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    Name:
    Retro X qRT PCR Titration Kit
    Description:
    The Retro X qRT PCR Titration Kit provides a fast and simple method for measuring retrovirus titer The kits use a quick RNA purification step before quantifying the number of retroviral genome copies using qRT PCR and TB Green technologies
    Catalog Number:
    631453
    Price:
    None
    Size:
    200 Rxns
    Category:
    Titration kits Retrovirus Viral transduction Gene function
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    Structured Review

    TaKaRa qrt pcr kits
    Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by <t>ChIP-PCR.</t> b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e <t>qRT-PCR</t> and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P
    The Retro X qRT PCR Titration Kit provides a fast and simple method for measuring retrovirus titer The kits use a quick RNA purification step before quantifying the number of retroviral genome copies using qRT PCR and TB Green technologies
    https://www.bioz.com/result/qrt pcr kits/product/TaKaRa
    Average 94 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    qrt pcr kits - by Bioz Stars, 2020-08
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    1) Product Images from "Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver"

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0216-z

    Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P
    Figure Legend Snippet: Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P

    Techniques Used: Knock-Out, Mouse Assay, Small Interfering RNA, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Concentration Assay, Quantitative RT-PCR, Western Blot, Expressing, Infection

    Hcy induces hepatic autophagy in CBS +/- mice. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy (100 μmol/L) for 24 h. a The concentration of Hcy in plasma of mice was measured by automatic biochemical analyzer. b Contents of ALT and AST in serum of CBS +/- mice were analyzed using automatic biochemical analyzer. c Hematoxylin and eosin (H E) and Oil Red O staining of CBS +/- mice liver. d Transmission electron microscope (TEM) was used to analyse cell autophagy in liver tissues of CBS +/− mice. The arrows indicate the double-membrane vacuoles digesting organelles or cytosolic contents. e and f mRNA and protein expression of p62, BECN1, LC3 and Atg12 in the liver tissue of CBS +/− mice by qRT-PCR and western blot, respectively. g Hepatocytes were treated with different concentrations of Hcy (50–500 μmol/L) for 24 h before MTT assay. h The activities of AST and ALT in hepatocytes after treatment with Hcy were detected by ELISA. ( I ) Confocal fluorescent microscopy analysis of hepatocytes overexpressing mRFP-GFP-LC3, treated with 100 µmol/L Hcy for 24 h. Quantification of mean red and green fluorescent puncta of at least 10 cells per condition is shown. The efficiency of transfection was also shown. ( J ) and ( K ) mRNA and protein expression of p62, BECN1, LC3 and Atg12 in hepatic cells treated with 100 µmol/L L-Hcy by qRT-PCR and western blot. Densitometry analysis of the proteins was performed for each sample (mean ± s.d.). * P
    Figure Legend Snippet: Hcy induces hepatic autophagy in CBS +/- mice. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy (100 μmol/L) for 24 h. a The concentration of Hcy in plasma of mice was measured by automatic biochemical analyzer. b Contents of ALT and AST in serum of CBS +/- mice were analyzed using automatic biochemical analyzer. c Hematoxylin and eosin (H E) and Oil Red O staining of CBS +/- mice liver. d Transmission electron microscope (TEM) was used to analyse cell autophagy in liver tissues of CBS +/− mice. The arrows indicate the double-membrane vacuoles digesting organelles or cytosolic contents. e and f mRNA and protein expression of p62, BECN1, LC3 and Atg12 in the liver tissue of CBS +/− mice by qRT-PCR and western blot, respectively. g Hepatocytes were treated with different concentrations of Hcy (50–500 μmol/L) for 24 h before MTT assay. h The activities of AST and ALT in hepatocytes after treatment with Hcy were detected by ELISA. ( I ) Confocal fluorescent microscopy analysis of hepatocytes overexpressing mRFP-GFP-LC3, treated with 100 µmol/L Hcy for 24 h. Quantification of mean red and green fluorescent puncta of at least 10 cells per condition is shown. The efficiency of transfection was also shown. ( J ) and ( K ) mRNA and protein expression of p62, BECN1, LC3 and Atg12 in hepatic cells treated with 100 µmol/L L-Hcy by qRT-PCR and western blot. Densitometry analysis of the proteins was performed for each sample (mean ± s.d.). * P

    Techniques Used: Mouse Assay, Knock-Out, Concentration Assay, AST Assay, Staining, Transmission Assay, Microscopy, Transmission Electron Microscopy, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay, Enzyme-linked Immunosorbent Assay, Transfection

