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  • 99
    Thermo Fisher taqman gene expression assays
    Akt regulates p21 Cip levels by transcriptional regulation and protein stability. a Assessment of p21 Cip mRNA levels in MIN6 GFP and MIN6 caAkt cells using <t>TaqMan</t> <t>RT-PCR.</t> b p21 Cip mRNA stability assays from MIN6 and MIN6 caAkt cells treated with 5 μg/ml
    Taqman Gene Expression Assays, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 50697 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman universal pcr master mix
    Relative expression levels of selected C. burnetii genes during morphological differentiation as detected by quantitative <t>RT-PCR.</t> Assays were performed using <t>TaqMan</t> primers and probes specific for each gene. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Total RNA was extracted at the indicated times. Transcriptional activity is expressed as relative expression, with transcript copy number normalized to the number of C. burnetii genomes present in each sample. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.
    Taqman Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman microrna reverse transcription kit
    LTP regulates <t>microRNA</t> expression in the dentate gyrus middle molecular layer 5 h post-tetanisation. Regulation of microRNA expression in the middle molecular layer revealed by <t>TaqMan</t> Low Density microRNA Arrays. Eight microRNAs were found to be differentially expressed using dual selection criteria (unadjusted two-tailed Student’s t-test p
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29866 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman gene expression master mix
    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by <t>TaqMan</t> <t>PCR</t> in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.
    Taqman Gene Expression Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21089 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman probes
    Expression of COX4/1 and PGC1α in SK-N-SH cells and HepG2 cells after incubation of the cells with PQQ, PQQH 2 and IPQ. (A) COX4/1 expression in SK-N-SH cells, (B) COX4/1 expression in HepG2 cells (C) PGC1α expression in SK-N-SH cells, (D) PGC1α expression in HepG2 cells. Total mRNAs were extracted from SK-N-SH cells and HepG2 cells that had been cultured for 24 h after the addition PQQ, PQQH 2 and IPQ. One hundred nM and 1 μM of PQQ, PQQH 2 or IPQ were added to SK-N-SH cells and HepG2 cells, respectively. Total RNAs were isolated from cultured cells. Total RNAs (3–5 μg) were transcribed into cDNA by real-time quantitative polymerase chain reactions with <t>TaqMan</t> probes for COX4/1 and PGC1α. The relative quantities of COX4/1 and PGC1α transcripts were normalized to the level of GAPDH message. Each value is the mean ± SEM (n = 3–5). ***p
    Taqman Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 32140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher taqman microrna assay
    Expression of COX4/1 and PGC1α in SK-N-SH cells and HepG2 cells after incubation of the cells with PQQ, PQQH 2 and IPQ. (A) COX4/1 expression in SK-N-SH cells, (B) COX4/1 expression in HepG2 cells (C) PGC1α expression in SK-N-SH cells, (D) PGC1α expression in HepG2 cells. Total mRNAs were extracted from SK-N-SH cells and HepG2 cells that had been cultured for 24 h after the addition PQQ, PQQH 2 and IPQ. One hundred nM and 1 μM of PQQ, PQQH 2 or IPQ were added to SK-N-SH cells and HepG2 cells, respectively. Total RNAs were isolated from cultured cells. Total RNAs (3–5 μg) were transcribed into cDNA by real-time quantitative polymerase chain reactions with <t>TaqMan</t> probes for COX4/1 and PGC1α. The relative quantities of COX4/1 and PGC1α transcripts were normalized to the level of GAPDH message. Each value is the mean ± SEM (n = 3–5). ***p
    Taqman Microrna Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 5442 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taqman reverse transcription reagents
    TALAM1 promotes the 3′ end processing of MALAT1 <t>RNA.</t> ( A ) Cutoff assay of cleaved and uncleaved MALAT1 in HeLa cells transfected with empty vector (V) or with TALAM1-overexpression construct (TALAM1-OE). ( B ) Analyses of mascRNA levels in V or TALAM1-OE samples by (Ba) northern blot, and (Bb) <t>Taqman</t> small RNA assay. ( C ) In vivo RNA decay assay. (Ca) Schematic representation of the βΔ1,2 construct. (Cb) Stability of the intronless β-globin transcript in U2OS Tet-off cells transfected with empty vector (V) or with TALAM1-overexpression construct (TALAM1-OE). Trendlines are single-exponential regression of the percentage of RNA remaining versus time. Dotted lines represent the half-life of reporter RNA. ( D ) In vitro processing assay of MALAT1 3′ end sequence. Internally 32 P-labeled MALAT1 ENE+A+mascRNA RNA substrate (filled arrow in Db) was incubated with HeLa total cell extract in absence or presence of in vitro transcribed YFP, TALAM1-F1R1, F2R2 RNA or 2′MOE targeting the mascRNA site, and its processing to mascRNA (open arrow in Db) is monitored at 40 min and 60 min time points. (Da) Schematic diagrams shown for the relative positions of the TALAM1-F1R1 and TALAM1-F2R2 fragments generated by in vitro transcription. (Db) A representative autoadiogram is shown. (Dc) The quantification of the mascRNA product levels measured from independent experiments, with ImageJ software. At 40 min time point, levels of mascRNA produced under different conditions were normalized to the condition with no extra RNA, which was set as one. Bar data in (A) and (Bb) are mean values with SD, N A = 4, N Bb = 3; Data in (Cb) and (Dc) are mean values with SEM, N Cb = 4, N Dc = 6 (biological replicates, * P
    Taqman Reverse Transcription Reagents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10913 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman universal master mix ii
    Real-time PCR. The plot shows the amplification obtained using <t>TaqMan</t> probes with the same <t>DNA</t> sources as those used for the nested PCR that yielded positive results. The probes are based in a different region of the env gene, namely from 5943 to 6011 (AF033807). Each line represents a sample, positive control, or negative control. The PCR cycles are shown on the x-axis, and the ΔRn values (representing the fluorescent units) are shown on the y-axis. All of the assays were performed in 48-well plates.
    Taqman Universal Master Mix Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman fast advanced master mix
    NF90 modulates the expression level of a subset of miRNAs in HepG2 cells. ( A ) Extracts of HepG2 cells transfected with non-targeting control siRNAs (Scr, Scr#2) or siRNA targeting NF90 (NF90, NF90#2) as indicated were analyzed by immunoblot using the antibodies indicated. ( B ) Total RNA extracted from cells transfected with siScr or siNF90 were analyzed by small RNA-seq. Results are shown as log 2 fold change versus –log 10 P -value. ( C ) Table summarizing the number of mature miRNAs and pri-miRNAs modulated in HepG2 cell line upon loss of NF90, according to small-RNA seq. ( D ) Total RNA extracted from cells described in (A) were analyzed by <t>Taqman</t> <t>RT-qPCR</t> as indicated. Results were normalized by those obtained for U6 abundance in the same samples. ND indicates ‘not detected’. Data represent mean ± SEM obtained from three independent experiments (*** P
    Taqman Fast Advanced Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6898 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman mirna assay kit
    miRNAs Are Differentially Expressed in RGCs during Development (A) A representative heatmap of <t>miRNA</t> expression profiles constructed from a microarray analysis of retinal ganglion cells (RGCs) purified from embryonic day 21 (E21, n = 3 biological replicates), postnatal day 6 (P6, n = 3 biological replicates), and P30 (n = 2 biological replicates) Sprague-Dawley (SD) rats, showing 76 endogenously expressed miRNAs with significant differential expression (≥4-fold changes in expression levels) during development. Developmental ages (as biological replicates) are indicated in columns, and differentially expressed miRNAs are indicated in rows. All six members of the miR-17-92 cluster (red asterisks) were found to have significant downregulation from E21 to P30 (right panel). Blue (−8.5) and red (+8.5) in the color-coding scale represent relative low and high normalized miRNA expressions, respectively. A two-tailed moderated t test with a Benjamini-Hochberg correction for false discovery rate was used to compare the means of miRNA microarray expression values between E21 and P6 and between E21 and P30. A list of the 76 miRNAs can be found in Table S1 , and all other microarray data that support the findings of this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO: GSE102458 ). (B) Illustration of the polycistronic miR-17-92 cluster region on human chromosome 13 (chromosome 15 for rats). Precursor transcripts derived from the miR-17-92 gene cluster contains six tandem stem-loop hairpin structures that yield six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. The miRNAs can be categorized into four miRNA families according to their conserved seed sequences (underlined). (C) Downregulation of the six miRNAs from E21 to P30 and from E21 to P6 was confirmed by <t>TaqMan</t> qRT-PCR (E21 vs. P6, miR-17-5p, p = 0.005; miR-18a, p = 0.003; miR-19a, p = 0.014; miR-19b, p = 0.013; miR-20a, p = 0.005; miR-92a, p = 0.014; E21 vs. P30, miR-17-5p, p = 0.0004; miR-18a, p = 0.001; miR-19a, p = 0.007; miR-19b, p = 0.005; miR-20a, p = 0.001; miR-92a, p = 0.003; n = 3 biological replicates for each age group). An unpaired two-tailed Student’s t test was used for TaqMan comparisons. All values are shown as mean ± SEM.
    Taqman Mirna Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1486 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Akt regulates p21 Cip levels by transcriptional regulation and protein stability. a Assessment of p21 Cip mRNA levels in MIN6 GFP and MIN6 caAkt cells using TaqMan RT-PCR. b p21 Cip mRNA stability assays from MIN6 and MIN6 caAkt cells treated with 5 μg/ml

