain reaction, Protein Simple Western and western blot analysis.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: PLoS ONE

Article Title: From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows

doi: 10.1371/journal.pone.0124671

Figure Lengend Snippet: Quantitative PCR and sequencing results of the sample screening assay. Quantitative PCR curves indicating the presence of DNA and the degree of inhibition (LEFT) with agarose gel electrophoresis clearly indicating the presence of DNA post amplification via means of strong bands (INSET ON GRAPH) . Samples were subsequently sequenced and the percentage abundance of two fish genera is indicated, where, based on taxa-specific quantitative PCR results, Sardinops (specifically S . sagax —Australian pilchard) should be in the highest abundance, with Engraulis (specifically E . australis— Australian anchovy) being in the lowest abundance. (RIGHT) .

Article Snippet: Common methods of screening samples include quantitative PCR (qPCR) and PCR end-point assays such as gel electrophoresis or capillary electrophoresis (e.g. Agilent Bioanalyzer).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Screening Assay, Inhibition, Agarose Gel Electrophoresis, Amplification, Fluorescence In Situ Hybridization