    Hcy promotes the autophagy of hepatocytes by downregulation of cystic fibrosis trans membrane conductance regulator ( CFTR ). Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy, VX-770 and CFTR(inh)-172, respectively, for 24 h. a , b qRT-PCR and western blot detected the mRNA and protein expression of CFTR in the liver of CBS +/- mice. c , d qRT-PCR and western blot detected the mRNA and protein expression of CFTR in HL-7702 cells treated with 100 μmol/L Hcy. e , f Effect of CFTR activation on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with VX-770. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. g , h Effect of CFTR inhibition on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with CFTR(inh)-172. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. i , j, k An adenoviral vector carrying either a scrambled sequence or CFTR plasmid infected hepatocytes and protein expression of CFTR was detected by western blot, and the effect of CFTR overexpression and activation on the ratio of LC3-II/I and the expression of autophagy related proteins p62, BECN1 and Atg12 was examined in hepatocytes infected with Ad-CFTR. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. Results represent the means ± s.d. of independent experiments. * P
    Figure Legend Snippet: Hcy promotes the autophagy of hepatocytes by downregulation of cystic fibrosis trans membrane conductance regulator ( CFTR ). Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy, VX-770 and CFTR(inh)-172, respectively, for 24 h. a , b qRT-PCR and western blot detected the mRNA and protein expression of CFTR in the liver of CBS +/- mice. c , d qRT-PCR and western blot detected the mRNA and protein expression of CFTR in HL-7702 cells treated with 100 μmol/L Hcy. e , f Effect of CFTR activation on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with VX-770. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. g , h Effect of CFTR inhibition on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with CFTR(inh)-172. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. i , j, k An adenoviral vector carrying either a scrambled sequence or CFTR plasmid infected hepatocytes and protein expression of CFTR was detected by western blot, and the effect of CFTR overexpression and activation on the ratio of LC3-II/I and the expression of autophagy related proteins p62, BECN1 and Atg12 was examined in hepatocytes infected with Ad-CFTR. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. Results represent the means ± s.d. of independent experiments. * P

    Techniques Used: Knock-Out, Mouse Assay, Quantitative RT-PCR, Western Blot, Expressing, Activation Assay, Inhibition, Plasmid Preparation, Sequencing, Infection, Over Expression

    Hcy induces liver autophagy by downregulation of CFTR expression via DNA methylation. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring DNMT1 small interfering RNA (si-DNMT1) respectively for 24 h. a A schematic drawing of the putative CpG islands in the 5’-flank region of CFTR . (B, C) The total methylation rate of CpG rich region (−572 bp/−262 bp) within the proximal promoter of the CFTR gene was detected by bisulfite-sequencing PCR (BSP) method in liver of CBS +/- mice and ( d , E ) HL-7702 cells. Black and white circles represent methylated and unmethylated CpGs respectively. f qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in HL-7702 cells treated with Hcy (100 μmol/L) or Hcy (100 μmol/L) plus 5-azacytidine (AZC) (10 μmol/L). g , h The mRNA and protein expression of DNMTs in liver tissues of mice were detected by qRT-PCR and western blot. i The mRNA and protein expression of DNMT1 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-DNMT1. j Bisulfite sequencing detected the DNA methylation of CFTR in HL-7702 cells infected with the adenovirus si-DNMT1. k Western blot was performed to detect the protein expression of autophagy related proteins p62, BECN1, LC3 and Atg12 in hepatocytes infected with adenovirus CFTR and adenovirus si-DNMT1. Mean ± s.d. of three experiments performed in triplicate. * P
    Figure Legend Snippet: Hcy induces liver autophagy by downregulation of CFTR expression via DNA methylation. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring DNMT1 small interfering RNA (si-DNMT1) respectively for 24 h. a A schematic drawing of the putative CpG islands in the 5’-flank region of CFTR . (B, C) The total methylation rate of CpG rich region (−572 bp/−262 bp) within the proximal promoter of the CFTR gene was detected by bisulfite-sequencing PCR (BSP) method in liver of CBS +/- mice and ( d , E ) HL-7702 cells. Black and white circles represent methylated and unmethylated CpGs respectively. f qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in HL-7702 cells treated with Hcy (100 μmol/L) or Hcy (100 μmol/L) plus 5-azacytidine (AZC) (10 μmol/L). g , h The mRNA and protein expression of DNMTs in liver tissues of mice were detected by qRT-PCR and western blot. i The mRNA and protein expression of DNMT1 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-DNMT1. j Bisulfite sequencing detected the DNA methylation of CFTR in HL-7702 cells infected with the adenovirus si-DNMT1. k Western blot was performed to detect the protein expression of autophagy related proteins p62, BECN1, LC3 and Atg12 in hepatocytes infected with adenovirus CFTR and adenovirus si-DNMT1. Mean ± s.d. of three experiments performed in triplicate. * P

    Techniques Used: Expressing, DNA Methylation Assay, Knock-Out, Mouse Assay, Small Interfering RNA, Methylation, Methylation Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Infection

    CFTR alters the levels of liver injury biomarker and autophagy in CBS +/ − mice. CBS +/− mice injected with lentivirus Lv-CFTR or Lv-GFP through the tail vein (see Materials and Methods). a CFTR mRNA and protein levels were determined by qRT-PCR and western blot respectively. Data are presented as mean ± s.d. relative to Lv-GFP-injected mice. b Circulating levels of ALT and AST were analyzed. Results are means ± s.d. ( n = 6). * P
    Figure Legend Snippet: CFTR alters the levels of liver injury biomarker and autophagy in CBS +/ − mice. CBS +/− mice injected with lentivirus Lv-CFTR or Lv-GFP through the tail vein (see Materials and Methods). a CFTR mRNA and protein levels were determined by qRT-PCR and western blot respectively. Data are presented as mean ± s.d. relative to Lv-GFP-injected mice. b Circulating levels of ALT and AST were analyzed. Results are means ± s.d. ( n = 6). * P

    Techniques Used: Biomarker Assay, Mouse Assay, Injection, Quantitative RT-PCR, Western Blot, AST Assay

    2) Product Images from "Long noncoding RNA NEAT1, regulated by LIN28B, promotes cell proliferation and migration through sponging miR-506 in high-grade serous ovarian cancer"