    Journal: Diabetologia

    Article Title: Enhanced beta cell proliferation in mice overexpressing a constitutively active form of Akt and one allele of p21Cip *

    doi: 10.1007/s00125-012-2465-9

    Figure Lengend Snippet: Akt regulates p21 Cip levels by transcriptional regulation and protein stability. a Assessment of p21 Cip mRNA levels in MIN6 GFP and MIN6 caAkt cells using TaqMan RT-PCR. b p21 Cip mRNA stability assays from MIN6 and MIN6 caAkt cells treated with 5 μg/ml

    Article Snippet: Real-time PCR was performed on an ABI 7000 sequence detection system using Taqman gene expression assays (Applied Biosystems, Foster City, CA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    mTORC1 inhibition destabilizes ISCU protein. A , 3T3-L1 cells control ( Con ) or serum-starved overnight ( SS ) were analyzed by Western blot and Taqman RT-PCR assays. B , 3T3-L1 cells were incubated with 200 n m rapamycin ( Rapa ) or dimethyl sulfoxide ( DMSO

    Journal: The Journal of Biological Chemistry

    Article Title: Mammalian Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation Stabilizes ISCU Protein

    doi: 10.1074/jbc.M112.424499

    Figure Lengend Snippet: mTORC1 inhibition destabilizes ISCU protein. A , 3T3-L1 cells control ( Con ) or serum-starved overnight ( SS ) were analyzed by Western blot and Taqman RT-PCR assays. B , 3T3-L1 cells were incubated with 200 n m rapamycin ( Rapa ) or dimethyl sulfoxide ( DMSO

    Article Snippet: Using the Taqman Gene expression assay (Applied Biosystems), ISCU mRNA levels were analyzed and further normalized to GAPDH mRNA levels.

    Techniques: Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation

    Relative expression levels of selected C. burnetii genes during morphological differentiation as detected by quantitative RT-PCR. Assays were performed using TaqMan primers and probes specific for each gene. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Total RNA was extracted at the indicated times. Transcriptional activity is expressed as relative expression, with transcript copy number normalized to the number of C. burnetii genomes present in each sample. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.

    Journal: Journal of Bacteriology

    Article Title: Temporal Analysis of Coxiella burnetii Morphological Differentiation

    doi: 10.1128/JB.186.21.7344-7352.2004

    Figure Lengend Snippet: Relative expression levels of selected C. burnetii genes during morphological differentiation as detected by quantitative RT-PCR. Assays were performed using TaqMan primers and probes specific for each gene. Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers, and fresh medium was added. This time was designated as 0 h p.i. Total RNA was extracted at the indicated times. Transcriptional activity is expressed as relative expression, with transcript copy number normalized to the number of C. burnetii genomes present in each sample. The results are expressed as the mean from three experiments, with error bars representing the standard error of the mean.

    Article Snippet: QPCR and reverse transcriptase PCR (RT-PCR) (see below) were performed using TaqMan Universal PCR Master Mix and a Prism 7000 sequence detection system (Applied Biosystems).

    Techniques: Expressing, Quantitative RT-PCR, Incubation, Purification, Activity Assay

    Quantitative RT-PCR validation of microarray data. ( A ) Quantitative RT-PCR was performed using SYBR green detection for the indicated genes. Bars show the mean (log 2 ) +/− S.E.M. of at least three independent experimental replicates from 3–6 biological replicates per group. All data is represented relative to the expression of actin (ΔC t ). In order to facilitate visualization on a log 2 scale, values were transformed as indicated on the y-axis label. ( B ) Microarray results for the same genes interrogated in ( A ). ( C ) Taqman qPCR quantification of pan-IFNα, Ifnb1 , or Ifng throughout diabetogenesis. Bars represent the mean of the normalized probe intensity +/− S.E.M. of 3–6 biological replicates per group. Asterisks indicate statistical significance (P

    Journal: PLoS ONE

    Article Title: Defining the Transcriptional and Cellular Landscape of Type 1 Diabetes in the NOD Mouse

    doi: 10.1371/journal.pone.0059701

    Figure Lengend Snippet: Quantitative RT-PCR validation of microarray data. ( A ) Quantitative RT-PCR was performed using SYBR green detection for the indicated genes. Bars show the mean (log 2 ) +/− S.E.M. of at least three independent experimental replicates from 3–6 biological replicates per group. All data is represented relative to the expression of actin (ΔC t ). In order to facilitate visualization on a log 2 scale, values were transformed as indicated on the y-axis label. ( B ) Microarray results for the same genes interrogated in ( A ). ( C ) Taqman qPCR quantification of pan-IFNα, Ifnb1 , or Ifng throughout diabetogenesis. Bars represent the mean of the normalized probe intensity +/− S.E.M. of 3–6 biological replicates per group. Asterisks indicate statistical significance (P

    Article Snippet: TaqMan PCR was performed using TaqMan Fast Universal PCR Master Mix (Life Technologies).

    Techniques: Quantitative RT-PCR, Microarray, SYBR Green Assay, Expressing, Transformation Assay, Real-time Polymerase Chain Reaction

    LTP regulates microRNA expression in the dentate gyrus middle molecular layer 5 h post-tetanisation. Regulation of microRNA expression in the middle molecular layer revealed by TaqMan Low Density microRNA Arrays. Eight microRNAs were found to be differentially expressed using dual selection criteria (unadjusted two-tailed Student’s t-test p

    Journal: PLoS ONE

    Article Title: MicroRNAs, miR-23a-3p and miR-151-3p, Are Regulated in Dentate Gyrus Neuropil following Induction of Long-Term Potentiation In Vivo

    doi: 10.1371/journal.pone.0170407

    Figure Lengend Snippet: LTP regulates microRNA expression in the dentate gyrus middle molecular layer 5 h post-tetanisation. Regulation of microRNA expression in the middle molecular layer revealed by TaqMan Low Density microRNA Arrays. Eight microRNAs were found to be differentially expressed using dual selection criteria (unadjusted two-tailed Student’s t-test p

    Article Snippet: Total RNA (10 ng per 15 μL reaction) was converted to cDNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems).

    Techniques: Expressing, Selection, Two Tailed Test

    miR-198 functions to repress Cyclin T1 protein expression in primary monocytes. (A) Expression levels of Cyclin T1 and β-actin proteins in freshly isolated monocytes (d0) or macrophages allowed to differentiate for five days (d5) were examined in immunoblots for two donors. (B) Expression levels of Cyclin T1 mRNA relative to β-actin mRNA were measured by RT-real-time PCR assay; expression levels of miR-198 were measured using TaqMan MicroRNA assays. The relative abundance was calculated by normalizing of β-actin mRNA or miR-198 levels. Bars represent range in three measurements. (C) miR-198 inhibitor (anti-miR-198) or control inhibitor (anti-miR-Control) were transfected into freshly isolated monocytes from two donors (Donor 3, 4). miR-198 precursor (pre-miR-198) or control precursor (pre-miR-Control) were transfected into freshly isolated monocytes and differentiation was induced with GM-CSF treatment (Donor 5). Cells were harvested by direct lysis at times indicated after transfection and Cyclin T1 protein expression was examined in immunoblots. Quantification of Cyclin T1 protein levels is shown below each band (normalized to β-actin).

    Journal: PLoS Pathogens

    Article Title: miR-198 Inhibits HIV-1 Gene Expression and Replication in Monocytes and Its Mechanism of Action Appears To Involve Repression of Cyclin T1

    doi: 10.1371/journal.ppat.1000263

    Figure Lengend Snippet: miR-198 functions to repress Cyclin T1 protein expression in primary monocytes. (A) Expression levels of Cyclin T1 and β-actin proteins in freshly isolated monocytes (d0) or macrophages allowed to differentiate for five days (d5) were examined in immunoblots for two donors. (B) Expression levels of Cyclin T1 mRNA relative to β-actin mRNA were measured by RT-real-time PCR assay; expression levels of miR-198 were measured using TaqMan MicroRNA assays. The relative abundance was calculated by normalizing of β-actin mRNA or miR-198 levels. Bars represent range in three measurements. (C) miR-198 inhibitor (anti-miR-198) or control inhibitor (anti-miR-Control) were transfected into freshly isolated monocytes from two donors (Donor 3, 4). miR-198 precursor (pre-miR-198) or control precursor (pre-miR-Control) were transfected into freshly isolated monocytes and differentiation was induced with GM-CSF treatment (Donor 5). Cells were harvested by direct lysis at times indicated after transfection and Cyclin T1 protein expression was examined in immunoblots. Quantification of Cyclin T1 protein levels is shown below each band (normalized to β-actin).

    Article Snippet: As quantified by TaqMan MicroRNA assays (Applied Biosystems), miR-198 was expressed > 400-fold in cell pools expressing shmiR-198 relative to pools expressing shGFP.

