qrt pcr analysis Thermo Fisher Search Results


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  • 90
    Thermo Fisher qrt pcr fast sybr green master mix
    Transcript distribution profile of Trica- SKR1 and Trica -SKR2. Quantification of transcript levels by <t>qRT-PCR</t> in seven different tissues from adult T. castaneum . The data represent samples of central brain (n = 15), optic lobes (n = 15), salivary glands (n = 15), gut (n = 15), fat body (n = 20), testes (n = 50) and ovaries (n = 50), normalized relative to β-actin and ribosomal protein 3 (RPs3) transcript levels. Abbreviations: B = central brain, OL = optic lobes, SG = salivary glands, G = gut, FB = fat body, Te = testes, Ov = ovaria.
    Qrt Pcr Fast Sybr Green Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr assay kits
    Transcript distribution profile of Trica- SKR1 and Trica -SKR2. Quantification of transcript levels by <t>qRT-PCR</t> in seven different tissues from adult T. castaneum . The data represent samples of central brain (n = 15), optic lobes (n = 15), salivary glands (n = 15), gut (n = 15), fat body (n = 20), testes (n = 50) and ovaries (n = 50), normalized relative to β-actin and ribosomal protein 3 (RPs3) transcript levels. Abbreviations: B = central brain, OL = optic lobes, SG = salivary glands, G = gut, FB = fat body, Te = testes, Ov = ovaria.
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Assay Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reverse transcription polymerase chain reaction qrt pcr analysis
    Effect of 4 weeks of exenatide treatment on insulin clearance. Mice were fed an RD or HF diet for 2 months and injected daily with exenatide in the last month. (A) (i) Cc1 +/+ liver lysates were analyzed by <t>qRT‐PCR</t> to measure Ceacam1 mRNA levels normalized to 18S (n = 5/each group in duplicate). Values are expressed as mean ± SE; * P
    Reverse Transcription Polymerase Chain Reaction Qrt Pcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr analysis taqman mirna assays
    Quantitative real-time polymerase chain reaction <t>(qRT-PCR)</t> and Locked nucleic acid in situ hybridization (LNA-ISH) analysis of expression levels of <t>microRNAs.</t> (A) qRT-PCR analysis of relative expression levels of 4 microRNAs (miR-205, miR-210, miR-492, and miR-1247) in pancreatic juice samples from patients with pancreatic ductal adenocarcinoma (PDAC), patients with chronic pancreatitis (CP), and non-pancreatic, non-healthy (NPNH) controls. The relative abundance of four <t>miRNAs,</t> normalized to the level of RNU6B, significantly varied in pancreatic juice samples from patients with PDAC, CP and NPNH. (B) LNA-ISH-IHC images showing miR-492 expression in 3 (J101, J16, J39) pancreatic ductal adenocarcinoma tissue samples (magnification ×100). (C) The graph shows qRT-PCR results for miR-492 in pancreatic juice samples from the same patients.
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Analysis Taqman Mirna Assays, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii platinum sybr green one step qrt pcr kit
    Lack of correlation between telomere length and TERRA levels in cells with stable telomeres. Two independent, exponentially-dividing strains of each genotype were split and used to isolate RNA or DNA. ( A ) A map of single copy loci amplified during TERRA measurements (B-E) and Southern blot probes (F). Distance from the centromeric end of the TG repeats is indicated. Primers used in the <t>qRT-PCR</t> and to make the probes are described in S2 Table. ( B–E ) TERRA measured as in Figure 1B . ( F ) Telomeric Southern blots performed after XhoI (top two gels) or HindIII (bottom two gels) digestion. One membrane was first probed for telomere 15L and then Y’+TG sequences ( Supplementary Figure S3A ). The other membrane was first probed for telomere 10R and finally 13R. Loading control corresponds to <t>SYBR</t> Safe staining of the gel probed for telomeres 10R and 13R.
    Superscript Iii Platinum Sybr Green One Step Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1485 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PolyIC/dsRBEC induces the expression and secretion of pro-inflammatory cytokines in MDA-MB-468 cells. A) <t>qRT-PCR</t> analysis of IFN-β, CCL5, IP10 and TNFα mRNA expression following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 2 and 4 hours. Data were normalized to GAPDH and are expressed as fold change relative to vehicle-treated samples. A representative experiment out of 3 experiments is shown. Error bars represent RQ max. B) Protein levels of IFN-β, CCL5, IP10 and TNFα were measured by ELISA following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 24 hours. Values are averages of triplicate biological samples from one representative experiment. (***,P
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    Regulation of Smpd3 expression in chondrocytes by BMP-2. (A and B) Gene expression analyses of 3-month-old fractured WT bones showing a significant upregulation of Bmp-2 expression (A) and Smpd3 expression (B) in the callus. Expression analyses by <t>qRT-PCR</t> were performed 13 days postsurgery. (C) Immunohistochemistry with anti-SMPD3 antibody shows an increased presence of SMPD3 in the callus of WT fractured tibia at 14 and 28 days postsurgery. A bone section of the unfractured contralateral tibia was used as a control. (D) ATDC5 chondrogenic cells treated with 300 ng/ml of BMP-2 for 48 h show a significant upregulation of chondrogenic marker Col2a1 , Col10a1 , Sox6 , Sox9 , Osx , Mef2c , and Smpd3 expression. (E) Differentiated ATDC5 cells (cultured in the presence of ascorbic acid and β-glycerophosphate for 6 days) when treated with 2 μM dorsomorphin (Dorso) show significant downregulation of Col2a1 , Col10a1 , Sox5 , Sox6 , Osx , Mef2c , and Smpd3 expression. Cells were treated with dorsomorphin for 30 min every 2 days, before the medium was changed. Error bars represent standard deviations. *, P
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    Thermo Fisher qrt pcr analyses
    Relative expression of eng mRNA in P. pastoris CL2 in response to cold-shock at 4 °C. CL2 cells were grown in medium BMGH (30 °C for 4 h), from OD 600 = 1 inoculum, then subjected to cold-shock for the induction of eng mRNA transcript expression (4 °C for 6 h). Samples were taken at 0, 2 and 6 h for analysis by <t>qRT-PCR;</t> and finally, they were transferred to 30 °C for 4 h for ENG product synthesis, stage in which the corresponding sample at 2 h was taken for analysis by qRT-PCR. The values of relative expression of eng mRNA were calculated in relation to eng and the housekeeping constitutive gene act1 (control) expression using method 2 −ΔΔCt
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    Thermo Fisher qrt pcr analysis trizol
    Vezf1 is expressed in adult cardiomyocytes and regulates cardiomyocyte growth and cardiomyopathy related genes. (A) <t>qRT-PCR</t> analysis of the expression of endothelial nitric oxide synthase (eNOS), collagen type I alpha 1 chain (Col1a1), and α myosin heavy chain 6 (α-MHC) mRNAs in pools of fractionated resident mouse cardiac cells. eNOS, Col1a1 and α-MHC were used as markers for endothelial cells (Endo), fibroblasts (Fibro), and cardiomyocytes (Myo), respectively. n = 5. (B) qPCR analysis of Vezf1 mRNA levels in cardiomyocytes and fibroblasts relative to that in the endothelial cells. n = 5. (C) Adult rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or Control siRNA (100 nM) and 3 days later RNA samples were collected. Shown is qRT-PCR analysis for expression of Vezf1. Results are normalized to expression of 18S (18S ribosomal RNA). n = 6. (D and E) Adult rat ventricular cardiomyocytes were transfected with two distinct Vezf1 siRNAs (100 nM) or Control siRNA (100 nM) and 1 day later cells were stimulated with isoprenaline (Iso, 1 µM) for 48 h where indicated. (D) Shown is microscopy analysis for cardiomyocyte size. Ctrl siRNA n = 8, Vezf1 A siRNA n = 9, Vezf1 B siRNA n = 10; Ctrl siRNA + Iso n = 10, Vezf1 A siRNA + Iso n = 15, Vezf1 B siRNA + Iso n = 13. (E) Shown is microscopy analysis for sarcomere length (µm). Ctrl siRNA n = 14, Vezf1 B siRNA n = 18; Ctrl siRNA + Iso n = 22, Vezf1 B siRNA + Iso n = 25. (F) Adult rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA and 3 days later Ca 2+ cycling and cardiomyocyte (CM) shortening were analyzed. Ctrl n = 44, Vezf1 siRNA n = 43. (G) Neonatal rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA and 2 days later cells were treated with Iso (1 µM) for 24 h. Shown is qRT-PCR analysis for expression of Vezf1, β-MHC (myosin heavy chain beta-subunit), Ska (skeletal alpha actin), ANP (atrial natriuretic peptide) and β-MHC versus Ska ratio. Results are normalized to expression of 18S (18S ribosomal RNA). n = 4. (H-I) Neonatal rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA (100 nM) and 3 days later cells were treated with either vehicle, phenylephrine (PE, 100 µM) or basic fibroblast growth factor (FGF, 20 ng/ml) for 48 h. (H) Shown is immunoblot analysis for β-MHC, ANP, GAPDH, Ska and Vinculin. (I) Shown is β-MHC versus Ska ratio from immunoblot quantification. n = 4. * P
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    Identification of FOXF1 target genes in endothelial cells from regenerating lungs. ( A ) FACS-sorting strategy to isolate populations of endothelial (CD45 − CD31 + CD326 − ) and epithelial (CD45 − CD31 − CD326 + ) cells, showing GFP expression in PDGFb-iCre expressing mice. ( B ) Foxf1 mRNA was highly expressed in endothelial cells but not in epithelial cells. Endothelial cells from PDGFb-iCre/Foxf1 fl /+ mice had significantly less Foxf1 mRNA than endothelial cells from control mice. ( C ) Heat map showing significant changes in expression of 1047 genes in endothelial cells from Foxf1 fl /+ and PDGFb-iCre/Foxf1 fl /+ lungs after PNX. ( D ) Western blot analysis showed increased protein levels for CDKN1A (P21 Cip1 ) and CDKN2B (P15 Ink4b ) in PDGFb-iCre/Foxf1 fl /+ lungs in sham mice and 3 days after PNX. Cropped gels are presented here with full gel available in Supplemental Fig. 8 . ( E ) <t>qRT-PCR</t> analysis showed significant changes in mRNA expression of several FOXF1 target genes in endothelial cells from PDGFb-iCre/Foxf1 fl /+ lungs. Cdkn2b was not detectable (N.D.) in control samples. *p
    Stepone Qrt Pcr Machine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Knockdown of TCTN1 expression in thyroid cancer cells by shTCTN1. (A) Representative pictures of green fluorescent protein (GFP) expression recorded under a fluorescence microscope in CAL62 and 8305C cells. (B) <t>qRT-PCR</t> analysis of TCTN1 mRNA levels in CAL62 and 8305C cells following shCon or shTCTN1 infection. Data are expressed as mean ± SD of 3 independent experiments. *P
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    Thermo Fisher taqman qrt pcr analysis system
    Knockdown of TCTN1 expression in thyroid cancer cells by shTCTN1. (A) Representative pictures of green fluorescent protein (GFP) expression recorded under a fluorescence microscope in CAL62 and 8305C cells. (B) <t>qRT-PCR</t> analysis of TCTN1 mRNA levels in CAL62 and 8305C cells following shCon or shTCTN1 infection. Data are expressed as mean ± SD of 3 independent experiments. *P
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    Knockdown of TCTN1 expression in thyroid cancer cells by shTCTN1. (A) Representative pictures of green fluorescent protein (GFP) expression recorded under a fluorescence microscope in CAL62 and 8305C cells. (B) <t>qRT-PCR</t> analysis of TCTN1 mRNA levels in CAL62 and 8305C cells following shCon or shTCTN1 infection. Data are expressed as mean ± SD of 3 independent experiments. *P
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    In the Postnatal Suture, PRX1-Expressing Cells Overlap with GLI1-Expressing Cells (A) Comparative evaluation of tdTOMATO expression in 4-week-old Prx1-creER-EGFP +/− ;tdTOMATO +/− and Gli1-creER +/− ;tdTOMATO +/− mice treated with tamoxifen for 5 days (short-pulsing). S, suture space (n = 2 mice). Scale bars, 100 μm. (B) Ex vivo <t>qRT-PCR</t> analysis of Gli1 and Axin2 expression in pnPRX1 + cells (EGFP + ), (EGFP − ), and in pnCOL1 + cells isolated from the sutures (n = 3 independent experiments). Relative expression values ± SD are reported. ∗∗ p