    Article Title: Long noncoding RNA NEAT1, regulated by LIN28B, promotes cell proliferation and migration through sponging miR-506 in high-grade serous ovarian cancer

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0908-z

    Knockdown of NEAT1 inhibits ovarian tumor growth in vivo. a NEAT1-knockdown stable cell lines were constructed, and the knockdown efficiency was tested by qRT-PCR. b Downregulation of NEAT1 expression attenuated tumor growth in nude mice. c , d The effect of NEAT1 on EOC tumor growth was evaluated based on the tumor volumes and tumor weights in the two groups. e An immunofluorescence assay was used to detect the Ki-67 expression levels in frozen sections. Scale bar is 50 μm. * P
    Figure Legend Snippet: Knockdown of NEAT1 inhibits ovarian tumor growth in vivo. a NEAT1-knockdown stable cell lines were constructed, and the knockdown efficiency was tested by qRT-PCR. b Downregulation of NEAT1 expression attenuated tumor growth in nude mice. c , d The effect of NEAT1 on EOC tumor growth was evaluated based on the tumor volumes and tumor weights in the two groups. e An immunofluorescence assay was used to detect the Ki-67 expression levels in frozen sections. Scale bar is 50 μm. * P

    Techniques Used: In Vivo, Stable Transfection, Construct, Quantitative RT-PCR, Expressing, Mouse Assay, Immunofluorescence

    NEAT1 is upregulated in HGSOC and is correlated with patient prognosis. a NEAT1 expression levels in HGSOC and unpaired normal ovarian tissues determined using the NEAT1 primer (which can detect both transcripts). b NEAT1 expression in HGSOC and normal tissues was determined using the NEAT1-2 primer (which can detect the long transcript). β-actin was used as an endogenous control to normalize the data. c A total of 75 HGSOC patients were divided into high- and low-expression groups based on the median expression value. d Kaplan–Meier analysis of overall survival in all patients with HGSOC according to NEAT1 expression. e Kaplan–Meier curves for PFS of patients based on NEAT1 expression. f NEAT1 expression was quantitated in seven EOC cell lines using qRT-PCR
    Figure Legend Snippet: NEAT1 is upregulated in HGSOC and is correlated with patient prognosis. a NEAT1 expression levels in HGSOC and unpaired normal ovarian tissues determined using the NEAT1 primer (which can detect both transcripts). b NEAT1 expression in HGSOC and normal tissues was determined using the NEAT1-2 primer (which can detect the long transcript). β-actin was used as an endogenous control to normalize the data. c A total of 75 HGSOC patients were divided into high- and low-expression groups based on the median expression value. d Kaplan–Meier analysis of overall survival in all patients with HGSOC according to NEAT1 expression. e Kaplan–Meier curves for PFS of patients based on NEAT1 expression. f NEAT1 expression was quantitated in seven EOC cell lines using qRT-PCR

    Techniques Used: Expressing, Quantitative RT-PCR

    Attenuation of NEAT1 expression inhibits cell proliferation, invasion, and migration in vitro. a The EOC cell lines OVCAR3 and A2780 were transfected with siRNAs, and the efficiency of knockdown was verified by qRT-PCR using two NEAT1 primers. b CCK-8 assays were performed to determine the proliferation of NEAT1-knockdown cells. c The cell colony formation ability of OVCAR3 and A2780 cells was assessed to determine the effects of NEAT1 knockdown on cell growth. d The cell invasion potential of the OVCAR3 and A2780 cells was assessed using a Transwell assay. Scale bar is 50 μm. e The cell migration ability was evaluated using a wound-healing assay; images of OVCAR3 and A2780 cells were taken at 0 and 36 h postscratch. Scale bar is 200 μm. f Western blotting analysis was used to determine the MMP2, MMP9, N-cadherin, and E-cadherin expression levels. Actin was used as a reference. The data are shown as the mean ± SD of three replicates; * P
    Figure Legend Snippet: Attenuation of NEAT1 expression inhibits cell proliferation, invasion, and migration in vitro. a The EOC cell lines OVCAR3 and A2780 were transfected with siRNAs, and the efficiency of knockdown was verified by qRT-PCR using two NEAT1 primers. b CCK-8 assays were performed to determine the proliferation of NEAT1-knockdown cells. c The cell colony formation ability of OVCAR3 and A2780 cells was assessed to determine the effects of NEAT1 knockdown on cell growth. d The cell invasion potential of the OVCAR3 and A2780 cells was assessed using a Transwell assay. Scale bar is 50 μm. e The cell migration ability was evaluated using a wound-healing assay; images of OVCAR3 and A2780 cells were taken at 0 and 36 h postscratch. Scale bar is 200 μm. f Western blotting analysis was used to determine the MMP2, MMP9, N-cadherin, and E-cadherin expression levels. Actin was used as a reference. The data are shown as the mean ± SD of three replicates; * P

    Techniques Used: Expressing, Migration, In Vitro, Transfection, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay, Wound Healing Assay, Western Blot