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

    Dynamic range of eight TaqMan miRNA assays using OP9 cell lysates. The number of cell input ranged from 3 to 2500 cells per RT. A Caenorhabditis elegans miRNA (miR-2) was used as a negative control.

    Journal: Nucleic Acids Research

    Article Title: Real-time quantification of microRNAs by stem-loop RT-PCR

    doi: 10.1093/nar/gni178

    Figure Lengend Snippet: Dynamic range of eight TaqMan miRNA assays using OP9 cell lysates. The number of cell input ranged from 3 to 2500 cells per RT. A Caenorhabditis elegans miRNA (miR-2) was used as a negative control.

    Article Snippet: All TaqMan miRNA assays are available through Applied Biosystems (P/N: 4365409).

    Techniques: Negative Control

    Dynamic range and sensitivity of the TaqMan lin-4 miRNA assay. ( A ) Amplification plot of synthetic lin-4 miRNA over seven orders of magnitude. Synthetic RNA input ranged from 1.3 × 10 −3 fM (equivalent to 7 copies per reaction) to 1.3 × 10 4 fM (7 × 10 7 copies per reaction) in PCR; ( B ) Standard curve of the lin-4 miRNA.

    Journal: Nucleic Acids Research

    Article Title: Real-time quantification of microRNAs by stem-loop RT-PCR

    doi: 10.1093/nar/gni178

    Figure Lengend Snippet: Dynamic range and sensitivity of the TaqMan lin-4 miRNA assay. ( A ) Amplification plot of synthetic lin-4 miRNA over seven orders of magnitude. Synthetic RNA input ranged from 1.3 × 10 −3 fM (equivalent to 7 copies per reaction) to 1.3 × 10 4 fM (7 × 10 7 copies per reaction) in PCR; ( B ) Standard curve of the lin-4 miRNA.

    Article Snippet: All TaqMan miRNA assays are available through Applied Biosystems (P/N: 4365409).

    Techniques: Amplification, Polymerase Chain Reaction

    Specificity of TaqMan miRNA assays between stem–loop and linear RT primers. Mature let-7a-specific assay was tested against let-7a, let-7e and pri-miR precursor let-7a-3. Δ C T represents the C T difference between two targets or methods. A total of 1.5 × 10 8 copies of synthetic targets were added to each RT reaction.

    Journal: Nucleic Acids Research

    Article Title: Real-time quantification of microRNAs by stem-loop RT-PCR

    doi: 10.1093/nar/gni178

    Figure Lengend Snippet: Specificity of TaqMan miRNA assays between stem–loop and linear RT primers. Mature let-7a-specific assay was tested against let-7a, let-7e and pri-miR precursor let-7a-3. Δ C T represents the C T difference between two targets or methods. A total of 1.5 × 10 8 copies of synthetic targets were added to each RT reaction.

    Article Snippet: All TaqMan miRNA assays are available through Applied Biosystems (P/N: 4365409).

    Techniques:

    Schematic description of TaqMan miRNA assays, TaqMan-based real-time quantification of miRNAs includes two steps, stem–loop RT and real-time PCR. Stem–loop RT primers bind to at the 3′ portion of miRNA molecules and are reverse transcribed with reverse transcriptase. Then, the RT product is quantified using conventional TaqMan PCR that includes miRNA-specific forward primer, reverse primer and a dye-labeled TaqMan probes. The purpose of tailed forward primer at 5′ is to increase its melting temperature (Tm) depending on the sequence composition of miRNA molecules.

    Journal: Nucleic Acids Research

    Article Title: Real-time quantification of microRNAs by stem-loop RT-PCR

    doi: 10.1093/nar/gni178

    Figure Lengend Snippet: Schematic description of TaqMan miRNA assays, TaqMan-based real-time quantification of miRNAs includes two steps, stem–loop RT and real-time PCR. Stem–loop RT primers bind to at the 3′ portion of miRNA molecules and are reverse transcribed with reverse transcriptase. Then, the RT product is quantified using conventional TaqMan PCR that includes miRNA-specific forward primer, reverse primer and a dye-labeled TaqMan probes. The purpose of tailed forward primer at 5′ is to increase its melting temperature (Tm) depending on the sequence composition of miRNA molecules.

    Article Snippet: All TaqMan miRNA assays are available through Applied Biosystems (P/N: 4365409).

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

    Comparison of TaqMan miRNA miR-16 assay to solution-based northern hybridization analysis. Total RNAs from mouse kidney, liver, lung, spleen and testicle tissues were used.

    Journal: Nucleic Acids Research

    Article Title: Real-time quantification of microRNAs by stem-loop RT-PCR

    doi: 10.1093/nar/gni178

    Figure Lengend Snippet: Comparison of TaqMan miRNA miR-16 assay to solution-based northern hybridization analysis. Total RNAs from mouse kidney, liver, lung, spleen and testicle tissues were used.

    Article Snippet: All TaqMan miRNA assays are available through Applied Biosystems (P/N: 4365409).

    Techniques: Northern Blot, Hybridization

    Correlation between OpenArray and single assay results. A technical replication of the four selected miRNAs was performed. Per miRNA, all ADA ( a and b ) or ETN ( c and d ) samples from the discovery cohort were re-analyzed using TaqMan single miRNA assays. The normalized values for the OpenArray (ΔCrt) and single assay (ΔCt) for all 40 samples was plotted and the Spearman correlation (r) was calculated

    Journal: Arthritis Research & Therapy

    Article Title: Can baseline serum microRNAs predict response to TNF-alpha inhibitors in rheumatoid arthritis?

    doi: 10.1186/s13075-016-1085-z

    Figure Lengend Snippet: Correlation between OpenArray and single assay results. A technical replication of the four selected miRNAs was performed. Per miRNA, all ADA ( a and b ) or ETN ( c and d ) samples from the discovery cohort were re-analyzed using TaqMan single miRNA assays. The normalized values for the OpenArray (ΔCrt) and single assay (ΔCt) for all 40 samples was plotted and the Spearman correlation (r) was calculated

    Article Snippet: Briefly, 2.5ul of isolated serum RNA was reverse-transcribed by using the miRNA multiplex RT primers pools, either v2.1 for pool A or v3.0 for pool B, and the TaqMan miRNA reverse transcription kit (Life Technologies).

    Techniques:

    RECQL5 mRNA is increased in UCC and is associated with poor prognosis Taqman qRT-PCR quantification of mRNA from 197 primary bladder tumour and 20 normal tissue samples using a RECQL5 probe and plotted as fold change [ 52 ]. A. Stratified by normal compared to malignant tissue and B. according to tumour grade; * and *** indicate p

    Journal: Oncotarget

    Article Title: Altered RECQL5 expression in urothelial bladder carcinoma increases cellular proliferation and makes RECQL5 helicase activity a novel target for chemotherapy

    doi: 10.18632/oncotarget.12683

    Figure Lengend Snippet: RECQL5 mRNA is increased in UCC and is associated with poor prognosis Taqman qRT-PCR quantification of mRNA from 197 primary bladder tumour and 20 normal tissue samples using a RECQL5 probe and plotted as fold change [ 52 ]. A. Stratified by normal compared to malignant tissue and B. according to tumour grade; * and *** indicate p

    Article Snippet: RT-PCR was performed using TaqMan probe and primers (Applied Biosystems, Grand Island, NY) for RECQL5β (Hs00696986_g1) and RECQL5 non isotype specific (Hs00188633_m1), Heat shock protein 90kDa alpha (cytosolic), class B member 1 (HSP90AB1) (Hs03043878_g1), Testis enhanced gene transcript protein (TEGT) (Hs01012085_m1) and Mitochondrial ATP synthase H+ transporting F1 complex beta subunit (ATP5B) (Hs00969573_mH) with TaqMan Universal PCR Mix (Applied Biosystems) as recommended by the manufacturer.