    Single Cell To Ct Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi7300 96 well qrt pcr system
    Iron can complement the defect of the putA mutant in production of cytochromes c . ( A) Iron influences heme c levels in ∆ putA . Cultures (∼0.6 of OD 600 ) of WT and ∆ putA grown in LB with iron at varying concentrations were pelletted and photographed, then were lysed for quantition of heme c levels as above. Note that Fe(III) was reduced extracellularly to form Fe 3 O 4 particles in both WT and ∆ putA when its concentrations were high. (B) Expression of the hem genes in ∆ putA analyzed by <t>qRT-PCR.</t> Enzymes for heme biosynthesis are shown above. Cells of mid-log phase grown in LB (WC) and MS (RC) were prepared as described in Methods. The averaged expression level of each gene in mutants was normalized to that of the arcA gene, which is relatively constant. (C) Nitrite susceptibility of ∆ putA . Nitrite susceptibility of S. oneidensis is dictated by cytochrome bd oxidase. Cells at 10 8 cfu/ml were serial diluted and 5 µl of each dilution was dropped on LB plates containing 5 mM nitrite. All experiments were performed at least three times and presented either as means ± SEM or by a representative of similar results.
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    Thermo Fisher cellsdirect one step qrt pcr kit
    A novel mouse model for inducible interleukin (IL)-10 expression: pMT-10 mice. (A) Schematic representation showing the targeting vector and insertion site. (B) Kinetics of IL-10 overexpression in the serum at different time points post Zn administration and Zn withdrawal. pMT-10 mice were fed with normal (pMT-10-Zn) or Zn-enriched (pMT-10 + Zn) water and at the indicated time points blood was harvested and the amount of IL-10 in serum measured by immunoassay. (C) <t>qRT-PCR</t> identified CD45 − TER119 − cell subsets from skin, bone marrow, and small intestine (SI) as the main producers of IL-10 in pMT-10 mice fed for 8 days with Zn-enriched water. In both (B,C) , each point or bar represents the mean ± SEM for three independent mice. Data were analyzed with (B) two-way analysis of variance (Sidak’s multiple comparisons test) or (C) Student’s t -test, * p
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    Expression of the mea-1 allele during early seed development. Amplification of MEA-1 ( top ) and ACTIN-11 ( ACT11 ) as a control for cDNA synthesis. <t>RNA</t> was isolated from siliques derived from self-pollinated mea-1 homozygous pistils ( mea × mea ), self-pollinated wild-type pistils (wt × wt), a cross between a homozygous mea-1 female and wild-type male ( mea × wt), and a cross between wild-type female and homozygous mea-1 male (wt × mea ). Primers that specifically amplify the mea-1 allele under these <t>PCR</t> conditions were used for RT–PCR. (M) Marker lane; (G) genomic DNA as a control.
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    Expression of the mea-1 allele during early seed development. Amplification of MEA-1 ( top ) and ACTIN-11 ( ACT11 ) as a control for cDNA synthesis. <t>RNA</t> was isolated from siliques derived from self-pollinated mea-1 homozygous pistils ( mea × mea ), self-pollinated wild-type pistils (wt × wt), a cross between a homozygous mea-1 female and wild-type male ( mea × wt), and a cross between wild-type female and homozygous mea-1 male (wt × mea ). Primers that specifically amplify the mea-1 allele under these <t>PCR</t> conditions were used for RT–PCR. (M) Marker lane; (G) genomic DNA as a control.
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    The VEGF–miR-1–Mpl axis in human endothelial cells. (a) HUVECs were stimulated with 10 ng/ml recombinant human VEGF and treated with SU5416 or vehicle (DMSO). miR-1 relative expression was measured by <t>TaqMan</t> <t>qRT-PCR,</t> normalized to vehicle controls, and expressed as 2 −ΔΔCt (three experiments; n = 3 or more in each group; VEGF-treated groups were compared; *, P
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    15 microRNA <t>(miRNA)</t> differentially expressed in the plasma of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) patients, and healthy control (HC). Expressions of the selected <t>miRNAs</t> in the plasma obtained from patients with SLE ( n = 50), RA ( n = 16), and HC ( n = 20) were determined by <t>qRT-PCR.</t> The expression levels of miRNAs were normalized to cel-miR-39.
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    Western blot analysis of ribonucleotide reductase. A) Cellular lysates from different treatment groups were analyzed for ribonucleotide reductase R2 and p53R2 subunits. Afterwards, blots were stripped and re-probed for actin. Freshly isolated monocytes (Mo), day 13 maturated GM-CSF (GM-CSF) and Leishmania (Leish) MDMs are shown. B) Quantitiation of western blots was done. Freshly isolated monocytes were set to 1 and increases in R2 and p53R2 expression levels for GM-CSF- and Leishmania -maturated MDMs groups are shown. Mean and SEM are displayed for four independent donors. C) <t>qRT-PCR</t> analysis was done on total cellular RNA extracts. mRNA fold changes for the different treatment groups (n = 3) are graphed as mean and SEM. Significantly different groups (p
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    The expression of osteogenesis-related genes was significantly upregulated. After culture with conditioned medium, the relative expressions of differentiation markers Runx2, ALP, and Osterix in MG-63 cells were upregulated. Gene expression was detected by <t>qRT-PCR.</t> The expression of proliferation marker cyclin-D1 was also upregulated. Expression of these genes after 40 minutes of LIPUS treatment was significantly higher than after 20 minutes of LIPUS treatment.
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    The expression of osteogenesis-related genes was significantly upregulated. After culture with conditioned medium, the relative expressions of differentiation markers Runx2, ALP, and Osterix in MG-63 cells were upregulated. Gene expression was detected by <t>qRT-PCR.</t> The expression of proliferation marker cyclin-D1 was also upregulated. Expression of these genes after 40 minutes of LIPUS treatment was significantly higher than after 20 minutes of LIPUS treatment.
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    Zika Virus Sampling and Sequencing in Central America and Mexico (A) Map of Central America and Mexico. Colored circles indicate the sampling locations of Zika virus sequences generated in this study, and the locations of publicly available sequences from Central America and Mexico. (B) The temporal and geographic distribution of Zika virus <t>qRT-PCR-positive</t> samples tested in this study. Samples are colored according to their sampling location. (C) Consensus genome coverage of the Zika virus sequences generated in this study. Sequences are colored according to their sampling location and the Zika virus genome structure is shown above the plot.
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    Zika Virus Sampling and Sequencing in Central America and Mexico (A) Map of Central America and Mexico. Colored circles indicate the sampling locations of Zika virus sequences generated in this study, and the locations of publicly available sequences from Central America and Mexico. (B) The temporal and geographic distribution of Zika virus <t>qRT-PCR-positive</t> samples tested in this study. Samples are colored according to their sampling location. (C) Consensus genome coverage of the Zika virus sequences generated in this study. Sequences are colored according to their sampling location and the Zika virus genome structure is shown above the plot.
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    Expression of SHBG by lymphocytic cells. Box-plots (whiskers: min to max) represent SHBG / Shbg mRNA expression levels, measured by <t>qRT-PCR,</t> in ( A ) human B cells (BL41), T cells (Jurkat), as well as in ( B ) mouse B cells (A20), T cells (IP12-7), splenocytes, spleen. Liver served as positive control in human and negative control in mouse. Relative expression level (−ΔCt with an arbitrary zero point) is denoted on the Y-axis. Three independent qRT-PCR experiments were performed in duplicates. Asterisks denote expression under the detection limit. ( C ) Representative Western blot images show SHBG protein in the cell lysate of human (BL41, Jurkat) and mouse (A20, IP12-7) lymphocytes. The two bands represent monomeric (~45 kDa) and dimeric (~90 kDa) forms of the protein.
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    Expression of SHBG by lymphocytic cells. Box-plots (whiskers: min to max) represent SHBG / Shbg mRNA expression levels, measured by <t>qRT-PCR,</t> in ( A ) human B cells (BL41), T cells (Jurkat), as well as in ( B ) mouse B cells (A20), T cells (IP12-7), splenocytes, spleen. Liver served as positive control in human and negative control in mouse. Relative expression level (−ΔCt with an arbitrary zero point) is denoted on the Y-axis. Three independent qRT-PCR experiments were performed in duplicates. Asterisks denote expression under the detection limit. ( C ) Representative Western blot images show SHBG protein in the cell lysate of human (BL41, Jurkat) and mouse (A20, IP12-7) lymphocytes. The two bands represent monomeric (~45 kDa) and dimeric (~90 kDa) forms of the protein.
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    Expression of SHBG by lymphocytic cells. Box-plots (whiskers: min to max) represent SHBG / Shbg mRNA expression levels, measured by <t>qRT-PCR,</t> in ( A ) human B cells (BL41), T cells (Jurkat), as well as in ( B ) mouse B cells (A20), T cells (IP12-7), splenocytes, spleen. Liver served as positive control in human and negative control in mouse. Relative expression level (−ΔCt with an arbitrary zero point) is denoted on the Y-axis. Three independent qRT-PCR experiments were performed in duplicates. Asterisks denote expression under the detection limit. ( C ) Representative Western blot images show SHBG protein in the cell lysate of human (BL41, Jurkat) and mouse (A20, IP12-7) lymphocytes. The two bands represent monomeric (~45 kDa) and dimeric (~90 kDa) forms of the protein.
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    Rhythmic expression of DBP in the liver of female Gα i3 -/- mice is phase advanced ( A , B ) Quantitative real-time <t>PCR</t> analysis of rhythmic expression of DBP <t>mRNA</t> in the liver of female (A) and male (B) Gα i3 deficient mice as compared to wild-type control animals. Dbp transcript levels were normalized to the endogenous control Gapdh. Shown are 2 -ΔCt values. ( C , D ) Representative immunoblots of rhythmic expression of the DBP protein in the liver of female (C) and male (D) Gα i3 -deficient mice and wild-type controls. GAPDH and β-Actin were employed as loading controls. Relative protein levels (DBP/GAPDH) were determined by densitometric analysis using ImageJ software (lower panels in C and D). Mice were sacrificed and analyzed every six hours at the indicated time points (ZT 0 to ZT 18). Results are expressed as mean ± s.d. of six animals (mRNA) or four animals (immunoblot) analyzed per genotype and time point ( * p
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    AMPK activation contributes to the induction of UCP2 and the down-regulation of MPC1. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 1 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) <t>qRT-PCR</t> analysis of UCP2 <t>mRNA</t> expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) for 1 and 48 h or with Compound C (CC, 5 μM) for 1 h. (C) qRT-PCR analysis of MPC1 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) or with Compound C (CC, 5 μM), both for 1 h. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p
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    Image Search Results