    LIN28B enhances the stability of NEAT1. a Immunoblotting to evaluate the LIN28B protein levels after NEAT1 downregulation in OVCAR3 and A2780 cells. b The qRT-PCR analysis of LIN28B interference in EOC cells. c The EOC cell lines A2780 and OVCAR3 were transfected with either LIN28B or a vector control, and LIN28B overexpression was verified by qRT-PCR. Relative RNA levels of NEAT1 in OVCAR3 and A2780 cells with LIN28B knockdown ( d ) or overexpression ( e ) based on qPCR. f The half-life of NEAT1 after treatment with 2.5 μM actinomycin D for the indicated times with LIN28B overexpression in A2780 cells. * P
    Figure Legend Snippet: LIN28B enhances the stability of NEAT1. a Immunoblotting to evaluate the LIN28B protein levels after NEAT1 downregulation in OVCAR3 and A2780 cells. b The qRT-PCR analysis of LIN28B interference in EOC cells. c The EOC cell lines A2780 and OVCAR3 were transfected with either LIN28B or a vector control, and LIN28B overexpression was verified by qRT-PCR. Relative RNA levels of NEAT1 in OVCAR3 and A2780 cells with LIN28B knockdown ( d ) or overexpression ( e ) based on qPCR. f The half-life of NEAT1 after treatment with 2.5 μM actinomycin D for the indicated times with LIN28B overexpression in A2780 cells. * P

    Techniques Used: Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression, Real-time Polymerase Chain Reaction

    3) Product Images from "Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver"

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0216-z

    Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P
    Figure Legend Snippet: Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P

    Techniques Used: Knock-Out, Mouse Assay, Small Interfering RNA, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Concentration Assay, Quantitative RT-PCR, Western Blot, Expressing, Infection

    Hcy induces hepatic autophagy in CBS +/- mice. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy (100 μmol/L) for 24 h. a The concentration of Hcy in plasma of mice was measured by automatic biochemical analyzer. b Contents of ALT and AST in serum of CBS +/- mice were analyzed using automatic biochemical analyzer. c Hematoxylin and eosin (H E) and Oil Red O staining of CBS +/- mice liver. d Transmission electron microscope (TEM) was used to analyse cell autophagy in liver tissues of CBS +/− mice. The arrows indicate the double-membrane vacuoles digesting organelles or cytosolic contents. e and f mRNA and protein expression of p62, BECN1, LC3 and Atg12 in the liver tissue of CBS +/− mice by qRT-PCR and western blot, respectively. g Hepatocytes were treated with different concentrations of Hcy (50–500 μmol/L) for 24 h before MTT assay. h The activities of AST and ALT in hepatocytes after treatment with Hcy were detected by ELISA. ( I ) Confocal fluorescent microscopy analysis of hepatocytes overexpressing mRFP-GFP-LC3, treated with 100 µmol/L Hcy for 24 h. Quantification of mean red and green fluorescent puncta of at least 10 cells per condition is shown. The efficiency of transfection was also shown. ( J ) and ( K ) mRNA and protein expression of p62, BECN1, LC3 and Atg12 in hepatic cells treated with 100 µmol/L L-Hcy by qRT-PCR and western blot. Densitometry analysis of the proteins was performed for each sample (mean ± s.d.). * P
    Figure Legend Snippet: Hcy induces hepatic autophagy in CBS +/- mice. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy (100 μmol/L) for 24 h. a The concentration of Hcy in plasma of mice was measured by automatic biochemical analyzer. b Contents of ALT and AST in serum of CBS +/- mice were analyzed using automatic biochemical analyzer. c Hematoxylin and eosin (H E) and Oil Red O staining of CBS +/- mice liver. d Transmission electron microscope (TEM) was used to analyse cell autophagy in liver tissues of CBS +/− mice. The arrows indicate the double-membrane vacuoles digesting organelles or cytosolic contents. e and f mRNA and protein expression of p62, BECN1, LC3 and Atg12 in the liver tissue of CBS +/− mice by qRT-PCR and western blot, respectively. g Hepatocytes were treated with different concentrations of Hcy (50–500 μmol/L) for 24 h before MTT assay. h The activities of AST and ALT in hepatocytes after treatment with Hcy were detected by ELISA. ( I ) Confocal fluorescent microscopy analysis of hepatocytes overexpressing mRFP-GFP-LC3, treated with 100 µmol/L Hcy for 24 h. Quantification of mean red and green fluorescent puncta of at least 10 cells per condition is shown. The efficiency of transfection was also shown. ( J ) and ( K ) mRNA and protein expression of p62, BECN1, LC3 and Atg12 in hepatic cells treated with 100 µmol/L L-Hcy by qRT-PCR and western blot. Densitometry analysis of the proteins was performed for each sample (mean ± s.d.). * P

    Techniques Used: Mouse Assay, Knock-Out, Concentration Assay, AST Assay, Staining, Transmission Assay, Microscopy, Transmission Electron Microscopy, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay, Enzyme-linked Immunosorbent Assay, Transfection