    Techniques: Quantitative RT-PCR

    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Polymerase Chain Reaction

    FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Journal: eLife

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    doi: 10.7554/eLife.35012

    Figure Lengend Snippet: FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

    Techniques: Activity Assay, Transfection, Western Blot, Staining, Polymerase Chain Reaction, Knock-Out, Immunostaining, Flow Cytometry, Cytometry, Fluorescence, Software, Incubation, MANN-WHITNEY

    Expression of COX4/1 and PGC1α in SK-N-SH cells and HepG2 cells after incubation of the cells with PQQ, PQQH 2 and IPQ. (A) COX4/1 expression in SK-N-SH cells, (B) COX4/1 expression in HepG2 cells (C) PGC1α expression in SK-N-SH cells, (D) PGC1α expression in HepG2 cells. Total mRNAs were extracted from SK-N-SH cells and HepG2 cells that had been cultured for 24 h after the addition PQQ, PQQH 2 and IPQ. One hundred nM and 1 μM of PQQ, PQQH 2 or IPQ were added to SK-N-SH cells and HepG2 cells, respectively. Total RNAs were isolated from cultured cells. Total RNAs (3–5 μg) were transcribed into cDNA by real-time quantitative polymerase chain reactions with TaqMan probes for COX4/1 and PGC1α. The relative quantities of COX4/1 and PGC1α transcripts were normalized to the level of GAPDH message. Each value is the mean ± SEM (n = 3–5). ***p

    Journal: Heliyon

    Article Title: Effects of pyrroloquinoline quinone and imidazole pyrroloquinoline on biological activities and neural functions

    doi: 10.1016/j.heliyon.2020.e03240

    Figure Lengend Snippet: Expression of COX4/1 and PGC1α in SK-N-SH cells and HepG2 cells after incubation of the cells with PQQ, PQQH 2 and IPQ. (A) COX4/1 expression in SK-N-SH cells, (B) COX4/1 expression in HepG2 cells (C) PGC1α expression in SK-N-SH cells, (D) PGC1α expression in HepG2 cells. Total mRNAs were extracted from SK-N-SH cells and HepG2 cells that had been cultured for 24 h after the addition PQQ, PQQH 2 and IPQ. One hundred nM and 1 μM of PQQ, PQQH 2 or IPQ were added to SK-N-SH cells and HepG2 cells, respectively. Total RNAs were isolated from cultured cells. Total RNAs (3–5 μg) were transcribed into cDNA by real-time quantitative polymerase chain reactions with TaqMan probes for COX4/1 and PGC1α. The relative quantities of COX4/1 and PGC1α transcripts were normalized to the level of GAPDH message. Each value is the mean ± SEM (n = 3–5). ***p

    Article Snippet: Real-time quantitative polymerase chain reactions were performed in an Eco real-time PCR system (Illumia, San Diego, CA) in combination with TaqMan probes (Applied Biosystems) for COX4/1 and PGC1 α.

    Techniques: Expressing, Incubation, Cell Culture, Isolation

    Inhibition of single miRNA has no effect on entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with either control miRNA inhibitor no. 1 or the indicated miRNA inhibitor (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μ mol/l) for another 24 h. ( a ) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. Bars , S.D. Data shows the representative of three independent experiments. ( b ) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β -actin

    Journal: Cell Death & Disease

    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells

    doi: 10.1038/cddis.2013.79

    Figure Lengend Snippet: Inhibition of single miRNA has no effect on entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with either control miRNA inhibitor no. 1 or the indicated miRNA inhibitor (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μ mol/l) for another 24 h. ( a ) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. Bars , S.D. Data shows the representative of three independent experiments. ( b ) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β -actin

    Article Snippet: The expression levels of these miRNAs were assessed by qRT-PCR using TaqMan (Applied Biosystems, Foster City, CA, USA) miRNA assays.

    Techniques: Inhibition, Multiple Displacement Amplification, Transfection, RNA Extraction, Expressing, Quantitative RT-PCR, Western Blot

    Simultaneous inhibition of two miRNAs reduces entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with combinations of either the two control miRNA inhibitors no. 1 and no. 2 or the two indicated miRNA inhibitors (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μ mol/l) for another 24 h. ( a ) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. ( b ) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β -actin. The bar graph underneath was obtained by densitometry analysis. The relative signal intensities of erbB2 or erbB3 were measured by the Bio-Rad Gel Documentation System. Bars, S.D. The data are representative of three independent experiments

    Journal: Cell Death & Disease

    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells

    doi: 10.1038/cddis.2013.79

    Figure Lengend Snippet: Simultaneous inhibition of two miRNAs reduces entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with combinations of either the two control miRNA inhibitors no. 1 and no. 2 or the two indicated miRNA inhibitors (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μ mol/l) for another 24 h. ( a ) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. ( b ) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β -actin. The bar graph underneath was obtained by densitometry analysis. The relative signal intensities of erbB2 or erbB3 were measured by the Bio-Rad Gel Documentation System. Bars, S.D. The data are representative of three independent experiments

    Article Snippet: The expression levels of these miRNAs were assessed by qRT-PCR using TaqMan (Applied Biosystems, Foster City, CA, USA) miRNA assays.

    Techniques: Inhibition, Multiple Displacement Amplification, Transfection, RNA Extraction, Expressing, Quantitative RT-PCR, Western Blot

    Entinostat upregulates the expression levels of miR-125a, miR-125b, and miR-205 in erbB2-overexpressing breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells untreated or treated with entinostat (0.5 and 2 μ mol/l, respectively) for 8, 16, 24 h were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. All results were normalized with the internal control RNU6B. Bars, S.D. The data are representative of three independent experiments

    Journal: Cell Death & Disease

    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells

    doi: 10.1038/cddis.2013.79

    Figure Lengend Snippet: Entinostat upregulates the expression levels of miR-125a, miR-125b, and miR-205 in erbB2-overexpressing breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells untreated or treated with entinostat (0.5 and 2 μ mol/l, respectively) for 8, 16, 24 h were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. All results were normalized with the internal control RNU6B. Bars, S.D. The data are representative of three independent experiments

    Article Snippet: The expression levels of these miRNAs were assessed by qRT-PCR using TaqMan (Applied Biosystems, Foster City, CA, USA) miRNA assays.

    Techniques: Expressing, Multiple Displacement Amplification, RNA Extraction, Quantitative RT-PCR

    EPT1 cells have undergone EMT. A. Representative light microscopic images show morphological changes of EPT1 cells derived from EP156T cells at both low density (a, d) and high density cultures (b, c, e, f). EPT1 cells are fusiform and much longer than EP156T cells. Overlapping growth of EPT1 cells is also shown (d, e, f). B. Real-time qPCR showing the cadherin switch of mRNA levels in EPT1 compared to EP156T using TaqMan assays for E-cadherin (Hs00170423_m1) and N-cadherin (Hs00169953_m1). C. Western blots show the cadherin switch of protein levels. Both primary antibodies against E-cadherin and N-cadherin (BD Transduction Labs.) were diluted 1∶2500. The anti-actin mouse Mab was diluted 1∶250. The HRP-conjugated anti-Ig was diluted 1∶8000. Molecular weights (kD) were estimated based upon the ECL DualVue Marker in wells labelled M. D. Indirect immunofluorescence images (20×) show decreased E-cadherin and increased N-cadherin in EPT1 cells (right panels). The primary antibody against E-cadherin was diluted 1∶50 and against N-cadherin (BD Transduction Labs.) 1∶25 and the FITC-conjugated secondary antibody 1∶250.

    Journal: PLoS ONE

    Article Title: Epithelial to Mesenchymal Transition of a Primary Prostate Cell Line with Switches of Cell Adhesion Modules but without Malignant Transformation

    doi: 10.1371/journal.pone.0003368

    Figure Lengend Snippet: EPT1 cells have undergone EMT. A. Representative light microscopic images show morphological changes of EPT1 cells derived from EP156T cells at both low density (a, d) and high density cultures (b, c, e, f). EPT1 cells are fusiform and much longer than EP156T cells. Overlapping growth of EPT1 cells is also shown (d, e, f). B. Real-time qPCR showing the cadherin switch of mRNA levels in EPT1 compared to EP156T using TaqMan assays for E-cadherin (Hs00170423_m1) and N-cadherin (Hs00169953_m1). C. Western blots show the cadherin switch of protein levels. Both primary antibodies against E-cadherin and N-cadherin (BD Transduction Labs.) were diluted 1∶2500. The anti-actin mouse Mab was diluted 1∶250. The HRP-conjugated anti-Ig was diluted 1∶8000. Molecular weights (kD) were estimated based upon the ECL DualVue Marker in wells labelled M. D. Indirect immunofluorescence images (20×) show decreased E-cadherin and increased N-cadherin in EPT1 cells (right panels). The primary antibody against E-cadherin was diluted 1∶50 and against N-cadherin (BD Transduction Labs.) 1∶25 and the FITC-conjugated secondary antibody 1∶250.

    Article Snippet: Differentially expressed genes in microarray data were confirmed by real-time quantitative reverse transcription PCR (qPCR) using TaqMan assays (Applied Biosystems, Foster City, CA, USA) and mean quantitative values were normalized according to ACTB expression as described .

    Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Transduction, Marker, Immunofluorescence

    In vitro acquired resistance to osimertinib associates with emergence of the C797S mutation and resistance to afatinib Shown are survival assays of PC9ER and derivative PC9ER‐AZDR cells, which were selected in vitro for resistance to osimertinib. Cells were treated for 72 h with increasing concentrations of erlotinib or afatinib, and MTT signals were determined. Data are means ± SD values of three independent experiments. Genomic DNA was isolated from PC9ER and from PC9ER‐AZDR cells and then subjected to dPCR analysis using the Fluidigm Biomark platform. Thereafter, DNA stocks were diluted to 10 ng/μl. In parallel, DNA was isolated from control pcDNA3 plasmids, encoding either wild‐type EGFR or the triple mutant del746/50, T790M, and C797S. Five serial dilutions of the two genomic DNA samples were prepared and loaded onto the dPCR chip, side by side with DNA from plasmids (1 pg). The results obtained with one serial dilution (5 ng DNA) and C797S‐specific primers are presented alongside the results from control plasmids. Analysis was performed utilizing dPCR software from Fluidigm. Amplifications with mutation positive and mutation negative wells appear in red and in blue, respectively. Histograms depicting results of digital PCR (dPCR) analysis performed three times with genomic DNA isolated from PC9ER and PC9ER‐AZDR cells. Average numbers of copies per 1 ng of genomic DNA (± SD) were obtained from serial dilutions of DNA. The TaqMan genotyping assay was applied on genomic DNA isolated from PC9, PC9ER, and PC9ER‐AZDR cells. As reference, we used DNA from normal human lung epithelial cells (NL20), along with a plasmid DNA encoding EGFR harboring the indicated mutations. Fluorescent signals corresponding to cellular DNA samples are shown as small colored dots, whereas the larger dots represent signals obtained from plasmid DNAs. Note that VIC‐labeled primers were used for amplification of wild‐type alleles, whereas FAM‐labeled primers were used for the mutant alleles. Each sample was run in duplicates. Data were analyzed using TaqMan Genotyper Software (Life technologies). As indicated, PC9ER‐AZDR cells scored positive for three mutant alleles: del746/750, T790M, and C797S. NTC, no‐template control. Genomic DNA was extracted from PC9ER‐AZDR cells. The region corresponding to EGFR's exon 20 was sequenced and aligned with the WT sequence. Note that the C to T (T790M) mutation (red) and the T to A (C797S) mutation (blue) are allelic. Genomic DNA isolated from PC9ER and from PC9ER‐AZDR cells was subjected to dPCR analysis using the Fluidigm Biomark platform (gDNA, 2.5 ng/sample). Exact copy number of the indicated EGFR mutated alleles, as well as the WT alleles, was then assessed. The RNase P specific set of probes was used as normalizer. The analysis was performed utilizing the dPCR software from Fluidigm. Amplifications with mutation positive and mutation negative wells appear in red and in blue, respectively.

    Journal: EMBO Molecular Medicine

    Article Title: An oligoclonal antibody durably overcomes resistance of lung cancer to third‐generation EGFR inhibitors

    doi: 10.15252/emmm.201708076

    Figure Lengend Snippet: In vitro acquired resistance to osimertinib associates with emergence of the C797S mutation and resistance to afatinib Shown are survival assays of PC9ER and derivative PC9ER‐AZDR cells, which were selected in vitro for resistance to osimertinib. Cells were treated for 72 h with increasing concentrations of erlotinib or afatinib, and MTT signals were determined. Data are means ± SD values of three independent experiments. Genomic DNA was isolated from PC9ER and from PC9ER‐AZDR cells and then subjected to dPCR analysis using the Fluidigm Biomark platform. Thereafter, DNA stocks were diluted to 10 ng/μl. In parallel, DNA was isolated from control pcDNA3 plasmids, encoding either wild‐type EGFR or the triple mutant del746/50, T790M, and C797S. Five serial dilutions of the two genomic DNA samples were prepared and loaded onto the dPCR chip, side by side with DNA from plasmids (1 pg). The results obtained with one serial dilution (5 ng DNA) and C797S‐specific primers are presented alongside the results from control plasmids. Analysis was performed utilizing dPCR software from Fluidigm. Amplifications with mutation positive and mutation negative wells appear in red and in blue, respectively. Histograms depicting results of digital PCR (dPCR) analysis performed three times with genomic DNA isolated from PC9ER and PC9ER‐AZDR cells. Average numbers of copies per 1 ng of genomic DNA (± SD) were obtained from serial dilutions of DNA. The TaqMan genotyping assay was applied on genomic DNA isolated from PC9, PC9ER, and PC9ER‐AZDR cells. As reference, we used DNA from normal human lung epithelial cells (NL20), along with a plasmid DNA encoding EGFR harboring the indicated mutations. Fluorescent signals corresponding to cellular DNA samples are shown as small colored dots, whereas the larger dots represent signals obtained from plasmid DNAs. Note that VIC‐labeled primers were used for amplification of wild‐type alleles, whereas FAM‐labeled primers were used for the mutant alleles. Each sample was run in duplicates. Data were analyzed using TaqMan Genotyper Software (Life technologies). As indicated, PC9ER‐AZDR cells scored positive for three mutant alleles: del746/750, T790M, and C797S. NTC, no‐template control. Genomic DNA was extracted from PC9ER‐AZDR cells. The region corresponding to EGFR's exon 20 was sequenced and aligned with the WT sequence. Note that the C to T (T790M) mutation (red) and the T to A (C797S) mutation (blue) are allelic. Genomic DNA isolated from PC9ER and from PC9ER‐AZDR cells was subjected to dPCR analysis using the Fluidigm Biomark platform (gDNA, 2.5 ng/sample). Exact copy number of the indicated EGFR mutated alleles, as well as the WT alleles, was then assessed. The RNase P specific set of probes was used as normalizer. The analysis was performed utilizing the dPCR software from Fluidigm. Amplifications with mutation positive and mutation negative wells appear in red and in blue, respectively.

    Article Snippet: Three TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA, USA) were custom designed to detect specific EGFR mutations: del476/750, T790M, and C797S (see a list of primers and probes in ).

    Techniques: In Vitro, Mutagenesis, MTT Assay, Isolation, Digital PCR, Chromatin Immunoprecipitation, Serial Dilution, Software, Genotyping Assay, Plasmid Preparation, Labeling, Amplification, Sequencing

    Expression of α-synuclein ( a ), PINK1 ( b ) and Parkin ( c ) in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the ΔΔCt method and normalization to 18S expression. Results

    Journal: Neurochemical research

    Article Title: iPLA2• Knockout Mouse, a Genetic Model for Progressive Human Motor Disorders, Develops Age-Related Neuropathology

    doi: 10.1007/s11064-014-1342-y

    Figure Lengend Snippet: Expression of α-synuclein ( a ), PINK1 ( b ) and Parkin ( c ) in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the ΔΔCt method and normalization to 18S expression. Results

    Article Snippet: Gene expression was determined by real time-polymerase chain reaction (RT-PCR) using the Taqman® Universal PCR Mastermix and specific Taqman® primers and probe (Applied Biosystems).

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

    Expression of microglial, astrocytic, synaptic markers and BNDF in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the ΔΔCt method and normalization to 18S expression. Results

    Journal: Neurochemical research

    Article Title: iPLA2• Knockout Mouse, a Genetic Model for Progressive Human Motor Disorders, Develops Age-Related Neuropathology

    doi: 10.1007/s11064-014-1342-y

    Figure Lengend Snippet: Expression of microglial, astrocytic, synaptic markers and BNDF in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the ΔΔCt method and normalization to 18S expression. Results

    Article Snippet: Gene expression was determined by real time-polymerase chain reaction (RT-PCR) using the Taqman® Universal PCR Mastermix and specific Taqman® primers and probe (Applied Biosystems).

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

    mRNA expression of iPLA 2 β in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the DDCt method and normalization to 18S expression. Results are expressed as mean ± SEM (n

    Journal: Neurochemical research

    Article Title: iPLA2• Knockout Mouse, a Genetic Model for Progressive Human Motor Disorders, Develops Age-Related Neuropathology

    doi: 10.1007/s11064-014-1342-y

    Figure Lengend Snippet: mRNA expression of iPLA 2 β in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the DDCt method and normalization to 18S expression. Results are expressed as mean ± SEM (n

    Article Snippet: Gene expression was determined by real time-polymerase chain reaction (RT-PCR) using the Taqman® Universal PCR Mastermix and specific Taqman® primers and probe (Applied Biosystems).

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

    Expression of cPLA 2 α ( a ), sPLA 2 -V ( b ) and iPLA 2 c in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the ΔΔCt method and normalization to 18S expression. Results

    Journal: Neurochemical research

    Article Title: iPLA2• Knockout Mouse, a Genetic Model for Progressive Human Motor Disorders, Develops Age-Related Neuropathology

    doi: 10.1007/s11064-014-1342-y

    Figure Lengend Snippet: Expression of cPLA 2 α ( a ), sPLA 2 -V ( b ) and iPLA 2 c in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the ΔΔCt method and normalization to 18S expression. Results

    Article Snippet: Gene expression was determined by real time-polymerase chain reaction (RT-PCR) using the Taqman® Universal PCR Mastermix and specific Taqman® primers and probe (Applied Biosystems).

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

    Expression of the pro-inflammatory cytokines TNF-α ( a ) and Il-1β ( b ) and the iNOS ( c ) in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the ΔΔCt method

    Journal: Neurochemical research

    Article Title: iPLA2• Knockout Mouse, a Genetic Model for Progressive Human Motor Disorders, Develops Age-Related Neuropathology

    doi: 10.1007/s11064-014-1342-y

    Figure Lengend Snippet: Expression of the pro-inflammatory cytokines TNF-α ( a ) and Il-1β ( b ) and the iNOS ( c ) in WT and iPLA 2 β −/− mice. mRNA was quantified in duplicate with Taqman® RT-PCR using the ΔΔCt method

    Article Snippet: Gene expression was determined by real time-polymerase chain reaction (RT-PCR) using the Taqman® Universal PCR Mastermix and specific Taqman® primers and probe (Applied Biosystems).