    Transcript distribution profile of Trica- SKR1 and Trica -SKR2. Quantification of transcript levels by qRT-PCR in seven different tissues from adult T. castaneum . The data represent samples of central brain (n = 15), optic lobes (n = 15), salivary glands (n = 15), gut (n = 15), fat body (n = 20), testes (n = 50) and ovaries (n = 50), normalized relative to β-actin and ribosomal protein 3 (RPs3) transcript levels. Abbreviations: B = central brain, OL = optic lobes, SG = salivary glands, G = gut, FB = fat body, Te = testes, Ov = ovaria.

    Journal: PLoS ONE

    Article Title: Signaling Properties and Pharmacological Analysis of Two Sulfakinin Receptors from the Red Flour Beetle, Tribolium castaneum

    doi: 10.1371/journal.pone.0094502

    Figure Lengend Snippet: Transcript distribution profile of Trica- SKR1 and Trica -SKR2. Quantification of transcript levels by qRT-PCR in seven different tissues from adult T. castaneum . The data represent samples of central brain (n = 15), optic lobes (n = 15), salivary glands (n = 15), gut (n = 15), fat body (n = 20), testes (n = 50) and ovaries (n = 50), normalized relative to β-actin and ribosomal protein 3 (RPs3) transcript levels. Abbreviations: B = central brain, OL = optic lobes, SG = salivary glands, G = gut, FB = fat body, Te = testes, Ov = ovaria.

    Article Snippet: For qRT-PCR Fast SYBR Green Master Mix (Applied Biosystems) as per manufacturer’s instruction and the StepOnePlus Real-Time PCR system (Applied Biosystems) were used.

    Techniques: Quantitative RT-PCR

    Effect of 4 weeks of exenatide treatment on insulin clearance. Mice were fed an RD or HF diet for 2 months and injected daily with exenatide in the last month. (A) (i) Cc1 +/+ liver lysates were analyzed by qRT‐PCR to measure Ceacam1 mRNA levels normalized to 18S (n = 5/each group in duplicate). Values are expressed as mean ± SE; * P

    Journal: Hepatology Communications

    Article Title: Exenatide induces carcinoembryonic antigen‐related cell adhesion molecule 1 expression to prevent hepatic steatosis

    doi: 10.1002/hep4.1117

    Figure Lengend Snippet: Effect of 4 weeks of exenatide treatment on insulin clearance. Mice were fed an RD or HF diet for 2 months and injected daily with exenatide in the last month. (A) (i) Cc1 +/+ liver lysates were analyzed by qRT‐PCR to measure Ceacam1 mRNA levels normalized to 18S (n = 5/each group in duplicate). Values are expressed as mean ± SE; * P

    Article Snippet: Immunoprecipitated and input samples were evaluated by quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) analysis (StepOne Plus; Applied Biosystems) using primers for RPL30 (kit) as control and human CEACAM1 ( Supporting Fig. S1B,C ).

    Techniques: Mouse Assay, Injection, Quantitative RT-PCR

    Effect of exenatide on insulin uptake in murine primary hepatocytes. (A) Primary hepatocytes were isolated from Cc1 +/+ mice fed an RD or HF diet for 1 month (n = 6 mice/treatment) and treated with exenatide or saline for 24 hours before performing qRT‐PCR analysis in triplicate to measure (i) Ceacam1 , (ii) Pparα , and (iii) Pparγ mRNA levels normalized to 18S. Values are expressed as mean ± SEM; * P

    Journal: Hepatology Communications

    Article Title: Exenatide induces carcinoembryonic antigen‐related cell adhesion molecule 1 expression to prevent hepatic steatosis

    doi: 10.1002/hep4.1117

    Figure Lengend Snippet: Effect of exenatide on insulin uptake in murine primary hepatocytes. (A) Primary hepatocytes were isolated from Cc1 +/+ mice fed an RD or HF diet for 1 month (n = 6 mice/treatment) and treated with exenatide or saline for 24 hours before performing qRT‐PCR analysis in triplicate to measure (i) Ceacam1 , (ii) Pparα , and (iii) Pparγ mRNA levels normalized to 18S. Values are expressed as mean ± SEM; * P

    Article Snippet: Immunoprecipitated and input samples were evaluated by quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) analysis (StepOne Plus; Applied Biosystems) using primers for RPL30 (kit) as control and human CEACAM1 ( Supporting Fig. S1B,C ).

    Techniques: Isolation, Mouse Assay, Quantitative RT-PCR

    Effect of exenatide on CEACAM1 and GLP‐1R mRNA levels in HepG2 cells. (A) HepG2 cells were treated with saline (light gray) or exenatide (white) for 0‐24 hours before qRT‐PCR analysis to measure (i) hGLP‐1R and (ii) hCEACAM1 ( CC1 ) mRNA levels normalized to iGAPDH (n = 5/group in triplicate). Values are expressed as mean ± SEM; * P

    Journal: Hepatology Communications

    Article Title: Exenatide induces carcinoembryonic antigen‐related cell adhesion molecule 1 expression to prevent hepatic steatosis

    doi: 10.1002/hep4.1117

    Figure Lengend Snippet: Effect of exenatide on CEACAM1 and GLP‐1R mRNA levels in HepG2 cells. (A) HepG2 cells were treated with saline (light gray) or exenatide (white) for 0‐24 hours before qRT‐PCR analysis to measure (i) hGLP‐1R and (ii) hCEACAM1 ( CC1 ) mRNA levels normalized to iGAPDH (n = 5/group in triplicate). Values are expressed as mean ± SEM; * P

    Article Snippet: Immunoprecipitated and input samples were evaluated by quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) analysis (StepOne Plus; Applied Biosystems) using primers for RPL30 (kit) as control and human CEACAM1 ( Supporting Fig. S1B,C ).

    Techniques: Quantitative RT-PCR

    Quantitative real-time polymerase chain reaction (qRT-PCR) and Locked nucleic acid in situ hybridization (LNA-ISH) analysis of expression levels of microRNAs. (A) qRT-PCR analysis of relative expression levels of 4 microRNAs (miR-205, miR-210, miR-492, and miR-1247) in pancreatic juice samples from patients with pancreatic ductal adenocarcinoma (PDAC), patients with chronic pancreatitis (CP), and non-pancreatic, non-healthy (NPNH) controls. The relative abundance of four miRNAs, normalized to the level of RNU6B, significantly varied in pancreatic juice samples from patients with PDAC, CP and NPNH. (B) LNA-ISH-IHC images showing miR-492 expression in 3 (J101, J16, J39) pancreatic ductal adenocarcinoma tissue samples (magnification ×100). (C) The graph shows qRT-PCR results for miR-492 in pancreatic juice samples from the same patients.

    Journal: Journal of Cancer

    Article Title: Circulating microRNAs in Pancreatic Juice as Candidate Biomarkers of Pancreatic Cancer

    doi: 10.7150/jca.10094

    Figure Lengend Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) and Locked nucleic acid in situ hybridization (LNA-ISH) analysis of expression levels of microRNAs. (A) qRT-PCR analysis of relative expression levels of 4 microRNAs (miR-205, miR-210, miR-492, and miR-1247) in pancreatic juice samples from patients with pancreatic ductal adenocarcinoma (PDAC), patients with chronic pancreatitis (CP), and non-pancreatic, non-healthy (NPNH) controls. The relative abundance of four miRNAs, normalized to the level of RNU6B, significantly varied in pancreatic juice samples from patients with PDAC, CP and NPNH. (B) LNA-ISH-IHC images showing miR-492 expression in 3 (J101, J16, J39) pancreatic ductal adenocarcinoma tissue samples (magnification ×100). (C) The graph shows qRT-PCR results for miR-492 in pancreatic juice samples from the same patients.

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis Taqman miRNA assays (Applied Biosystems, Foster City, CA) were used to perform expression profiling of the miRNAs of interest.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, In Situ Hybridization, Expressing, Immunohistochemistry

    Lack of correlation between telomere length and TERRA levels in cells with stable telomeres. Two independent, exponentially-dividing strains of each genotype were split and used to isolate RNA or DNA. ( A ) A map of single copy loci amplified during TERRA measurements (B-E) and Southern blot probes (F). Distance from the centromeric end of the TG repeats is indicated. Primers used in the qRT-PCR and to make the probes are described in S2 Table. ( B–E ) TERRA measured as in Figure 1B . ( F ) Telomeric Southern blots performed after XhoI (top two gels) or HindIII (bottom two gels) digestion. One membrane was first probed for telomere 15L and then Y’+TG sequences ( Supplementary Figure S3A ). The other membrane was first probed for telomere 10R and finally 13R. Loading control corresponds to SYBR Safe staining of the gel probed for telomeres 10R and 13R.

    Journal: Nucleic Acids Research

    Article Title: Paf1 and Ctr9, core components of the PAF1 complex, maintain low levels of telomeric repeat containing RNA

    doi: 10.1093/nar/gkx1131

    Figure Lengend Snippet: Lack of correlation between telomere length and TERRA levels in cells with stable telomeres. Two independent, exponentially-dividing strains of each genotype were split and used to isolate RNA or DNA. ( A ) A map of single copy loci amplified during TERRA measurements (B-E) and Southern blot probes (F). Distance from the centromeric end of the TG repeats is indicated. Primers used in the qRT-PCR and to make the probes are described in S2 Table. ( B–E ) TERRA measured as in Figure 1B . ( F ) Telomeric Southern blots performed after XhoI (top two gels) or HindIII (bottom two gels) digestion. One membrane was first probed for telomere 15L and then Y’+TG sequences ( Supplementary Figure S3A ). The other membrane was first probed for telomere 10R and finally 13R. Loading control corresponds to SYBR Safe staining of the gel probed for telomeres 10R and 13R.

    Article Snippet: Next, one-step qRT-PCR was carried out in the presence of forward and reverse primers, directed to specific telomeres, using the Superscript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen).

    Techniques: Amplification, Southern Blot, Quantitative RT-PCR, Staining

    PolyIC/dsRBEC induces the expression and secretion of pro-inflammatory cytokines in MDA-MB-468 cells. A) qRT-PCR analysis of IFN-β, CCL5, IP10 and TNFα mRNA expression following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 2 and 4 hours. Data were normalized to GAPDH and are expressed as fold change relative to vehicle-treated samples. A representative experiment out of 3 experiments is shown. Error bars represent RQ max. B) Protein levels of IFN-β, CCL5, IP10 and TNFα were measured by ELISA following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 24 hours. Values are averages of triplicate biological samples from one representative experiment. (***,P

    Journal: PLoS ONE

    Article Title: Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

    doi: 10.1371/journal.pone.0162321

    Figure Lengend Snippet: PolyIC/dsRBEC induces the expression and secretion of pro-inflammatory cytokines in MDA-MB-468 cells. A) qRT-PCR analysis of IFN-β, CCL5, IP10 and TNFα mRNA expression following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 2 and 4 hours. Data were normalized to GAPDH and are expressed as fold change relative to vehicle-treated samples. A representative experiment out of 3 experiments is shown. Error bars represent RQ max. B) Protein levels of IFN-β, CCL5, IP10 and TNFα were measured by ELISA following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 24 hours. Values are averages of triplicate biological samples from one representative experiment. (***,P

    Article Snippet: 1 μg of total RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and the resulting cDNA was used for qRT-PCR analysis (Fast SYBR Green; Applied Biosystems) using the primer pairs listed in .

    Techniques: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Relative mRNA expression levels of osteoblast cell differentiation markers in BMCs from wild-type and PKD1 (+/−) pubescent male (A and C) and female (B and D) mice. Total RNA was isolated from 7- (CFU-F) and 18-day (CFU-OB) cultures and BSP, OCN, OSX, and TIC mRNA levels were measured by qRT-PCR as described in Materials and Methods . For each marker gene, values were normalized to the wild-type male and were set at 1. Values represent the mean ± SEM of five and 10 for males and females, respectively. *, P

    Journal: Endocrinology

    Article Title: Protein Kinase D1 Is Essential for Bone Acquisition during Pubertal Growth

    doi: 10.1210/en.2013-1376

    Figure Lengend Snippet: Relative mRNA expression levels of osteoblast cell differentiation markers in BMCs from wild-type and PKD1 (+/−) pubescent male (A and C) and female (B and D) mice. Total RNA was isolated from 7- (CFU-F) and 18-day (CFU-OB) cultures and BSP, OCN, OSX, and TIC mRNA levels were measured by qRT-PCR as described in Materials and Methods . For each marker gene, values were normalized to the wild-type male and were set at 1. Values represent the mean ± SEM of five and 10 for males and females, respectively. *, P

    Article Snippet: Reagents and enzymes for quantitative RT-PCR (qRT-PCR) analysis were purchased from Applied Biosystems.