    Hcy promotes the autophagy of hepatocytes by downregulation of cystic fibrosis trans membrane conductance regulator ( CFTR ). Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy, VX-770 and CFTR(inh)-172, respectively, for 24 h. a , b qRT-PCR and western blot detected the mRNA and protein expression of CFTR in the liver of CBS +/- mice. c , d qRT-PCR and western blot detected the mRNA and protein expression of CFTR in HL-7702 cells treated with 100 μmol/L Hcy. e , f Effect of CFTR activation on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with VX-770. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. g , h Effect of CFTR inhibition on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with CFTR(inh)-172. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. i , j, k An adenoviral vector carrying either a scrambled sequence or CFTR plasmid infected hepatocytes and protein expression of CFTR was detected by western blot, and the effect of CFTR overexpression and activation on the ratio of LC3-II/I and the expression of autophagy related proteins p62, BECN1 and Atg12 was examined in hepatocytes infected with Ad-CFTR. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. Results represent the means ± s.d. of independent experiments. * P
    Figure Legend Snippet: Hcy promotes the autophagy of hepatocytes by downregulation of cystic fibrosis trans membrane conductance regulator ( CFTR ). Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy, VX-770 and CFTR(inh)-172, respectively, for 24 h. a , b qRT-PCR and western blot detected the mRNA and protein expression of CFTR in the liver of CBS +/- mice. c , d qRT-PCR and western blot detected the mRNA and protein expression of CFTR in HL-7702 cells treated with 100 μmol/L Hcy. e , f Effect of CFTR activation on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with VX-770. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. g , h Effect of CFTR inhibition on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with CFTR(inh)-172. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. i , j, k An adenoviral vector carrying either a scrambled sequence or CFTR plasmid infected hepatocytes and protein expression of CFTR was detected by western blot, and the effect of CFTR overexpression and activation on the ratio of LC3-II/I and the expression of autophagy related proteins p62, BECN1 and Atg12 was examined in hepatocytes infected with Ad-CFTR. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. Results represent the means ± s.d. of independent experiments. * P

    Techniques Used: Knock-Out, Mouse Assay, Quantitative RT-PCR, Western Blot, Expressing, Activation Assay, Inhibition, Plasmid Preparation, Sequencing, Infection, Over Expression

    Hcy induces liver autophagy by downregulation of CFTR expression via DNA methylation. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring DNMT1 small interfering RNA (si-DNMT1) respectively for 24 h. a A schematic drawing of the putative CpG islands in the 5’-flank region of CFTR . (B, C) The total methylation rate of CpG rich region (−572 bp/−262 bp) within the proximal promoter of the CFTR gene was detected by bisulfite-sequencing PCR (BSP) method in liver of CBS +/- mice and ( d , E ) HL-7702 cells. Black and white circles represent methylated and unmethylated CpGs respectively. f qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in HL-7702 cells treated with Hcy (100 μmol/L) or Hcy (100 μmol/L) plus 5-azacytidine (AZC) (10 μmol/L). g , h The mRNA and protein expression of DNMTs in liver tissues of mice were detected by qRT-PCR and western blot. i The mRNA and protein expression of DNMT1 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-DNMT1. j Bisulfite sequencing detected the DNA methylation of CFTR in HL-7702 cells infected with the adenovirus si-DNMT1. k Western blot was performed to detect the protein expression of autophagy related proteins p62, BECN1, LC3 and Atg12 in hepatocytes infected with adenovirus CFTR and adenovirus si-DNMT1. Mean ± s.d. of three experiments performed in triplicate. * P
    Figure Legend Snippet: Hcy induces liver autophagy by downregulation of CFTR expression via DNA methylation. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring DNMT1 small interfering RNA (si-DNMT1) respectively for 24 h. a A schematic drawing of the putative CpG islands in the 5’-flank region of CFTR . (B, C) The total methylation rate of CpG rich region (−572 bp/−262 bp) within the proximal promoter of the CFTR gene was detected by bisulfite-sequencing PCR (BSP) method in liver of CBS +/- mice and ( d , E ) HL-7702 cells. Black and white circles represent methylated and unmethylated CpGs respectively. f qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in HL-7702 cells treated with Hcy (100 μmol/L) or Hcy (100 μmol/L) plus 5-azacytidine (AZC) (10 μmol/L). g , h The mRNA and protein expression of DNMTs in liver tissues of mice were detected by qRT-PCR and western blot. i The mRNA and protein expression of DNMT1 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-DNMT1. j Bisulfite sequencing detected the DNA methylation of CFTR in HL-7702 cells infected with the adenovirus si-DNMT1. k Western blot was performed to detect the protein expression of autophagy related proteins p62, BECN1, LC3 and Atg12 in hepatocytes infected with adenovirus CFTR and adenovirus si-DNMT1. Mean ± s.d. of three experiments performed in triplicate. * P

    Techniques Used: Expressing, DNA Methylation Assay, Knock-Out, Mouse Assay, Small Interfering RNA, Methylation, Methylation Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Infection

    CFTR alters the levels of liver injury biomarker and autophagy in CBS +/ − mice. CBS +/− mice injected with lentivirus Lv-CFTR or Lv-GFP through the tail vein (see Materials and Methods). a CFTR mRNA and protein levels were determined by qRT-PCR and western blot respectively. Data are presented as mean ± s.d. relative to Lv-GFP-injected mice. b Circulating levels of ALT and AST were analyzed. Results are means ± s.d. ( n = 6). * P
    Figure Legend Snippet: CFTR alters the levels of liver injury biomarker and autophagy in CBS +/ − mice. CBS +/− mice injected with lentivirus Lv-CFTR or Lv-GFP through the tail vein (see Materials and Methods). a CFTR mRNA and protein levels were determined by qRT-PCR and western blot respectively. Data are presented as mean ± s.d. relative to Lv-GFP-injected mice. b Circulating levels of ALT and AST were analyzed. Results are means ± s.d. ( n = 6). * P

    Techniques Used: Biomarker Assay, Mouse Assay, Injection, Quantitative RT-PCR, Western Blot, AST Assay

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    Real-time Polymerase Chain Reaction:

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    Functional Assay:

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    Quantitative RT-PCR:

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    Cell Culture:

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    Titration:

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    TaKaRa mir x mirna qrt pcr sybr kit
    Expression of screened <t>miRNAs</t> in serum exosomes of STZ treated mice. Serum was sampled from ICR mice at different time after STZ injection. Fasting tail blood glucose ( A ) and serum insulin ( B ) levels were measured. The expression of 4 serum exosomal miRNAs selected from microarray miR-375-3p ( C ), miR-129-5p ( D ), miR-378a-3p ( E ), miR-382-5p ( F ) were analyzed using <t>qRT-PCR.</t> Values represent means ± SEM (n≥9 mice of each time point). (* P
    Mir X Mirna Qrt Pcr Sybr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GBP1 is a direct target of miR-199b-5p. ( A ) Supposed miRNA binding sites in GBP1 sequences. ( B ) Direct target sites as verified by dual-luciferase reporter gene assay. ( C ) GBP1 expression in cervical cancer cell lines (SW756, C-33A, SiHa, HeLa and CaSki) and normal human cervical epithelial cell line HcerEpic based on <t>qRT-PCR.</t> ( D ) Western Blot for protein level of GBP1 in HcerEpic, HeLa and C-33A cell lines. ( E ) GBP1 expression in cervical cancer tissues and normal tissues on TCGA dataset. ( F ) The correlation between miR-199b-5p and GBP1 on TCGA dataset. * P
    Qrt Pcr Reverse Transcription Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FOXO1, which is directly targeted by miR-21 in addition to PTEN, controls Bim expression at a transcriptional level in DLBCLs A , The expression levels of miR-21, FOXO1, and PTEN were evaluated in GCB-DLBCL and ABC-DLBCL cell lines using <t>qRT-PCR.</t> The bar graph shows the relative expression levels (2 −ddCt ) of miR-21, FOXO1, and PTEN in each cell line compared with those of OCI-Ly1 cells. B , SU-DHL4 and SU-DHL5 cells were transfected with a miR-21 inhibitor or a negative (scramble) inhibitor using Lipofectamine 2000, and the cells were harvested at 24 hours after transfection. The expression levels of FOXO1 and PTEN in cells that were transfected with the miR-21 inhibitor were compared with those transfected with the negative inhibitor using qRT-PCR. C , OCI-Ly10 cells were transfected with miR-21 mimics or negative (scramble) mimics, and the differences in the expression levels of FOXO1 and PTEN between the negative- and miR-21 mimic-cells were assessed using qRT-PCR. D , The FOXO1 3′-untranslated region (UTR)-luciferase reporter construct containing many miR-21 binding motifs (5′-AGCUUAU-3′) was co-transfected into SU-DHL-4 and SU-DHL5 cells together with a miR-21 mimic, negative mimic, miR-21 inhibitor or negative inhibitor. At 24 hours post-transfection, the supernatant fractions of the lysed cells were recovered, and the luciferase activities were determined using a luminometer. E , At 24 hours after transfection of the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells, or miR-21 mimics or negative mimics into OCI-Ly10 cells, Western blotting was performed to determine FOXO1 level in the nucleus of tumor cells. F , Changes in the expression of FOXO1 target genes, including p27, p21, FasL and Bim, were evaluated using qRT-PCR after inhibiting the expression of miR-21 in SU-DHL4 and SU-DHL5 cells. G , A Bim promoter luciferase-reporter construct containing many copies of the FOXO1 binding motif (5′-GTAAACAA-3′) was co-transfected with either a miR-21 inhibitor or negative inhibitor into SU-DHL4 or SU-DHL5 cells. At 24 hours post-transfection, the supernatant fractions of the lysed cells were recovered, and the luciferase activities were measured using a luminometer. The values presented in the histogram are the mean values ± SD. Statistically significant differences are indicated by * and **, which signify P
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    Expression of screened miRNAs in serum exosomes of STZ treated mice. Serum was sampled from ICR mice at different time after STZ injection. Fasting tail blood glucose ( A ) and serum insulin ( B ) levels were measured. The expression of 4 serum exosomal miRNAs selected from microarray miR-375-3p ( C ), miR-129-5p ( D ), miR-378a-3p ( E ), miR-382-5p ( F ) were analyzed using qRT-PCR. Values represent means ± SEM (n≥9 mice of each time point). (* P

    Journal: Aging (Albany NY)

    Article Title: Injury factors alter miRNAs profiles of exosomes derived from islets and circulation

    doi: 10.18632/aging.101689

    Figure Lengend Snippet: Expression of screened miRNAs in serum exosomes of STZ treated mice. Serum was sampled from ICR mice at different time after STZ injection. Fasting tail blood glucose ( A ) and serum insulin ( B ) levels were measured. The expression of 4 serum exosomal miRNAs selected from microarray miR-375-3p ( C ), miR-129-5p ( D ), miR-378a-3p ( E ), miR-382-5p ( F ) were analyzed using qRT-PCR. Values represent means ± SEM (n≥9 mice of each time point). (* P

    Article Snippet: Expression of miRNA was analyzed using MiR-X miRNA qRT-PCR SYBR Kit and One Step SYBR PrimeScript RT-PCR Kit, respectively (Clontech Laboratories, Inc., Mountain View, USA).