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

    TALAM1 promotes the 3′ end processing of MALAT1 RNA. ( A ) Cutoff assay of cleaved and uncleaved MALAT1 in HeLa cells transfected with empty vector (V) or with TALAM1-overexpression construct (TALAM1-OE). ( B ) Analyses of mascRNA levels in V or TALAM1-OE samples by (Ba) northern blot, and (Bb) Taqman small RNA assay. ( C ) In vivo RNA decay assay. (Ca) Schematic representation of the βΔ1,2 construct. (Cb) Stability of the intronless β-globin transcript in U2OS Tet-off cells transfected with empty vector (V) or with TALAM1-overexpression construct (TALAM1-OE). Trendlines are single-exponential regression of the percentage of RNA remaining versus time. Dotted lines represent the half-life of reporter RNA. ( D ) In vitro processing assay of MALAT1 3′ end sequence. Internally 32 P-labeled MALAT1 ENE+A+mascRNA RNA substrate (filled arrow in Db) was incubated with HeLa total cell extract in absence or presence of in vitro transcribed YFP, TALAM1-F1R1, F2R2 RNA or 2′MOE targeting the mascRNA site, and its processing to mascRNA (open arrow in Db) is monitored at 40 min and 60 min time points. (Da) Schematic diagrams shown for the relative positions of the TALAM1-F1R1 and TALAM1-F2R2 fragments generated by in vitro transcription. (Db) A representative autoadiogram is shown. (Dc) The quantification of the mascRNA product levels measured from independent experiments, with ImageJ software. At 40 min time point, levels of mascRNA produced under different conditions were normalized to the condition with no extra RNA, which was set as one. Bar data in (A) and (Bb) are mean values with SD, N A = 4, N Bb = 3; Data in (Cb) and (Dc) are mean values with SEM, N Cb = 4, N Dc = 6 (biological replicates, * P

    Journal: Nucleic Acids Research

    Article Title: Natural antisense RNA promotes 3′ end processing and maturation of MALAT1 lncRNA

    doi: 10.1093/nar/gkw047

    Figure Lengend Snippet: TALAM1 promotes the 3′ end processing of MALAT1 RNA. ( A ) Cutoff assay of cleaved and uncleaved MALAT1 in HeLa cells transfected with empty vector (V) or with TALAM1-overexpression construct (TALAM1-OE). ( B ) Analyses of mascRNA levels in V or TALAM1-OE samples by (Ba) northern blot, and (Bb) Taqman small RNA assay. ( C ) In vivo RNA decay assay. (Ca) Schematic representation of the βΔ1,2 construct. (Cb) Stability of the intronless β-globin transcript in U2OS Tet-off cells transfected with empty vector (V) or with TALAM1-overexpression construct (TALAM1-OE). Trendlines are single-exponential regression of the percentage of RNA remaining versus time. Dotted lines represent the half-life of reporter RNA. ( D ) In vitro processing assay of MALAT1 3′ end sequence. Internally 32 P-labeled MALAT1 ENE+A+mascRNA RNA substrate (filled arrow in Db) was incubated with HeLa total cell extract in absence or presence of in vitro transcribed YFP, TALAM1-F1R1, F2R2 RNA or 2′MOE targeting the mascRNA site, and its processing to mascRNA (open arrow in Db) is monitored at 40 min and 60 min time points. (Da) Schematic diagrams shown for the relative positions of the TALAM1-F1R1 and TALAM1-F2R2 fragments generated by in vitro transcription. (Db) A representative autoadiogram is shown. (Dc) The quantification of the mascRNA product levels measured from independent experiments, with ImageJ software. At 40 min time point, levels of mascRNA produced under different conditions were normalized to the condition with no extra RNA, which was set as one. Bar data in (A) and (Bb) are mean values with SD, N A = 4, N Bb = 3; Data in (Cb) and (Dc) are mean values with SEM, N Cb = 4, N Dc = 6 (biological replicates, * P

    Article Snippet: Strand-specific RT-qPCR and Taqman small RNA assay For strand-specific RT-qPCR, reverse transcription was performed using gene specific reverse transcription primers with a linker sequence at 5′ end, and then qPCR was performed using gene specific forward primer and the linker as reverse primer.

    Techniques: Transfection, Plasmid Preparation, Over Expression, Construct, Northern Blot, In Vivo, In Vitro, Sequencing, Labeling, Incubation, Generated, Software, Produced

    L. monocytogenes -induced transcriptional regulation of miR-146a and miR-125a. Bone marrow derived macrophages (BMDMs) were infected (MOI 10) with L. monocytogenes ( Lm ) and the LLO-deficient mutant Δhly ( Lm Δhly ) for 3 h and 6 h. Total RNA was extracted and the expression levels of primary transcript (pri)-miR-125a (A) and pri-miR-146a (B) were quantified by RT-qPCR using TaqMan assays. Data was normalized to the endogenous control gene HPRT1 and fold changes of miRNA induction in infected compared to control cells of each genotype were calculated by the 2 −ΔΔCT method. Data represents the mean ± SEM from three biological replicates. Statistical significance of miRNA expression between infected and non infected WT BMDMs was determined by the Student t -test; * P

    Journal: PLoS ONE

    Article Title: Listeria monocytogenes Infection in Macrophages Induces Vacuolar-Dependent Host miRNA Response

    doi: 10.1371/journal.pone.0027435

    Figure Lengend Snippet: L. monocytogenes -induced transcriptional regulation of miR-146a and miR-125a. Bone marrow derived macrophages (BMDMs) were infected (MOI 10) with L. monocytogenes ( Lm ) and the LLO-deficient mutant Δhly ( Lm Δhly ) for 3 h and 6 h. Total RNA was extracted and the expression levels of primary transcript (pri)-miR-125a (A) and pri-miR-146a (B) were quantified by RT-qPCR using TaqMan assays. Data was normalized to the endogenous control gene HPRT1 and fold changes of miRNA induction in infected compared to control cells of each genotype were calculated by the 2 −ΔΔCT method. Data represents the mean ± SEM from three biological replicates. Statistical significance of miRNA expression between infected and non infected WT BMDMs was determined by the Student t -test; * P

    Article Snippet: Total RNA (800 ng) was reverse transcribed using miRNA-specific Megaplex RT Primer-Pools A and B with the TaqMan Reverse Transcription Kit (ABI, Life technologies).

    Techniques: Derivative Assay, Infection, Mutagenesis, Expressing, Quantitative RT-PCR

    Regulation of miR-125a-3p/5p, miR-146a and miR-155 expression in macrophages by NF-κB p65. Wild type (WT) and p65 MYEL KO BMDMs were treated with 100 ng/ml ultra-pure LPS for 4 h and 8 h (A–D, F) or infected (MOI 1) with L. monocytogenes ( Lm ) for 4 h (E–F). Total RNA was extracted and the expression levels of miR-125a-3p, miR-125a-5p, miR-146a and miR-155 were quantified by TaqMan miRNA assays. Data was normalized to sno202 or HPRT1, endogenous controls for miR or pri-miR expression, respectively, and fold changes were calculated by the 2 −ΔΔCT method and expressed relative to the mock-treated/mock-infected control for each timepoint/condition in each genotype. Data represents the mean ± SEM of at least two independent experiments, including minimum two mice from each genotype. Statistical significance of miR/pri-miR induction between WT and p65 MYEL KO BMDMs was determined by two way ANOVA; * P

    Journal: PLoS ONE

    Article Title: Listeria monocytogenes Infection in Macrophages Induces Vacuolar-Dependent Host miRNA Response

    doi: 10.1371/journal.pone.0027435

    Figure Lengend Snippet: Regulation of miR-125a-3p/5p, miR-146a and miR-155 expression in macrophages by NF-κB p65. Wild type (WT) and p65 MYEL KO BMDMs were treated with 100 ng/ml ultra-pure LPS for 4 h and 8 h (A–D, F) or infected (MOI 1) with L. monocytogenes ( Lm ) for 4 h (E–F). Total RNA was extracted and the expression levels of miR-125a-3p, miR-125a-5p, miR-146a and miR-155 were quantified by TaqMan miRNA assays. Data was normalized to sno202 or HPRT1, endogenous controls for miR or pri-miR expression, respectively, and fold changes were calculated by the 2 −ΔΔCT method and expressed relative to the mock-treated/mock-infected control for each timepoint/condition in each genotype. Data represents the mean ± SEM of at least two independent experiments, including minimum two mice from each genotype. Statistical significance of miR/pri-miR induction between WT and p65 MYEL KO BMDMs was determined by two way ANOVA; * P

    Article Snippet: Total RNA (800 ng) was reverse transcribed using miRNA-specific Megaplex RT Primer-Pools A and B with the TaqMan Reverse Transcription Kit (ABI, Life technologies).