    Techniques: Expressing, Cell Differentiation, Mouse Assay, Isolation, Quantitative RT-PCR, Marker

    Osteoclast cell differentiation marker expression in BMCs from wild-type and PKD1 (+/−) pubescent male (A) and female (B) mice. BMC cultures were grown in αMEM plus vitamin D3 (10 −8 M) for 7 days. Total RNA was isolated and mRNA expression levels for TRAP, RANKL, and OPG were measured by qRT-PCR as described in Materials and Methods . Values represent the mean ± SEM of three qRT-PCR determinations on three different cell preparations with four to seven mice per preparation. *, P

    Journal: Endocrinology

    Article Title: Protein Kinase D1 Is Essential for Bone Acquisition during Pubertal Growth

    doi: 10.1210/en.2013-1376

    Figure Lengend Snippet: Osteoclast cell differentiation marker expression in BMCs from wild-type and PKD1 (+/−) pubescent male (A) and female (B) mice. BMC cultures were grown in αMEM plus vitamin D3 (10 −8 M) for 7 days. Total RNA was isolated and mRNA expression levels for TRAP, RANKL, and OPG were measured by qRT-PCR as described in Materials and Methods . Values represent the mean ± SEM of three qRT-PCR determinations on three different cell preparations with four to seven mice per preparation. *, P

    Article Snippet: Reagents and enzymes for quantitative RT-PCR (qRT-PCR) analysis were purchased from Applied Biosystems.

    Techniques: Cell Differentiation, Marker, Expressing, Mouse Assay, Isolation, Quantitative RT-PCR

    Regulation of Smpd3 expression in chondrocytes by BMP-2. (A and B) Gene expression analyses of 3-month-old fractured WT bones showing a significant upregulation of Bmp-2 expression (A) and Smpd3 expression (B) in the callus. Expression analyses by qRT-PCR were performed 13 days postsurgery. (C) Immunohistochemistry with anti-SMPD3 antibody shows an increased presence of SMPD3 in the callus of WT fractured tibia at 14 and 28 days postsurgery. A bone section of the unfractured contralateral tibia was used as a control. (D) ATDC5 chondrogenic cells treated with 300 ng/ml of BMP-2 for 48 h show a significant upregulation of chondrogenic marker Col2a1 , Col10a1 , Sox6 , Sox9 , Osx , Mef2c , and Smpd3 expression. (E) Differentiated ATDC5 cells (cultured in the presence of ascorbic acid and β-glycerophosphate for 6 days) when treated with 2 μM dorsomorphin (Dorso) show significant downregulation of Col2a1 , Col10a1 , Sox5 , Sox6 , Osx , Mef2c , and Smpd3 expression. Cells were treated with dorsomorphin for 30 min every 2 days, before the medium was changed. Error bars represent standard deviations. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Role of SMPD3 during Bone Fracture Healing and Regulation of Its Expression

    doi: 10.1128/MCB.00370-18

    Figure Lengend Snippet: Regulation of Smpd3 expression in chondrocytes by BMP-2. (A and B) Gene expression analyses of 3-month-old fractured WT bones showing a significant upregulation of Bmp-2 expression (A) and Smpd3 expression (B) in the callus. Expression analyses by qRT-PCR were performed 13 days postsurgery. (C) Immunohistochemistry with anti-SMPD3 antibody shows an increased presence of SMPD3 in the callus of WT fractured tibia at 14 and 28 days postsurgery. A bone section of the unfractured contralateral tibia was used as a control. (D) ATDC5 chondrogenic cells treated with 300 ng/ml of BMP-2 for 48 h show a significant upregulation of chondrogenic marker Col2a1 , Col10a1 , Sox6 , Sox9 , Osx , Mef2c , and Smpd3 expression. (E) Differentiated ATDC5 cells (cultured in the presence of ascorbic acid and β-glycerophosphate for 6 days) when treated with 2 μM dorsomorphin (Dorso) show significant downregulation of Col2a1 , Col10a1 , Sox5 , Sox6 , Osx , Mef2c , and Smpd3 expression. Cells were treated with dorsomorphin for 30 min every 2 days, before the medium was changed. Error bars represent standard deviations. *, P

    Article Snippet: Gene expression analysis was performed using a qRT-PCR system (model 7500; Applied Biosystems).

    Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Marker, Cell Culture

    Relative expression of eng mRNA in P. pastoris CL2 in response to cold-shock at 4 °C. CL2 cells were grown in medium BMGH (30 °C for 4 h), from OD 600 = 1 inoculum, then subjected to cold-shock for the induction of eng mRNA transcript expression (4 °C for 6 h). Samples were taken at 0, 2 and 6 h for analysis by qRT-PCR; and finally, they were transferred to 30 °C for 4 h for ENG product synthesis, stage in which the corresponding sample at 2 h was taken for analysis by qRT-PCR. The values of relative expression of eng mRNA were calculated in relation to eng and the housekeeping constitutive gene act1 (control) expression using method 2 −ΔΔCt

    Journal: AMB Express

    Article Title: Autolysis of Pichia pastoris induced by cold

    doi: 10.1186/s13568-017-0397-y

    Figure Lengend Snippet: Relative expression of eng mRNA in P. pastoris CL2 in response to cold-shock at 4 °C. CL2 cells were grown in medium BMGH (30 °C for 4 h), from OD 600 = 1 inoculum, then subjected to cold-shock for the induction of eng mRNA transcript expression (4 °C for 6 h). Samples were taken at 0, 2 and 6 h for analysis by qRT-PCR; and finally, they were transferred to 30 °C for 4 h for ENG product synthesis, stage in which the corresponding sample at 2 h was taken for analysis by qRT-PCR. The values of relative expression of eng mRNA were calculated in relation to eng and the housekeeping constitutive gene act1 (control) expression using method 2 −ΔΔCt

    Article Snippet: Primers and probes used for qRT-PCR analyses were designed by Applied Biosystems (Foster City, CA, USA; Table ).

    Techniques: Expressing, Quantitative RT-PCR

    Vezf1 is expressed in adult cardiomyocytes and regulates cardiomyocyte growth and cardiomyopathy related genes. (A) qRT-PCR analysis of the expression of endothelial nitric oxide synthase (eNOS), collagen type I alpha 1 chain (Col1a1), and α myosin heavy chain 6 (α-MHC) mRNAs in pools of fractionated resident mouse cardiac cells. eNOS, Col1a1 and α-MHC were used as markers for endothelial cells (Endo), fibroblasts (Fibro), and cardiomyocytes (Myo), respectively. n = 5. (B) qPCR analysis of Vezf1 mRNA levels in cardiomyocytes and fibroblasts relative to that in the endothelial cells. n = 5. (C) Adult rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or Control siRNA (100 nM) and 3 days later RNA samples were collected. Shown is qRT-PCR analysis for expression of Vezf1. Results are normalized to expression of 18S (18S ribosomal RNA). n = 6. (D and E) Adult rat ventricular cardiomyocytes were transfected with two distinct Vezf1 siRNAs (100 nM) or Control siRNA (100 nM) and 1 day later cells were stimulated with isoprenaline (Iso, 1 µM) for 48 h where indicated. (D) Shown is microscopy analysis for cardiomyocyte size. Ctrl siRNA n = 8, Vezf1 A siRNA n = 9, Vezf1 B siRNA n = 10; Ctrl siRNA + Iso n = 10, Vezf1 A siRNA + Iso n = 15, Vezf1 B siRNA + Iso n = 13. (E) Shown is microscopy analysis for sarcomere length (µm). Ctrl siRNA n = 14, Vezf1 B siRNA n = 18; Ctrl siRNA + Iso n = 22, Vezf1 B siRNA + Iso n = 25. (F) Adult rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA and 3 days later Ca 2+ cycling and cardiomyocyte (CM) shortening were analyzed. Ctrl n = 44, Vezf1 siRNA n = 43. (G) Neonatal rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA and 2 days later cells were treated with Iso (1 µM) for 24 h. Shown is qRT-PCR analysis for expression of Vezf1, β-MHC (myosin heavy chain beta-subunit), Ska (skeletal alpha actin), ANP (atrial natriuretic peptide) and β-MHC versus Ska ratio. Results are normalized to expression of 18S (18S ribosomal RNA). n = 4. (H-I) Neonatal rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA (100 nM) and 3 days later cells were treated with either vehicle, phenylephrine (PE, 100 µM) or basic fibroblast growth factor (FGF, 20 ng/ml) for 48 h. (H) Shown is immunoblot analysis for β-MHC, ANP, GAPDH, Ska and Vinculin. (I) Shown is β-MHC versus Ska ratio from immunoblot quantification. n = 4. * P

    Journal: EBioMedicine

    Article Title: Vezf1 regulates cardiac structure and contractile function

    doi: 10.1016/j.ebiom.2019.102608

    Figure Lengend Snippet: Vezf1 is expressed in adult cardiomyocytes and regulates cardiomyocyte growth and cardiomyopathy related genes. (A) qRT-PCR analysis of the expression of endothelial nitric oxide synthase (eNOS), collagen type I alpha 1 chain (Col1a1), and α myosin heavy chain 6 (α-MHC) mRNAs in pools of fractionated resident mouse cardiac cells. eNOS, Col1a1 and α-MHC were used as markers for endothelial cells (Endo), fibroblasts (Fibro), and cardiomyocytes (Myo), respectively. n = 5. (B) qPCR analysis of Vezf1 mRNA levels in cardiomyocytes and fibroblasts relative to that in the endothelial cells. n = 5. (C) Adult rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or Control siRNA (100 nM) and 3 days later RNA samples were collected. Shown is qRT-PCR analysis for expression of Vezf1. Results are normalized to expression of 18S (18S ribosomal RNA). n = 6. (D and E) Adult rat ventricular cardiomyocytes were transfected with two distinct Vezf1 siRNAs (100 nM) or Control siRNA (100 nM) and 1 day later cells were stimulated with isoprenaline (Iso, 1 µM) for 48 h where indicated. (D) Shown is microscopy analysis for cardiomyocyte size. Ctrl siRNA n = 8, Vezf1 A siRNA n = 9, Vezf1 B siRNA n = 10; Ctrl siRNA + Iso n = 10, Vezf1 A siRNA + Iso n = 15, Vezf1 B siRNA + Iso n = 13. (E) Shown is microscopy analysis for sarcomere length (µm). Ctrl siRNA n = 14, Vezf1 B siRNA n = 18; Ctrl siRNA + Iso n = 22, Vezf1 B siRNA + Iso n = 25. (F) Adult rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA and 3 days later Ca 2+ cycling and cardiomyocyte (CM) shortening were analyzed. Ctrl n = 44, Vezf1 siRNA n = 43. (G) Neonatal rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA and 2 days later cells were treated with Iso (1 µM) for 24 h. Shown is qRT-PCR analysis for expression of Vezf1, β-MHC (myosin heavy chain beta-subunit), Ska (skeletal alpha actin), ANP (atrial natriuretic peptide) and β-MHC versus Ska ratio. Results are normalized to expression of 18S (18S ribosomal RNA). n = 4. (H-I) Neonatal rat ventricular cardiomyocytes were transfected with Vezf1 siRNA (100 nM) or control siRNA (100 nM) and 3 days later cells were treated with either vehicle, phenylephrine (PE, 100 µM) or basic fibroblast growth factor (FGF, 20 ng/ml) for 48 h. (H) Shown is immunoblot analysis for β-MHC, ANP, GAPDH, Ska and Vinculin. (I) Shown is β-MHC versus Ska ratio from immunoblot quantification. n = 4. * P

    Article Snippet: 2.12 RNA isolation, cDNA synthesis and qRT-PCR analysis Trizol (Thermo Fisher Scientific) was used for isolation of RNA from human cardiac tissue samples and an EZNA Total RNA kit I (Omega Bio-tek, Norcross, GA) from isolated cardiomyocytes and from crushed zebrafish larvae.