    Techniques: Expressing, Mouse Assay, Injection, Microarray, Quantitative RT-PCR

    Validation of exosomal miRNAs detected by microarray. Four miRNAs with top high FC values (FC > 1.5 in both STZ and cytokines treated groups) miR-375-3p ( A ), miR-129-5p ( B ), miR-378a-3p ( C ), miR-382-5p ( D ) were further verified by qRT-PCR in three independent samples. The data are presented as mean±SEM. (* P

    Journal: Aging (Albany NY)

    Article Title: Injury factors alter miRNAs profiles of exosomes derived from islets and circulation

    doi: 10.18632/aging.101689

    Figure Lengend Snippet: Validation of exosomal miRNAs detected by microarray. Four miRNAs with top high FC values (FC > 1.5 in both STZ and cytokines treated groups) miR-375-3p ( A ), miR-129-5p ( B ), miR-378a-3p ( C ), miR-382-5p ( D ) were further verified by qRT-PCR in three independent samples. The data are presented as mean±SEM. (* P

    Article Snippet: Expression of miRNA was analyzed using MiR-X miRNA qRT-PCR SYBR Kit and One Step SYBR PrimeScript RT-PCR Kit, respectively (Clontech Laboratories, Inc., Mountain View, USA).

    Techniques: Microarray, Quantitative RT-PCR

    Serum exosomal miRNA-375 and miRNA-129 expression in T1DM and T2DM patients with different β cell function. After standard mixed meal tolerance tests, the area under curve (AUC) of plasma glucose were measured ( A ), and the ratio of C-peptide AUC to glucose AUC were calculated to evaluate islet β cell function ( B ). Serum exosomal miRNA-375 ( E , F ) and miRNA-129 ( G , H ) expression in T1DM and T2DM patients with diverse diabetes course were detected by qRT-PCR. All measurements are mean ± SEM (n≥10 subjects of each group) (* P

    Journal: Aging (Albany NY)

    Article Title: Injury factors alter miRNAs profiles of exosomes derived from islets and circulation

    doi: 10.18632/aging.101689

    Figure Lengend Snippet: Serum exosomal miRNA-375 and miRNA-129 expression in T1DM and T2DM patients with different β cell function. After standard mixed meal tolerance tests, the area under curve (AUC) of plasma glucose were measured ( A ), and the ratio of C-peptide AUC to glucose AUC were calculated to evaluate islet β cell function ( B ). Serum exosomal miRNA-375 ( E , F ) and miRNA-129 ( G , H ) expression in T1DM and T2DM patients with diverse diabetes course were detected by qRT-PCR. All measurements are mean ± SEM (n≥10 subjects of each group) (* P

    Article Snippet: Expression of miRNA was analyzed using MiR-X miRNA qRT-PCR SYBR Kit and One Step SYBR PrimeScript RT-PCR Kit, respectively (Clontech Laboratories, Inc., Mountain View, USA).

    Techniques: Expressing, Cell Function Assay, Quantitative RT-PCR

    GBP1 is a direct target of miR-199b-5p. ( A ) Supposed miRNA binding sites in GBP1 sequences. ( B ) Direct target sites as verified by dual-luciferase reporter gene assay. ( C ) GBP1 expression in cervical cancer cell lines (SW756, C-33A, SiHa, HeLa and CaSki) and normal human cervical epithelial cell line HcerEpic based on qRT-PCR. ( D ) Western Blot for protein level of GBP1 in HcerEpic, HeLa and C-33A cell lines. ( E ) GBP1 expression in cervical cancer tissues and normal tissues on TCGA dataset. ( F ) The correlation between miR-199b-5p and GBP1 on TCGA dataset. * P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA LINC01783 Promotes the Progression of Cervical Cancer by Sponging miR-199b-5p to Mediate GBP1 Expression

    doi: 10.2147/CMAR.S230171

    Figure Lengend Snippet: GBP1 is a direct target of miR-199b-5p. ( A ) Supposed miRNA binding sites in GBP1 sequences. ( B ) Direct target sites as verified by dual-luciferase reporter gene assay. ( C ) GBP1 expression in cervical cancer cell lines (SW756, C-33A, SiHa, HeLa and CaSki) and normal human cervical epithelial cell line HcerEpic based on qRT-PCR. ( D ) Western Blot for protein level of GBP1 in HcerEpic, HeLa and C-33A cell lines. ( E ) GBP1 expression in cervical cancer tissues and normal tissues on TCGA dataset. ( F ) The correlation between miR-199b-5p and GBP1 on TCGA dataset. * P

    Article Snippet: RNA Extraction and qRT-PCR Reverse Transcription Kit (Takara, Tokyo, Japan) was utilized for reversely transcribing RNAs into cDNAs while 2−ΔΔCt method was used for RNA quantification via normalizing to GAPDH.

    Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Expressing, Quantitative RT-PCR, Western Blot

    LINC01783 is increased in cervical cancer. ( A ) LINC01783 expression in cervical cancer tissues and normal tissues on TCGA dataset. ( B ) Overall survival of cervical cancer patients stratified by LINC01783 expression based on TCGA dataset. ( C ) LINC01783 expression in cervical cancer cell lines (SW756, C-33A, SiHa, HeLa and CaSki) and normal human cervical epithelial cell line HcerEpic as determined by qRT-PCR. * P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA LINC01783 Promotes the Progression of Cervical Cancer by Sponging miR-199b-5p to Mediate GBP1 Expression

    doi: 10.2147/CMAR.S230171

    Figure Lengend Snippet: LINC01783 is increased in cervical cancer. ( A ) LINC01783 expression in cervical cancer tissues and normal tissues on TCGA dataset. ( B ) Overall survival of cervical cancer patients stratified by LINC01783 expression based on TCGA dataset. ( C ) LINC01783 expression in cervical cancer cell lines (SW756, C-33A, SiHa, HeLa and CaSki) and normal human cervical epithelial cell line HcerEpic as determined by qRT-PCR. * P

    Article Snippet: RNA Extraction and qRT-PCR Reverse Transcription Kit (Takara, Tokyo, Japan) was utilized for reversely transcribing RNAs into cDNAs while 2−ΔΔCt method was used for RNA quantification via normalizing to GAPDH.