    Techniques: Expressing, Infection, Mouse Assay

    Regulation of miR-125a-3p/5p and miR-146a expression upon TLR2 and TLR4 stimulation. Wild type (WT; n = 3) and MyD88 −/− (n = 2) mouse bone marrow derived macrophages (BMDMs) were treated with 1 µg/ml ultra-pure LPS or 2 µg/ml Pam3CSK4 for 6 h. Total RNA was extracted and miRNA expression levels were quantified by RT-qPCR using TaqMan miRNA assays. MiR-125a-3p (A), miR-125a-5p (B) and miR-146a (C) expression in MyD88 −/− BMDMs. Pri-miR-125a (D) and pri-miR-146a (E) expression upon TLR stimulation in WT and MyD88 −/− BMDMs. Data was normalized to sno202 or HPRT1, endogenous controls for miR or pri-miR expression, respectively, and fold changes were calculated by the 2 −ΔΔCT method and expressed relative to non-infected control of each genotype. Data represents the mean ± SEM of at least two biological replicates. Statistical significance of miR/pri-miR induction was determined by the Student t -test compared to non infected control (* P

    Journal: PLoS ONE

    Article Title: Listeria monocytogenes Infection in Macrophages Induces Vacuolar-Dependent Host miRNA Response

    doi: 10.1371/journal.pone.0027435

    Figure Lengend Snippet: Regulation of miR-125a-3p/5p and miR-146a expression upon TLR2 and TLR4 stimulation. Wild type (WT; n = 3) and MyD88 −/− (n = 2) mouse bone marrow derived macrophages (BMDMs) were treated with 1 µg/ml ultra-pure LPS or 2 µg/ml Pam3CSK4 for 6 h. Total RNA was extracted and miRNA expression levels were quantified by RT-qPCR using TaqMan miRNA assays. MiR-125a-3p (A), miR-125a-5p (B) and miR-146a (C) expression in MyD88 −/− BMDMs. Pri-miR-125a (D) and pri-miR-146a (E) expression upon TLR stimulation in WT and MyD88 −/− BMDMs. Data was normalized to sno202 or HPRT1, endogenous controls for miR or pri-miR expression, respectively, and fold changes were calculated by the 2 −ΔΔCT method and expressed relative to non-infected control of each genotype. Data represents the mean ± SEM of at least two biological replicates. Statistical significance of miR/pri-miR induction was determined by the Student t -test compared to non infected control (* P

    Article Snippet: Total RNA (800 ng) was reverse transcribed using miRNA-specific Megaplex RT Primer-Pools A and B with the TaqMan Reverse Transcription Kit (ABI, Life technologies).

    Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Infection

    Regulation of L. monocytogenes -induced miRNAs in macrophages. Bone marrow derived macrophages (BMDMs) from wild type (WT; n = 4) and MyD88 −/− (n = 4) mice were infected (MOI 10) with L. monocytogenes ( Lm ) and the LLO-deficient mutant Δhly ( Lm Δhly ) for 3 h and 6 h. Total RNA was extracted and the expression levels of miR-155 (A), miR-146a (B), miR-125a-3p (C), miR-125a-5p (D) and miR-149 (E) were quantified by RT-qPCR using TaqMan miRNA assays. Data was normalized to the endogenous control sno202 and fold changes of miRNA expression relative to the non-infected control of its own genotype was calculated by the 2 −ΔΔCT method. Data represents the mean ± SEM from three to four biological replicates. Statistical significance of miRNA expression between infected and non infected WT BMDMs was determined by the Student t -test; * P

    Journal: PLoS ONE

    Article Title: Listeria monocytogenes Infection in Macrophages Induces Vacuolar-Dependent Host miRNA Response

    doi: 10.1371/journal.pone.0027435

    Figure Lengend Snippet: Regulation of L. monocytogenes -induced miRNAs in macrophages. Bone marrow derived macrophages (BMDMs) from wild type (WT; n = 4) and MyD88 −/− (n = 4) mice were infected (MOI 10) with L. monocytogenes ( Lm ) and the LLO-deficient mutant Δhly ( Lm Δhly ) for 3 h and 6 h. Total RNA was extracted and the expression levels of miR-155 (A), miR-146a (B), miR-125a-3p (C), miR-125a-5p (D) and miR-149 (E) were quantified by RT-qPCR using TaqMan miRNA assays. Data was normalized to the endogenous control sno202 and fold changes of miRNA expression relative to the non-infected control of its own genotype was calculated by the 2 −ΔΔCT method. Data represents the mean ± SEM from three to four biological replicates. Statistical significance of miRNA expression between infected and non infected WT BMDMs was determined by the Student t -test; * P

    Article Snippet: Total RNA (800 ng) was reverse transcribed using miRNA-specific Megaplex RT Primer-Pools A and B with the TaqMan Reverse Transcription Kit (ABI, Life technologies).

    Techniques: Derivative Assay, Mouse Assay, Infection, Mutagenesis, Expressing, Quantitative RT-PCR

    Real-time PCR. The plot shows the amplification obtained using TaqMan probes with the same DNA sources as those used for the nested PCR that yielded positive results. The probes are based in a different region of the env gene, namely from 5943 to 6011 (AF033807). Each line represents a sample, positive control, or negative control. The PCR cycles are shown on the x-axis, and the ΔRn values (representing the fluorescent units) are shown on the y-axis. All of the assays were performed in 48-well plates.

    Journal: BMC Cancer

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women

    doi: 10.1186/1471-2407-14-942

    Figure Lengend Snippet: Real-time PCR. The plot shows the amplification obtained using TaqMan probes with the same DNA sources as those used for the nested PCR that yielded positive results. The probes are based in a different region of the env gene, namely from 5943 to 6011 (AF033807). Each line represents a sample, positive control, or negative control. The PCR cycles are shown on the x-axis, and the ΔRn values (representing the fluorescent units) are shown on the y-axis. All of the assays were performed in 48-well plates.

    Article Snippet: The reaction contained 100 ng of DNA, 900 nM of each primer, 250 nM probe, and the TaqMan Universal Master Mix II (Applied Biosystems).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Nested PCR, Positive Control, Negative Control, Polymerase Chain Reaction

    Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Journal: Nucleic Acids Research

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability

    doi: 10.1093/nar/gkv933

    Figure Lengend Snippet: Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Article Snippet: Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems).

    Techniques: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P

    Journal: Nucleic Acids Research

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability

    doi: 10.1093/nar/gkv933

    Figure Lengend Snippet: Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P

    Article Snippet: Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems).

    Techniques: Amplification, Fluorescence In Situ Hybridization, Labeling, Real-time Polymerase Chain Reaction, CNV Assay, Transduction

    Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Journal: Nucleic Acids Research

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability

    doi: 10.1093/nar/gkv933

    Figure Lengend Snippet: Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Article Snippet: Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems).

    Techniques: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    NF90 modulates the expression level of a subset of miRNAs in HepG2 cells. ( A ) Extracts of HepG2 cells transfected with non-targeting control siRNAs (Scr, Scr#2) or siRNA targeting NF90 (NF90, NF90#2) as indicated were analyzed by immunoblot using the antibodies indicated. ( B ) Total RNA extracted from cells transfected with siScr or siNF90 were analyzed by small RNA-seq. Results are shown as log 2 fold change versus –log 10 P -value. ( C ) Table summarizing the number of mature miRNAs and pri-miRNAs modulated in HepG2 cell line upon loss of NF90, according to small-RNA seq. ( D ) Total RNA extracted from cells described in (A) were analyzed by Taqman RT-qPCR as indicated. Results were normalized by those obtained for U6 abundance in the same samples. ND indicates ‘not detected’. Data represent mean ± SEM obtained from three independent experiments (*** P

    Journal: Nucleic Acids Research

    Article Title: NF90 modulates processing of a subset of human pri-miRNAs

    doi: 10.1093/nar/gkaa386

    Figure Lengend Snippet: NF90 modulates the expression level of a subset of miRNAs in HepG2 cells. ( A ) Extracts of HepG2 cells transfected with non-targeting control siRNAs (Scr, Scr#2) or siRNA targeting NF90 (NF90, NF90#2) as indicated were analyzed by immunoblot using the antibodies indicated. ( B ) Total RNA extracted from cells transfected with siScr or siNF90 were analyzed by small RNA-seq. Results are shown as log 2 fold change versus –log 10 P -value. ( C ) Table summarizing the number of mature miRNAs and pri-miRNAs modulated in HepG2 cell line upon loss of NF90, according to small-RNA seq. ( D ) Total RNA extracted from cells described in (A) were analyzed by Taqman RT-qPCR as indicated. Results were normalized by those obtained for U6 abundance in the same samples. ND indicates ‘not detected’. Data represent mean ± SEM obtained from three independent experiments (*** P

    Article Snippet: For RT-qPCR, RT was performed using TaqMan™ Reverse Transcription Reagent or TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher). qPCRs were performed using GoTaq® Probe qPCR Master Mix (Promega) or TaqMan® Fast Advanced Master Mix (Thermo Fisher).