    Techniques: Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Transfection, Microscopy, Aqueous Normal-phase Chromatography

    Identification of FOXF1 target genes in endothelial cells from regenerating lungs. ( A ) FACS-sorting strategy to isolate populations of endothelial (CD45 − CD31 + CD326 − ) and epithelial (CD45 − CD31 − CD326 + ) cells, showing GFP expression in PDGFb-iCre expressing mice. ( B ) Foxf1 mRNA was highly expressed in endothelial cells but not in epithelial cells. Endothelial cells from PDGFb-iCre/Foxf1 fl /+ mice had significantly less Foxf1 mRNA than endothelial cells from control mice. ( C ) Heat map showing significant changes in expression of 1047 genes in endothelial cells from Foxf1 fl /+ and PDGFb-iCre/Foxf1 fl /+ lungs after PNX. ( D ) Western blot analysis showed increased protein levels for CDKN1A (P21 Cip1 ) and CDKN2B (P15 Ink4b ) in PDGFb-iCre/Foxf1 fl /+ lungs in sham mice and 3 days after PNX. Cropped gels are presented here with full gel available in Supplemental Fig. 8 . ( E ) qRT-PCR analysis showed significant changes in mRNA expression of several FOXF1 target genes in endothelial cells from PDGFb-iCre/Foxf1 fl /+ lungs. Cdkn2b was not detectable (N.D.) in control samples. *p

    Journal: Scientific Reports

    Article Title: FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy

    doi: 10.1038/s41598-017-11175-3

    Figure Lengend Snippet: Identification of FOXF1 target genes in endothelial cells from regenerating lungs. ( A ) FACS-sorting strategy to isolate populations of endothelial (CD45 − CD31 + CD326 − ) and epithelial (CD45 − CD31 − CD326 + ) cells, showing GFP expression in PDGFb-iCre expressing mice. ( B ) Foxf1 mRNA was highly expressed in endothelial cells but not in epithelial cells. Endothelial cells from PDGFb-iCre/Foxf1 fl /+ mice had significantly less Foxf1 mRNA than endothelial cells from control mice. ( C ) Heat map showing significant changes in expression of 1047 genes in endothelial cells from Foxf1 fl /+ and PDGFb-iCre/Foxf1 fl /+ lungs after PNX. ( D ) Western blot analysis showed increased protein levels for CDKN1A (P21 Cip1 ) and CDKN2B (P15 Ink4b ) in PDGFb-iCre/Foxf1 fl /+ lungs in sham mice and 3 days after PNX. Cropped gels are presented here with full gel available in Supplemental Fig. 8 . ( E ) qRT-PCR analysis showed significant changes in mRNA expression of several FOXF1 target genes in endothelial cells from PDGFb-iCre/Foxf1 fl /+ lungs. Cdkn2b was not detectable (N.D.) in control samples. *p

    Article Snippet: Analysis of changes in gene expression was performed using a StepONE qRT-PCR machine (Applied Biosystems) and inventoried Taqman probes (Applied Biosystems, Supplemental Table ) as previously described – .

    Techniques: FACS, Expressing, Mouse Assay, Western Blot, Quantitative RT-PCR

    Decreased endothelial proliferation in PDGFb-iCre/Foxf1 fl /+ mice after PNX. ( A , B ) BrdU incorporation showed less proliferation in lungs from PDGFb-iCre/Foxf1 fl /+ mice than control following PNX. ( C , D ) Co-immunofluorescence experiments with SOX17 to mark endothelial cells and Ki-67 to mark proliferating cells showed that endothelial cell proliferation increased following PNX. Endothelial proliferation was attenuated in PDGFb-iCre/Foxf1 fl /+ mice compared to controls. ( E ) qRT-PCR analysis showed decreased Pecam-1 mRNA in lungs from PDGFb-iCre/Foxf1 fl /+ mice compared to controls, Sox17 mRNA expression was unaltered. ( F ) Western blot analysis demonstrated decreased PECAM-1 protein in PDGFb-iCre/Foxf1 fl /+ mice following PNX compared to controls. Cropped gels are presented here with full gel available in Supplemental Fig. 8 . *p

    Journal: Scientific Reports

    Article Title: FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy

    doi: 10.1038/s41598-017-11175-3

    Figure Lengend Snippet: Decreased endothelial proliferation in PDGFb-iCre/Foxf1 fl /+ mice after PNX. ( A , B ) BrdU incorporation showed less proliferation in lungs from PDGFb-iCre/Foxf1 fl /+ mice than control following PNX. ( C , D ) Co-immunofluorescence experiments with SOX17 to mark endothelial cells and Ki-67 to mark proliferating cells showed that endothelial cell proliferation increased following PNX. Endothelial proliferation was attenuated in PDGFb-iCre/Foxf1 fl /+ mice compared to controls. ( E ) qRT-PCR analysis showed decreased Pecam-1 mRNA in lungs from PDGFb-iCre/Foxf1 fl /+ mice compared to controls, Sox17 mRNA expression was unaltered. ( F ) Western blot analysis demonstrated decreased PECAM-1 protein in PDGFb-iCre/Foxf1 fl /+ mice following PNX compared to controls. Cropped gels are presented here with full gel available in Supplemental Fig. 8 . *p

    Article Snippet: Analysis of changes in gene expression was performed using a StepONE qRT-PCR machine (Applied Biosystems) and inventoried Taqman probes (Applied Biosystems, Supplemental Table ) as previously described – .

    Techniques: Mouse Assay, BrdU Incorporation Assay, Immunofluorescence, Quantitative RT-PCR, Expressing, Western Blot

    Knockdown of TCTN1 expression in thyroid cancer cells by shTCTN1. (A) Representative pictures of green fluorescent protein (GFP) expression recorded under a fluorescence microscope in CAL62 and 8305C cells. (B) qRT-PCR analysis of TCTN1 mRNA levels in CAL62 and 8305C cells following shCon or shTCTN1 infection. Data are expressed as mean ± SD of 3 independent experiments. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Silencing of TCTN1 inhibits proliferation, induces cell cycle arrest and apoptosis in human thyroid cancer

    doi: 10.3892/etm.2017.4940

    Figure Lengend Snippet: Knockdown of TCTN1 expression in thyroid cancer cells by shTCTN1. (A) Representative pictures of green fluorescent protein (GFP) expression recorded under a fluorescence microscope in CAL62 and 8305C cells. (B) qRT-PCR analysis of TCTN1 mRNA levels in CAL62 and 8305C cells following shCon or shTCTN1 infection. Data are expressed as mean ± SD of 3 independent experiments. *P

    Article Snippet: qRT-PCR analysis Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract RNA and reverse transcription was conducted with the revertra ace qPCR RT kit (TOYOBO Co., Ltd., Osaka, Japan).

    Techniques: Expressing, Fluorescence, Microscopy, Quantitative RT-PCR, Infection

    In the Postnatal Suture, PRX1-Expressing Cells Overlap with GLI1-Expressing Cells (A) Comparative evaluation of tdTOMATO expression in 4-week-old Prx1-creER-EGFP +/− ;tdTOMATO +/− and Gli1-creER +/− ;tdTOMATO +/− mice treated with tamoxifen for 5 days (short-pulsing). S, suture space (n = 2 mice). Scale bars, 100 μm. (B) Ex vivo qRT-PCR analysis of Gli1 and Axin2 expression in pnPRX1 + cells (EGFP + ), (EGFP − ), and in pnCOL1 + cells isolated from the sutures (n = 3 independent experiments). Relative expression values ± SD are reported. ∗∗ p

    Journal: Stem Cell Reports

    Article Title: Postnatal Calvarial Skeletal Stem Cells Expressing PRX1 Reside Exclusively in the Calvarial Sutures and Are Required for Bone Regeneration

    doi: 10.1016/j.stemcr.2017.03.002

    Figure Lengend Snippet: In the Postnatal Suture, PRX1-Expressing Cells Overlap with GLI1-Expressing Cells (A) Comparative evaluation of tdTOMATO expression in 4-week-old Prx1-creER-EGFP +/− ;tdTOMATO +/− and Gli1-creER +/− ;tdTOMATO +/− mice treated with tamoxifen for 5 days (short-pulsing). S, suture space (n = 2 mice). Scale bars, 100 μm. (B) Ex vivo qRT-PCR analysis of Gli1 and Axin2 expression in pnPRX1 + cells (EGFP + ), (EGFP − ), and in pnCOL1 + cells isolated from the sutures (n = 3 independent experiments). Relative expression values ± SD are reported. ∗∗ p

    Article Snippet: After 24 hr, EGFP+ cells were isolated from calvarial bones by FACS and qRT-PCR was performed using the Single Cell to CT kit (Thermo Fisher Scientific; cat. #4458237).

    Techniques: Expressing, Mouse Assay, Ex Vivo, Quantitative RT-PCR, Isolation

    Iron can complement the defect of the putA mutant in production of cytochromes c . ( A) Iron influences heme c levels in ∆ putA . Cultures (∼0.6 of OD 600 ) of WT and ∆ putA grown in LB with iron at varying concentrations were pelletted and photographed, then were lysed for quantition of heme c levels as above. Note that Fe(III) was reduced extracellularly to form Fe 3 O 4 particles in both WT and ∆ putA when its concentrations were high. (B) Expression of the hem genes in ∆ putA analyzed by qRT-PCR. Enzymes for heme biosynthesis are shown above. Cells of mid-log phase grown in LB (WC) and MS (RC) were prepared as described in Methods. The averaged expression level of each gene in mutants was normalized to that of the arcA gene, which is relatively constant. (C) Nitrite susceptibility of ∆ putA . Nitrite susceptibility of S. oneidensis is dictated by cytochrome bd oxidase. Cells at 10 8 cfu/ml were serial diluted and 5 µl of each dilution was dropped on LB plates containing 5 mM nitrite. All experiments were performed at least three times and presented either as means ± SEM or by a representative of similar results.

    Journal: Scientific Reports

    Article Title: Investigation of a spontaneous mutant reveals novel features of iron uptake in Shewanella oneidensis

    doi: 10.1038/s41598-017-11987-3

    Figure Lengend Snippet: Iron can complement the defect of the putA mutant in production of cytochromes c . ( A) Iron influences heme c levels in ∆ putA . Cultures (∼0.6 of OD 600 ) of WT and ∆ putA grown in LB with iron at varying concentrations were pelletted and photographed, then were lysed for quantition of heme c levels as above. Note that Fe(III) was reduced extracellularly to form Fe 3 O 4 particles in both WT and ∆ putA when its concentrations were high. (B) Expression of the hem genes in ∆ putA analyzed by qRT-PCR. Enzymes for heme biosynthesis are shown above. Cells of mid-log phase grown in LB (WC) and MS (RC) were prepared as described in Methods. The averaged expression level of each gene in mutants was normalized to that of the arcA gene, which is relatively constant. (C) Nitrite susceptibility of ∆ putA . Nitrite susceptibility of S. oneidensis is dictated by cytochrome bd oxidase. Cells at 10 8 cfu/ml were serial diluted and 5 µl of each dilution was dropped on LB plates containing 5 mM nitrite. All experiments were performed at least three times and presented either as means ± SEM or by a representative of similar results.

    Article Snippet: The analysis was carried out with an ABI7300 96-well qRT-PCR system (Applied Biosystems) as described previously .

    Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Mass Spectrometry

    Transcription read-through underlies the suppression. (A) Expression of fabF1 and acpP revealed by qRT-PCR in various strains. The abundance of mRNAs for fabF1 and acpP in indicated strains at the mid-log phase was assayed by qRT-PCR. Expression levels

    Journal: Journal of Bacteriology

    Article Title: Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis

    doi: 10.1128/JB.00463-16

    Figure Lengend Snippet: Transcription read-through underlies the suppression. (A) Expression of fabF1 and acpP revealed by qRT-PCR in various strains. The abundance of mRNAs for fabF1 and acpP in indicated strains at the mid-log phase was assayed by qRT-PCR. Expression levels

    Article Snippet: The analysis was carried out with an ABI 7300 96-well qRT-PCR system (Applied Biosystems) as described previously ( ).

    Techniques: Expressing, Quantitative RT-PCR

    A novel mouse model for inducible interleukin (IL)-10 expression: pMT-10 mice. (A) Schematic representation showing the targeting vector and insertion site. (B) Kinetics of IL-10 overexpression in the serum at different time points post Zn administration and Zn withdrawal. pMT-10 mice were fed with normal (pMT-10-Zn) or Zn-enriched (pMT-10 + Zn) water and at the indicated time points blood was harvested and the amount of IL-10 in serum measured by immunoassay. (C) qRT-PCR identified CD45 − TER119 − cell subsets from skin, bone marrow, and small intestine (SI) as the main producers of IL-10 in pMT-10 mice fed for 8 days with Zn-enriched water. In both (B,C) , each point or bar represents the mean ± SEM for three independent mice. Data were analyzed with (B) two-way analysis of variance (Sidak’s multiple comparisons test) or (C) Student’s t -test, * p

    Journal: Frontiers in Immunology

    Article Title: The Dynamics of Interleukin-10-Afforded Protection during Dextran Sulfate Sodium-Induced Colitis

    doi: 10.3389/fimmu.2018.00400

    Figure Lengend Snippet: A novel mouse model for inducible interleukin (IL)-10 expression: pMT-10 mice. (A) Schematic representation showing the targeting vector and insertion site. (B) Kinetics of IL-10 overexpression in the serum at different time points post Zn administration and Zn withdrawal. pMT-10 mice were fed with normal (pMT-10-Zn) or Zn-enriched (pMT-10 + Zn) water and at the indicated time points blood was harvested and the amount of IL-10 in serum measured by immunoassay. (C) qRT-PCR identified CD45 − TER119 − cell subsets from skin, bone marrow, and small intestine (SI) as the main producers of IL-10 in pMT-10 mice fed for 8 days with Zn-enriched water. In both (B,C) , each point or bar represents the mean ± SEM for three independent mice. Data were analyzed with (B) two-way analysis of variance (Sidak’s multiple comparisons test) or (C) Student’s t -test, * p

    Article Snippet: CD45.2+ CD11b+ Ly6C+ cells were sorted directly into a mix of 9 µl of CellsDirect One-Step qRT-PCR kit (Life Technologies), containing a mixture of diluted primers (0.05× final concentration, see Table S1 in Supplementary Material for references).

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

    Ly6C + cells preexposed to interleukin (IL)-10 reveal a less inflammatory profile upon DSS-induced colitis than those preexposed to Zn. (A) pMT-10 or BL/6 mice were fed with Zn-enriched water for 8 days, followed by 4 days of 3% DSS administration. (B) At the end of the DSS treatment, Lamina propria leukocytes (LPLs) were isolated and Ly6C + cells sort-purified. Shown is the gating strategy for Ly6C + cells purification. (C) Sort-purified Ly6C + cells ( n = 25 cells) were analyzed by qRT-PCR for a total of 22 genes using the BioMark HD system. Samples were normalized for Hprt expression. Represented is the expression heatmap compiling the genes which expression was detected in either mouse group. Each heatmap rectangle represents the mean of gene expression obtained for cells isolated from five independent mice. (D) The frequency of the different leukocyte subsets was determined upon staining of LPLs for Ly6C + cell sorting. Each dot represents one independent animal; represented is also mean ± SEM. Data were analyzed with Student’s t -test, * p

    Journal: Frontiers in Immunology

    Article Title: The Dynamics of Interleukin-10-Afforded Protection during Dextran Sulfate Sodium-Induced Colitis

    doi: 10.3389/fimmu.2018.00400

    Figure Lengend Snippet: Ly6C + cells preexposed to interleukin (IL)-10 reveal a less inflammatory profile upon DSS-induced colitis than those preexposed to Zn. (A) pMT-10 or BL/6 mice were fed with Zn-enriched water for 8 days, followed by 4 days of 3% DSS administration. (B) At the end of the DSS treatment, Lamina propria leukocytes (LPLs) were isolated and Ly6C + cells sort-purified. Shown is the gating strategy for Ly6C + cells purification. (C) Sort-purified Ly6C + cells ( n = 25 cells) were analyzed by qRT-PCR for a total of 22 genes using the BioMark HD system. Samples were normalized for Hprt expression. Represented is the expression heatmap compiling the genes which expression was detected in either mouse group. Each heatmap rectangle represents the mean of gene expression obtained for cells isolated from five independent mice. (D) The frequency of the different leukocyte subsets was determined upon staining of LPLs for Ly6C + cell sorting. Each dot represents one independent animal; represented is also mean ± SEM. Data were analyzed with Student’s t -test, * p

    Article Snippet: CD45.2+ CD11b+ Ly6C+ cells were sorted directly into a mix of 9 µl of CellsDirect One-Step qRT-PCR kit (Life Technologies), containing a mixture of diluted primers (0.05× final concentration, see Table S1 in Supplementary Material for references).

    Techniques: Mouse Assay, Isolation, Purification, Quantitative RT-PCR, Expressing, Staining, FACS

    Expression of the mea-1 allele during early seed development. Amplification of MEA-1 ( top ) and ACTIN-11 ( ACT11 ) as a control for cDNA synthesis. RNA was isolated from siliques derived from self-pollinated mea-1 homozygous pistils ( mea × mea ), self-pollinated wild-type pistils (wt × wt), a cross between a homozygous mea-1 female and wild-type male ( mea × wt), and a cross between wild-type female and homozygous mea-1 male (wt × mea ). Primers that specifically amplify the mea-1 allele under these PCR conditions were used for RT–PCR. (M) Marker lane; (G) genomic DNA as a control.

    Journal: Genes & Development

    Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity

    doi:

    Figure Lengend Snippet: Expression of the mea-1 allele during early seed development. Amplification of MEA-1 ( top ) and ACTIN-11 ( ACT11 ) as a control for cDNA synthesis. RNA was isolated from siliques derived from self-pollinated mea-1 homozygous pistils ( mea × mea ), self-pollinated wild-type pistils (wt × wt), a cross between a homozygous mea-1 female and wild-type male ( mea × wt), and a cross between wild-type female and homozygous mea-1 male (wt × mea ). Primers that specifically amplify the mea-1 allele under these PCR conditions were used for RT–PCR. (M) Marker lane; (G) genomic DNA as a control.

    Article Snippet: For RT–PCR, ∼5 μg of total RNA were treated with 5 units of RNase-free DNase (Boehringer-Mannheim) in 1× PCR buffer (GIBCO-BRL) containing 2.5 m m MgCl2 at 37°C for 30 min. After heat inactivation at 80°C for 5 min, samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated with ethanol.

    Techniques: Expressing, Microelectrode Array, Amplification, Isolation, Derivative Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Marker

    The VEGF–miR-1–Mpl axis in human endothelial cells. (a) HUVECs were stimulated with 10 ng/ml recombinant human VEGF and treated with SU5416 or vehicle (DMSO). miR-1 relative expression was measured by TaqMan qRT-PCR, normalized to vehicle controls, and expressed as 2 −ΔΔCt (three experiments; n = 3 or more in each group; VEGF-treated groups were compared; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: VEGF controls lung Th2 inflammation via the miR-1-Mpl (myeloproliferative leukemia virus oncogene)-P-selectin axis

    doi: 10.1084/jem.20121200

    Figure Lengend Snippet: The VEGF–miR-1–Mpl axis in human endothelial cells. (a) HUVECs were stimulated with 10 ng/ml recombinant human VEGF and treated with SU5416 or vehicle (DMSO). miR-1 relative expression was measured by TaqMan qRT-PCR, normalized to vehicle controls, and expressed as 2 −ΔΔCt (three experiments; n = 3 or more in each group; VEGF-treated groups were compared; *, P

    Article Snippet: miRNA and mRNA analysis by qRT-PCR. miRNA analyses were performed by TaqMan real-time qRT-PCR assay (Applied Biosystems) on a 7500 Fast Real-Time PCR machine and according to the manufacturer’s instructions.

    Techniques: Recombinant, Expressing, Quantitative RT-PCR

    The effect of lung Th2 inflammation of VEGFR2 siRNA on miR-1 expression. (a) IL-13 expression was induced in the lung epithelium of CC10-rtTA-Il-13 transgenic mice for 7 d, and miR-1 expression was measured by TaqMan qRT-PCR in the whole lung RNA from these mice and their WT litters (values normalized to WT; three experiments; n = 3 or more in each group; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: VEGF controls lung Th2 inflammation via the miR-1-Mpl (myeloproliferative leukemia virus oncogene)-P-selectin axis

    doi: 10.1084/jem.20121200

    Figure Lengend Snippet: The effect of lung Th2 inflammation of VEGFR2 siRNA on miR-1 expression. (a) IL-13 expression was induced in the lung epithelium of CC10-rtTA-Il-13 transgenic mice for 7 d, and miR-1 expression was measured by TaqMan qRT-PCR in the whole lung RNA from these mice and their WT litters (values normalized to WT; three experiments; n = 3 or more in each group; *, P

    Article Snippet: miRNA and mRNA analysis by qRT-PCR. miRNA analyses were performed by TaqMan real-time qRT-PCR assay (Applied Biosystems) on a 7500 Fast Real-Time PCR machine and according to the manufacturer’s instructions.

    Techniques: Expressing, Transgenic Assay, Mouse Assay, Quantitative RT-PCR

    Identification and validation of an miR-1 target. (a) MLECs were transfected with miR-1 or scrambled control (scr–miR-1) and stimulated with VEGF or PBS. RNA was extracted from the cells and hybridized to an mRNA microarray. Heat map shows the 10 genes that were selected as potential targets of miR-1 based on their response to VEGF stimulation and the effect of miR-1 transfection on this response. The dataset was log transformed, normalized, and median centered. The bar shows the color scale used in the heat map (values are in log base). (b–d) MLECs were transfected with miR-1 or scr–miR-1. Cells were lysed, and the lysate was immune-precipitated with anti-Ago2. (b) The level of miR-1 that coimmunoprecipitated with Ago2 was measured by TaqMan qRT-PCR and normalized to the scrambled control (data from two experiments; n = 4 in each group; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: VEGF controls lung Th2 inflammation via the miR-1-Mpl (myeloproliferative leukemia virus oncogene)-P-selectin axis

    doi: 10.1084/jem.20121200

    Figure Lengend Snippet: Identification and validation of an miR-1 target. (a) MLECs were transfected with miR-1 or scrambled control (scr–miR-1) and stimulated with VEGF or PBS. RNA was extracted from the cells and hybridized to an mRNA microarray. Heat map shows the 10 genes that were selected as potential targets of miR-1 based on their response to VEGF stimulation and the effect of miR-1 transfection on this response. The dataset was log transformed, normalized, and median centered. The bar shows the color scale used in the heat map (values are in log base). (b–d) MLECs were transfected with miR-1 or scr–miR-1. Cells were lysed, and the lysate was immune-precipitated with anti-Ago2. (b) The level of miR-1 that coimmunoprecipitated with Ago2 was measured by TaqMan qRT-PCR and normalized to the scrambled control (data from two experiments; n = 4 in each group; *, P

    Article Snippet: miRNA and mRNA analysis by qRT-PCR. miRNA analyses were performed by TaqMan real-time qRT-PCR assay (Applied Biosystems) on a 7500 Fast Real-Time PCR machine and according to the manufacturer’s instructions.