    Techniques: Expressing, Transfection, RNA Sequencing Assay, Quantitative RT-PCR

    Circulating levels of miR-7-5p and miR-26b-5p in hypertensive patients with left ventricular hypertrophy (LVH) and hypertensive patients without left ventricular hypertrophy (HAS) patients and controls subjects, evaluated by Taqman real-time PCR (arbitrary units). A , Circulating level of miR-7-5-p, and B , circulating level of miR-26b-5p in healthy controls, hypertensive patients and patients with left ventricular hypertrophy. Data are reported as means±SE of 8 to 28 subjects/group. *P

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: MicroRNA profiling identifies miR-7-5p and miR-26b-5p as differentially expressed in hypertensive patients with left ventricular hypertrophy

    doi: 10.1590/1414-431X20176211

    Figure Lengend Snippet: Circulating levels of miR-7-5p and miR-26b-5p in hypertensive patients with left ventricular hypertrophy (LVH) and hypertensive patients without left ventricular hypertrophy (HAS) patients and controls subjects, evaluated by Taqman real-time PCR (arbitrary units). A , Circulating level of miR-7-5-p, and B , circulating level of miR-26b-5p in healthy controls, hypertensive patients and patients with left ventricular hypertrophy. Data are reported as means±SE of 8 to 28 subjects/group. *P

    Article Snippet: Validation of miR-7-5p and miR-26b-5p expression by real-time PCR To validate the levels of miRNA selected from miRNA profile in wide range of samples, we used Taqman MicroRNA Assays (Applied Biosystems). qRT-PCR amplification mixtures contained 20 ηg template cDNA, Taqman master mix (10 μL; Applied Biosystems) and probes for MiR-7-5p (Cat. #4427975) and MiR-26b-5p (Cat. #4427975) in a final volume of 20 μL.

    Techniques: Real-time Polymerase Chain Reaction

    Tumor variants of LMP1 differentially regulate miR-155 and miR-193b from the B95.8 lab strain of LMP1. Two million EBV− BL41 cells expressing B95.8 lab strain or natural variant NGFR.LMP1 molecules were activated for 12 h prior to RNA isolation with the miRVana miRNA isolation kit. Relative expression of specific microRNAs was determined by quantitative PCR (qPCR) using TaqMan MicroRNA Assays. Target-specific cDNA was generated from 10 ng of total RNA using the TaqMan MicroRNA Reverse Transcription Kit and pre-amplified using the TaqMan PreAmp Master Mix. Finally, qPCR assays were performed using the TaqMan Universal Master Mix II, No AmpERASE UNG. The relative expression of (A) miR-155 and (B) miR-193b was calculated by first normalizing to the endogenous control U47 (ΔC t ) and then to unactivated samples (ΔΔ C t ). Fold-induction (2 −ΔΔ C t ) of each miR is shown. Each point represents an experimental replicate; two different lines expressing B95.8 NGFR.LMP1 were used. *** p ≤ 0.001, **** p ≤ 0.0001 by one-way ANOVA with post hoc multiple comparisons to activate B95.8 lab strain NGFR.LMP1.

    Journal: Frontiers in Microbiology

    Article Title: Epstein-Barr Virus Latent Membrane Protein 1 Regulates Host B Cell MicroRNA-155 and Its Target FOXO3a via PI3K p110α Activation

    doi: 10.3389/fmicb.2019.02692

    Figure Lengend Snippet: Tumor variants of LMP1 differentially regulate miR-155 and miR-193b from the B95.8 lab strain of LMP1. Two million EBV− BL41 cells expressing B95.8 lab strain or natural variant NGFR.LMP1 molecules were activated for 12 h prior to RNA isolation with the miRVana miRNA isolation kit. Relative expression of specific microRNAs was determined by quantitative PCR (qPCR) using TaqMan MicroRNA Assays. Target-specific cDNA was generated from 10 ng of total RNA using the TaqMan MicroRNA Reverse Transcription Kit and pre-amplified using the TaqMan PreAmp Master Mix. Finally, qPCR assays were performed using the TaqMan Universal Master Mix II, No AmpERASE UNG. The relative expression of (A) miR-155 and (B) miR-193b was calculated by first normalizing to the endogenous control U47 (ΔC t ) and then to unactivated samples (ΔΔ C t ). Fold-induction (2 −ΔΔ C t ) of each miR is shown. Each point represents an experimental replicate; two different lines expressing B95.8 NGFR.LMP1 were used. *** p ≤ 0.001, **** p ≤ 0.0001 by one-way ANOVA with post hoc multiple comparisons to activate B95.8 lab strain NGFR.LMP1.

    Article Snippet: After multiplexed reverse transcriptase reactions, cDNA was pre-amplified using the TaqMan PreAmp Master Mix (ThermoFisher Scientific).

    Techniques: Expressing, Variant Assay, Isolation, Real-time Polymerase Chain Reaction, Generated, Amplification

    miRNAs Are Differentially Expressed in RGCs during Development (A) A representative heatmap of miRNA expression profiles constructed from a microarray analysis of retinal ganglion cells (RGCs) purified from embryonic day 21 (E21, n = 3 biological replicates), postnatal day 6 (P6, n = 3 biological replicates), and P30 (n = 2 biological replicates) Sprague-Dawley (SD) rats, showing 76 endogenously expressed miRNAs with significant differential expression (≥4-fold changes in expression levels) during development. Developmental ages (as biological replicates) are indicated in columns, and differentially expressed miRNAs are indicated in rows. All six members of the miR-17-92 cluster (red asterisks) were found to have significant downregulation from E21 to P30 (right panel). Blue (−8.5) and red (+8.5) in the color-coding scale represent relative low and high normalized miRNA expressions, respectively. A two-tailed moderated t test with a Benjamini-Hochberg correction for false discovery rate was used to compare the means of miRNA microarray expression values between E21 and P6 and between E21 and P30. A list of the 76 miRNAs can be found in Table S1 , and all other microarray data that support the findings of this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO: GSE102458 ). (B) Illustration of the polycistronic miR-17-92 cluster region on human chromosome 13 (chromosome 15 for rats). Precursor transcripts derived from the miR-17-92 gene cluster contains six tandem stem-loop hairpin structures that yield six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. The miRNAs can be categorized into four miRNA families according to their conserved seed sequences (underlined). (C) Downregulation of the six miRNAs from E21 to P30 and from E21 to P6 was confirmed by TaqMan qRT-PCR (E21 vs. P6, miR-17-5p, p = 0.005; miR-18a, p = 0.003; miR-19a, p = 0.014; miR-19b, p = 0.013; miR-20a, p = 0.005; miR-92a, p = 0.014; E21 vs. P30, miR-17-5p, p = 0.0004; miR-18a, p = 0.001; miR-19a, p = 0.007; miR-19b, p = 0.005; miR-20a, p = 0.001; miR-92a, p = 0.003; n = 3 biological replicates for each age group). An unpaired two-tailed Student’s t test was used for TaqMan comparisons. All values are shown as mean ± SEM.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: MicroRNA-19a-PTEN Axis Is Involved in the Developmental Decline of Axon Regenerative Capacity in Retinal Ganglion Cells

    doi: 10.1016/j.omtn.2020.05.031

    Figure Lengend Snippet: miRNAs Are Differentially Expressed in RGCs during Development (A) A representative heatmap of miRNA expression profiles constructed from a microarray analysis of retinal ganglion cells (RGCs) purified from embryonic day 21 (E21, n = 3 biological replicates), postnatal day 6 (P6, n = 3 biological replicates), and P30 (n = 2 biological replicates) Sprague-Dawley (SD) rats, showing 76 endogenously expressed miRNAs with significant differential expression (≥4-fold changes in expression levels) during development. Developmental ages (as biological replicates) are indicated in columns, and differentially expressed miRNAs are indicated in rows. All six members of the miR-17-92 cluster (red asterisks) were found to have significant downregulation from E21 to P30 (right panel). Blue (−8.5) and red (+8.5) in the color-coding scale represent relative low and high normalized miRNA expressions, respectively. A two-tailed moderated t test with a Benjamini-Hochberg correction for false discovery rate was used to compare the means of miRNA microarray expression values between E21 and P6 and between E21 and P30. A list of the 76 miRNAs can be found in Table S1 , and all other microarray data that support the findings of this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO: GSE102458 ). (B) Illustration of the polycistronic miR-17-92 cluster region on human chromosome 13 (chromosome 15 for rats). Precursor transcripts derived from the miR-17-92 gene cluster contains six tandem stem-loop hairpin structures that yield six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. The miRNAs can be categorized into four miRNA families according to their conserved seed sequences (underlined). (C) Downregulation of the six miRNAs from E21 to P30 and from E21 to P6 was confirmed by TaqMan qRT-PCR (E21 vs. P6, miR-17-5p, p = 0.005; miR-18a, p = 0.003; miR-19a, p = 0.014; miR-19b, p = 0.013; miR-20a, p = 0.005; miR-92a, p = 0.014; E21 vs. P30, miR-17-5p, p = 0.0004; miR-18a, p = 0.001; miR-19a, p = 0.007; miR-19b, p = 0.005; miR-20a, p = 0.001; miR-92a, p = 0.003; n = 3 biological replicates for each age group). An unpaired two-tailed Student’s t test was used for TaqMan comparisons. All values are shown as mean ± SEM.

    Article Snippet: Newly synthesized cDNA was qPCR amplified using a TaqMan miRNA assay kit (Applied Biosystems) and Roche PCR machine (LC480 II), according to the manufacturers’ instructions.

    Techniques: Expressing, Construct, Microarray, Purification, Two Tailed Test, Derivative Assay, Quantitative RT-PCR