    Techniques: Transfection, Microarray, Transformation Assay, Quantitative RT-PCR

    The role of miR-1 in the lung Th2 inflammation. (a) C57BL/6 mice were sensitized and challenged with OVA as described in Fig. 2 c . Intranasal miR-1 or scrambled miR-1 (scr–miR-1) was delivered the day before the first challenge and every day afterward. The level of miR-1 was measured by TaqMan qRT-PCR in lung endothelial cells (normalized to the control group; n = 4 in each group; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: VEGF controls lung Th2 inflammation via the miR-1-Mpl (myeloproliferative leukemia virus oncogene)-P-selectin axis

    doi: 10.1084/jem.20121200

    Figure Lengend Snippet: The role of miR-1 in the lung Th2 inflammation. (a) C57BL/6 mice were sensitized and challenged with OVA as described in Fig. 2 c . Intranasal miR-1 or scrambled miR-1 (scr–miR-1) was delivered the day before the first challenge and every day afterward. The level of miR-1 was measured by TaqMan qRT-PCR in lung endothelial cells (normalized to the control group; n = 4 in each group; *, P

    Article Snippet: miRNA and mRNA analysis by qRT-PCR. miRNA analyses were performed by TaqMan real-time qRT-PCR assay (Applied Biosystems) on a 7500 Fast Real-Time PCR machine and according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Quantitative RT-PCR

    15 microRNA (miRNA) differentially expressed in the plasma of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) patients, and healthy control (HC). Expressions of the selected miRNAs in the plasma obtained from patients with SLE ( n = 50), RA ( n = 16), and HC ( n = 20) were determined by qRT-PCR. The expression levels of miRNAs were normalized to cel-miR-39.

    Journal: Frontiers in Immunology

    Article Title: B Cell-Related Circulating MicroRNAs With the Potential Value of Biomarkers in the Differential Diagnosis, and Distinguishment Between the Disease Activity and Lupus Nephritis for Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2018.01473

    Figure Lengend Snippet: 15 microRNA (miRNA) differentially expressed in the plasma of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) patients, and healthy control (HC). Expressions of the selected miRNAs in the plasma obtained from patients with SLE ( n = 50), RA ( n = 16), and HC ( n = 20) were determined by qRT-PCR. The expression levels of miRNAs were normalized to cel-miR-39.

    Article Snippet: The expressions of miRNAs in the 80 plama samples from Ruijin Hospital were detected using mirVana qRT-PCR miRNA Detection Kit (Cat#AM1558, Ambion, US).

    Techniques: Quantitative RT-PCR, Expressing

    Western blot analysis of ribonucleotide reductase. A) Cellular lysates from different treatment groups were analyzed for ribonucleotide reductase R2 and p53R2 subunits. Afterwards, blots were stripped and re-probed for actin. Freshly isolated monocytes (Mo), day 13 maturated GM-CSF (GM-CSF) and Leishmania (Leish) MDMs are shown. B) Quantitiation of western blots was done. Freshly isolated monocytes were set to 1 and increases in R2 and p53R2 expression levels for GM-CSF- and Leishmania -maturated MDMs groups are shown. Mean and SEM are displayed for four independent donors. C) qRT-PCR analysis was done on total cellular RNA extracts. mRNA fold changes for the different treatment groups (n = 3) are graphed as mean and SEM. Significantly different groups (p

    Journal: PLoS Pathogens

    Article Title: Leishmania Induces Survival, Proliferation and Elevated Cellular dNTP Levels in Human Monocytes Promoting Acceleration of HIV Co-Infection

    doi: 10.1371/journal.ppat.1002635

    Figure Lengend Snippet: Western blot analysis of ribonucleotide reductase. A) Cellular lysates from different treatment groups were analyzed for ribonucleotide reductase R2 and p53R2 subunits. Afterwards, blots were stripped and re-probed for actin. Freshly isolated monocytes (Mo), day 13 maturated GM-CSF (GM-CSF) and Leishmania (Leish) MDMs are shown. B) Quantitiation of western blots was done. Freshly isolated monocytes were set to 1 and increases in R2 and p53R2 expression levels for GM-CSF- and Leishmania -maturated MDMs groups are shown. Mean and SEM are displayed for four independent donors. C) qRT-PCR analysis was done on total cellular RNA extracts. mRNA fold changes for the different treatment groups (n = 3) are graphed as mean and SEM. Significantly different groups (p

    Article Snippet: Template RNA was diluted to 80 ng/µl and 4 µl from each sample, mixed with Express One-Step SuperScript qRT-PCR reagents, was ran in triplicate using an Applied Biosystems 7300 Real Time thermocycler.

    Techniques: Western Blot, Isolation, Expressing, Quantitative RT-PCR

    The expression of osteogenesis-related genes was significantly upregulated. After culture with conditioned medium, the relative expressions of differentiation markers Runx2, ALP, and Osterix in MG-63 cells were upregulated. Gene expression was detected by qRT-PCR. The expression of proliferation marker cyclin-D1 was also upregulated. Expression of these genes after 40 minutes of LIPUS treatment was significantly higher than after 20 minutes of LIPUS treatment.

    Journal: Medicine

    Article Title: Low-intensity pulsed ultrasound promotes endothelial cell-mediated osteogenesis in a conditioned medium coculture system with osteoblasts

    doi: 10.1097/MD.0000000000008397

    Figure Lengend Snippet: The expression of osteogenesis-related genes was significantly upregulated. After culture with conditioned medium, the relative expressions of differentiation markers Runx2, ALP, and Osterix in MG-63 cells were upregulated. Gene expression was detected by qRT-PCR. The expression of proliferation marker cyclin-D1 was also upregulated. Expression of these genes after 40 minutes of LIPUS treatment was significantly higher than after 20 minutes of LIPUS treatment.

    Article Snippet: 2.4 RNA extraction and qRT-PCR analyses Trizol reagent (Invitrogen, CA) was used according to the manufacturer's protocol to extract MG-63 total RNA.

    Techniques: Expressing, ALP Assay, Quantitative RT-PCR, Marker

    Zika Virus Sampling and Sequencing in Central America and Mexico (A) Map of Central America and Mexico. Colored circles indicate the sampling locations of Zika virus sequences generated in this study, and the locations of publicly available sequences from Central America and Mexico. (B) The temporal and geographic distribution of Zika virus qRT-PCR-positive samples tested in this study. Samples are colored according to their sampling location. (C) Consensus genome coverage of the Zika virus sequences generated in this study. Sequences are colored according to their sampling location and the Zika virus genome structure is shown above the plot.

    Journal: Cell Host & Microbe

    Article Title: Genomic Epidemiology Reconstructs the Introduction and Spread of Zika Virus in Central America and Mexico

    doi: 10.1016/j.chom.2018.04.017

    Figure Lengend Snippet: Zika Virus Sampling and Sequencing in Central America and Mexico (A) Map of Central America and Mexico. Colored circles indicate the sampling locations of Zika virus sequences generated in this study, and the locations of publicly available sequences from Central America and Mexico. (B) The temporal and geographic distribution of Zika virus qRT-PCR-positive samples tested in this study. Samples are colored according to their sampling location. (C) Consensus genome coverage of the Zika virus sequences generated in this study. Sequences are colored according to their sampling location and the Zika virus genome structure is shown above the plot.

    Article Snippet: The ZCD assay qRT-PCR reactions were performed with primers and probes previously described by , using 25ìL master mix reactions of the SuperScript III Platinum One-Step qRT-PCR kit (Invitrogen) and 5 ìL of RNA.

    Techniques: Sampling, Sequencing, Generated, Quantitative RT-PCR

    Expression of SHBG by lymphocytic cells. Box-plots (whiskers: min to max) represent SHBG / Shbg mRNA expression levels, measured by qRT-PCR, in ( A ) human B cells (BL41), T cells (Jurkat), as well as in ( B ) mouse B cells (A20), T cells (IP12-7), splenocytes, spleen. Liver served as positive control in human and negative control in mouse. Relative expression level (−ΔCt with an arbitrary zero point) is denoted on the Y-axis. Three independent qRT-PCR experiments were performed in duplicates. Asterisks denote expression under the detection limit. ( C ) Representative Western blot images show SHBG protein in the cell lysate of human (BL41, Jurkat) and mouse (A20, IP12-7) lymphocytes. The two bands represent monomeric (~45 kDa) and dimeric (~90 kDa) forms of the protein.

    Journal: Scientific Reports

    Article Title: Sex hormone-binding globulin provides a novel entry pathway for estradiol and influences subsequent signaling in lymphocytes via membrane receptor

    doi: 10.1038/s41598-018-36882-3

    Figure Lengend Snippet: Expression of SHBG by lymphocytic cells. Box-plots (whiskers: min to max) represent SHBG / Shbg mRNA expression levels, measured by qRT-PCR, in ( A ) human B cells (BL41), T cells (Jurkat), as well as in ( B ) mouse B cells (A20), T cells (IP12-7), splenocytes, spleen. Liver served as positive control in human and negative control in mouse. Relative expression level (−ΔCt with an arbitrary zero point) is denoted on the Y-axis. Three independent qRT-PCR experiments were performed in duplicates. Asterisks denote expression under the detection limit. ( C ) Representative Western blot images show SHBG protein in the cell lysate of human (BL41, Jurkat) and mouse (A20, IP12-7) lymphocytes. The two bands represent monomeric (~45 kDa) and dimeric (~90 kDa) forms of the protein.

    Article Snippet: The cycle threshold (Ct) value of each reaction was obtained by the StepOne qRT-PCR analysis software (ThermoFisher Scientific-Applied Biosystems).

    Techniques: Expressing, Quantitative RT-PCR, Positive Control, Negative Control, Western Blot

    Rhythmic expression of DBP in the liver of female Gα i3 -/- mice is phase advanced ( A , B ) Quantitative real-time PCR analysis of rhythmic expression of DBP mRNA in the liver of female (A) and male (B) Gα i3 deficient mice as compared to wild-type control animals. Dbp transcript levels were normalized to the endogenous control Gapdh. Shown are 2 -ΔCt values. ( C , D ) Representative immunoblots of rhythmic expression of the DBP protein in the liver of female (C) and male (D) Gα i3 -deficient mice and wild-type controls. GAPDH and β-Actin were employed as loading controls. Relative protein levels (DBP/GAPDH) were determined by densitometric analysis using ImageJ software (lower panels in C and D). Mice were sacrificed and analyzed every six hours at the indicated time points (ZT 0 to ZT 18). Results are expressed as mean ± s.d. of six animals (mRNA) or four animals (immunoblot) analyzed per genotype and time point ( * p

    Journal: Oncotarget

    Article Title: Gαi3 signaling is associated with sexual dimorphic expression of the clock-controlled output gene Dbp in murine liver

    doi: 10.18632/oncotarget.25727

    Figure Lengend Snippet: Rhythmic expression of DBP in the liver of female Gα i3 -/- mice is phase advanced ( A , B ) Quantitative real-time PCR analysis of rhythmic expression of DBP mRNA in the liver of female (A) and male (B) Gα i3 deficient mice as compared to wild-type control animals. Dbp transcript levels were normalized to the endogenous control Gapdh. Shown are 2 -ΔCt values. ( C , D ) Representative immunoblots of rhythmic expression of the DBP protein in the liver of female (C) and male (D) Gα i3 -deficient mice and wild-type controls. GAPDH and β-Actin were employed as loading controls. Relative protein levels (DBP/GAPDH) were determined by densitometric analysis using ImageJ software (lower panels in C and D). Mice were sacrificed and analyzed every six hours at the indicated time points (ZT 0 to ZT 18). Results are expressed as mean ± s.d. of six animals (mRNA) or four animals (immunoblot) analyzed per genotype and time point ( * p

    Article Snippet: Relative quantification of mRNA was carried out using quantitative real-time PCR (qRT-PCR; 7500 Real-Time PCR System; Applied Biosystems) and specific TaqMan probes (Applied Biosystems) for core clock genes and clock regulated genes [ ].

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

    AMPK activation contributes to the induction of UCP2 and the down-regulation of MPC1. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 1 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) qRT-PCR analysis of UCP2 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) for 1 and 48 h or with Compound C (CC, 5 μM) for 1 h. (C) qRT-PCR analysis of MPC1 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) or with Compound C (CC, 5 μM), both for 1 h. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p

    Journal: PLoS ONE

    Article Title: Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

    doi: 10.1371/journal.pone.0144273

    Figure Lengend Snippet: AMPK activation contributes to the induction of UCP2 and the down-regulation of MPC1. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 1 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) qRT-PCR analysis of UCP2 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) for 1 and 48 h or with Compound C (CC, 5 μM) for 1 h. (C) qRT-PCR analysis of MPC1 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR (1 mM) or with Compound C (CC, 5 μM), both for 1 h. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p

    Article Snippet: To quantify mRNA levels, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Activation Assay, Western Blot, Negative Control, Quantitative RT-PCR, Expressing

    shGTPBP3 cells show increased antioxidant capacity. (A) Determination of the mitochondrial membrane potential by flow cytometry in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells with the fluorescent probe MitoTracker Red. NC cells treated for 30 min with sodium azide (SA) at 25 mM were included in the analysis as a positive control for the membrane potential drop. (B) Determination of oxygen consumption rate with a Clark-type oxygen electrode in shGTPBP3-1, shGTPBP3-2 and NC cells. (C) Determination of ROS by flow cytometry in shGTPBP3-1, shGTPBP3-2 and NC cells with hydroethidine. (D) Determination of ROS by flow cytometry in shGTPBP3-1, shGTPBP3-2 and NC cells treated (+) or not (-) for 2 h with 0.3 mM H 2 O 2 with dihydrorhodamine 123. (E) Measurements of antioxidant enzyme activities: Catalase, SOD (superoxide dismutase) and GSH-Px (glutathione peroxidase). Data in A, C, D and E are expressed as % of NC. (F) qRT-PCR analysis of Thioredoxin-1 , Thioredoxin-2 , Peroxiredoxin-3 , Peroxiredoxin-5 and Uncoupling protein-2 ( UCP2 ) mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells. (G) Western blot analysis of UCP2 in shGTPBP3-1, shGTPBP3-2 and NC cells. The filter was also probed with porin as a loading control. (H) Densitometric analysis of UCP2 normalized to loading control and represented as % of NC. All data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

    Journal: PLoS ONE

    Article Title: Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

    doi: 10.1371/journal.pone.0144273

    Figure Lengend Snippet: shGTPBP3 cells show increased antioxidant capacity. (A) Determination of the mitochondrial membrane potential by flow cytometry in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells with the fluorescent probe MitoTracker Red. NC cells treated for 30 min with sodium azide (SA) at 25 mM were included in the analysis as a positive control for the membrane potential drop. (B) Determination of oxygen consumption rate with a Clark-type oxygen electrode in shGTPBP3-1, shGTPBP3-2 and NC cells. (C) Determination of ROS by flow cytometry in shGTPBP3-1, shGTPBP3-2 and NC cells with hydroethidine. (D) Determination of ROS by flow cytometry in shGTPBP3-1, shGTPBP3-2 and NC cells treated (+) or not (-) for 2 h with 0.3 mM H 2 O 2 with dihydrorhodamine 123. (E) Measurements of antioxidant enzyme activities: Catalase, SOD (superoxide dismutase) and GSH-Px (glutathione peroxidase). Data in A, C, D and E are expressed as % of NC. (F) qRT-PCR analysis of Thioredoxin-1 , Thioredoxin-2 , Peroxiredoxin-3 , Peroxiredoxin-5 and Uncoupling protein-2 ( UCP2 ) mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells. (G) Western blot analysis of UCP2 in shGTPBP3-1, shGTPBP3-2 and NC cells. The filter was also probed with porin as a loading control. (H) Densitometric analysis of UCP2 normalized to loading control and represented as % of NC. All data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

    Article Snippet: To quantify mRNA levels, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Flow Cytometry, Cytometry, Negative Control, Positive Control, Quantitative RT-PCR, Expressing, Western Blot

    Stable knock-down of GTPBP3 disturbs Complex I assembly and reduces the expression of Complex I assembly factors NDUFAF3 and NDUFAF4 (A) Western blot analysis of OXPHOS subunits ND1, NDUFS3 and NDUFB8 (Complex I), SDHA (Complex II), COXII and COXIV (Complex IV), and β-subunit (Complex V) in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. The filter was also probed with porin as a loading control. (B) Densitometric analysis of OXPHOS subunits normalized to porin and represented as % of NC. (C) Representative Blue Native-PAGE of OXPHOS complexes in shGTPBP3-1, shGTPBP3-2 and NC cells. (D) Densitometric analysis of OXPHOS Complexes normalized to Complex-II (loading control) and represented as % of NC. (E) qRT-PCR analysis of C20ORF7 , NUBPL , NDUFAF3 and NDUFAF4 mRNA expression in shGTPBP3 and NC cells. All data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

    Journal: PLoS ONE

    Article Title: Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

    doi: 10.1371/journal.pone.0144273

    Figure Lengend Snippet: Stable knock-down of GTPBP3 disturbs Complex I assembly and reduces the expression of Complex I assembly factors NDUFAF3 and NDUFAF4 (A) Western blot analysis of OXPHOS subunits ND1, NDUFS3 and NDUFB8 (Complex I), SDHA (Complex II), COXII and COXIV (Complex IV), and β-subunit (Complex V) in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. The filter was also probed with porin as a loading control. (B) Densitometric analysis of OXPHOS subunits normalized to porin and represented as % of NC. (C) Representative Blue Native-PAGE of OXPHOS complexes in shGTPBP3-1, shGTPBP3-2 and NC cells. (D) Densitometric analysis of OXPHOS Complexes normalized to Complex-II (loading control) and represented as % of NC. (E) qRT-PCR analysis of C20ORF7 , NUBPL , NDUFAF3 and NDUFAF4 mRNA expression in shGTPBP3 and NC cells. All data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

    Article Snippet: To quantify mRNA levels, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Expressing, Western Blot, Negative Control, Blue Native PAGE, Quantitative RT-PCR

    Increased mRNA expression in shGTPBP3 cells of genes involved in glycolysis and fatty acid oxidation. qRT-PCR analysis of mRNA expression of genes related to: 1) glycolysis ( GLUT1 : glucose transporter 1, PKF1 : phosphofructokinase, and LDHA and LDHB : lactate dehydrogenase A and B, respectively), 2) fatty acid oxidation ( CPT1 : carnitine palmitoyltransferase I, LCAD : long-chain acyl-CoA dehydrogenase, and MCAD : medium-chain acyl-CoA dehydrogenase), and 3) glutaminolysis ( ASCT2 : glutamine/amino acid transporter 2, SN2 : glutamine/amino acid transporter system N, and GLS : glutaminase) in shGTPBP3-1, shGTPBP3-2 and NC cells. Data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

    Journal: PLoS ONE

    Article Title: Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

    doi: 10.1371/journal.pone.0144273

    Figure Lengend Snippet: Increased mRNA expression in shGTPBP3 cells of genes involved in glycolysis and fatty acid oxidation. qRT-PCR analysis of mRNA expression of genes related to: 1) glycolysis ( GLUT1 : glucose transporter 1, PKF1 : phosphofructokinase, and LDHA and LDHB : lactate dehydrogenase A and B, respectively), 2) fatty acid oxidation ( CPT1 : carnitine palmitoyltransferase I, LCAD : long-chain acyl-CoA dehydrogenase, and MCAD : medium-chain acyl-CoA dehydrogenase), and 3) glutaminolysis ( ASCT2 : glutamine/amino acid transporter 2, SN2 : glutamine/amino acid transporter system N, and GLS : glutaminase) in shGTPBP3-1, shGTPBP3-2 and NC cells. Data are the mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

    Article Snippet: To quantify mRNA levels, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Expressing, Quantitative RT-PCR

    Expression of GTPBP3 is down regulated in shGTPBP3 cells. (A) qRT-PCR analysis of GTPBP3 mRNA expression in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. (B) Western blot analysis of GTPBP3 protein in shGTPBP3-1, shGTPBP3-2 and NC cells, using porin as a loading control. Positions of molecular-mass markers (in kDa) are indicated on the left. The arrow denotes a non-specific band. (C) Densitometric analysis of GTPBP3 protein normalized to loading control and represented as % of NC. In A and C, mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at ***p

    Journal: PLoS ONE

    Article Title: Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

    doi: 10.1371/journal.pone.0144273

    Figure Lengend Snippet: Expression of GTPBP3 is down regulated in shGTPBP3 cells. (A) qRT-PCR analysis of GTPBP3 mRNA expression in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. (B) Western blot analysis of GTPBP3 protein in shGTPBP3-1, shGTPBP3-2 and NC cells, using porin as a loading control. Positions of molecular-mass markers (in kDa) are indicated on the left. The arrow denotes a non-specific band. (C) Densitometric analysis of GTPBP3 protein normalized to loading control and represented as % of NC. In A and C, mean ± SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at ***p

    Article Snippet: To quantify mRNA levels, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Expressing, Quantitative RT-PCR, Negative Control, Western Blot

    AMPK contributes to the down regulation of NDUFAF4. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 48 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) qRT-PCR analysis of NDUFAF4 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR or with Compound C (CC) as in (A). (C and D) Blue Native-PAGE analysis of Complex I in shGTPBP3-1, shGTPBP3-2 and NC cells treated (+) or not (-) with AICAR (C) or with Compound C (D) as in (A). (E) Densitometric analysis of Complex I normalized to the loading control and represented as % of NC. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p

    Journal: PLoS ONE

    Article Title: Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier

    doi: 10.1371/journal.pone.0144273

    Figure Lengend Snippet: AMPK contributes to the down regulation of NDUFAF4. (A) Western blot analysis of phospho-Thr172-AMPKα in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells treated (+) or not (-) with AMPKα activator AICAR (1 mM) for 48 h or with AMPKα inhibitor Compound C (5 μM) for 1 h. The filter was also probed with AMPKα. (B) qRT-PCR analysis of NDUFAF4 mRNA expression in shGTPBP3-1, shGTPBP3-2 and NC cells treated or not with AICAR or with Compound C (CC) as in (A). (C and D) Blue Native-PAGE analysis of Complex I in shGTPBP3-1, shGTPBP3-2 and NC cells treated (+) or not (-) with AICAR (C) or with Compound C (D) as in (A). (E) Densitometric analysis of Complex I normalized to the loading control and represented as % of NC. All data are the mean ± SEM of at least three independent biological replicates. Differences were found to be statistically significant at *p

    Article Snippet: To quantify mRNA levels, one-step qRT-PCRs were performed in an Applied Biosystems Step-One Real-Time PCR System.

    Techniques: Western Blot, Negative Control, Quantitative RT-PCR, Expressing, Blue Native PAGE