    Techniques: Expressing, Quantitative RT-PCR

    Direct interaction of LINC01783 with miR-199b-5p. ( A ) Cytoplasmic and nuclear level of LINC01783 in HeLa and C-33A cells as determined by qRT-PCR. ( B ) MiR-199b-5p expression in cervical cancer cell lines (SW756, C-33A, SiHa, HeLa and CaSki) and normal human cervical epithelial cell line HcerEpic as indicated by qRT-PCR. ( C ) MiR-199b-5p expression in cervical cancer tissues and normal tissues on TCGA dataset. ( D ) The correlation between miR-199b-5p and LINC01783 on TCGA dataset. ( E ) Bioinformatics evidences of miR-199b-5p binding onto 3ʹ-UTR of LINC01783. ( F ) Dual-luciferase reporter gene assay in HeLa and C-33A cells post transfection with miR-NC or miR-199b-5p mimics. ( G ) Amount of LINC01783 and miR-199b-5p in HeLa and C-33A cells as detected by RIP experiments. * P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA LINC01783 Promotes the Progression of Cervical Cancer by Sponging miR-199b-5p to Mediate GBP1 Expression

    doi: 10.2147/CMAR.S230171

    Figure Lengend Snippet: Direct interaction of LINC01783 with miR-199b-5p. ( A ) Cytoplasmic and nuclear level of LINC01783 in HeLa and C-33A cells as determined by qRT-PCR. ( B ) MiR-199b-5p expression in cervical cancer cell lines (SW756, C-33A, SiHa, HeLa and CaSki) and normal human cervical epithelial cell line HcerEpic as indicated by qRT-PCR. ( C ) MiR-199b-5p expression in cervical cancer tissues and normal tissues on TCGA dataset. ( D ) The correlation between miR-199b-5p and LINC01783 on TCGA dataset. ( E ) Bioinformatics evidences of miR-199b-5p binding onto 3ʹ-UTR of LINC01783. ( F ) Dual-luciferase reporter gene assay in HeLa and C-33A cells post transfection with miR-NC or miR-199b-5p mimics. ( G ) Amount of LINC01783 and miR-199b-5p in HeLa and C-33A cells as detected by RIP experiments. * P

    Article Snippet: RNA Extraction and qRT-PCR Reverse Transcription Kit (Takara, Tokyo, Japan) was utilized for reversely transcribing RNAs into cDNAs while 2−ΔΔCt method was used for RNA quantification via normalizing to GAPDH.

    Techniques: Quantitative RT-PCR, Expressing, Binding Assay, Luciferase, Reporter Gene Assay, Transfection

    LINC01783/miR-199b-5p axis is critical for GBP1 expression. ( A ) Transfection of MiR-199b-5p inhibitor with or without LINC01783 siRNA into HeLa cells and qRT-PCR evaluation for RNA level of GBP1. ( B ) Western blot of GBP1 protein level after treatment of HeLa cells, GAPDH as the control. ( C ) Transfection of C-33A cells with miR-199b-5p mimics with or without LINC01783 overexpression plasmid and relative RNA levels of GBP1 as detected by qRT-PCR. ( D ) Relative protein level of GBP1 for transfection with miR-199b-5p mimics and reversion by LINC01783 expression plasmid. ( E ) Relative RNA level of GBP1 for transfection with LINC01783 MUT overexpression plasmid or LINC01783 WT overexpression plasmid. ( F ) Relative protein level of GBP1 for transfection with LINC01783 WT overexpression plasmid or LINC01783 MUT overexpression plasmid. ** P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA LINC01783 Promotes the Progression of Cervical Cancer by Sponging miR-199b-5p to Mediate GBP1 Expression

    doi: 10.2147/CMAR.S230171

    Figure Lengend Snippet: LINC01783/miR-199b-5p axis is critical for GBP1 expression. ( A ) Transfection of MiR-199b-5p inhibitor with or without LINC01783 siRNA into HeLa cells and qRT-PCR evaluation for RNA level of GBP1. ( B ) Western blot of GBP1 protein level after treatment of HeLa cells, GAPDH as the control. ( C ) Transfection of C-33A cells with miR-199b-5p mimics with or without LINC01783 overexpression plasmid and relative RNA levels of GBP1 as detected by qRT-PCR. ( D ) Relative protein level of GBP1 for transfection with miR-199b-5p mimics and reversion by LINC01783 expression plasmid. ( E ) Relative RNA level of GBP1 for transfection with LINC01783 MUT overexpression plasmid or LINC01783 WT overexpression plasmid. ( F ) Relative protein level of GBP1 for transfection with LINC01783 WT overexpression plasmid or LINC01783 MUT overexpression plasmid. ** P

    Article Snippet: RNA Extraction and qRT-PCR Reverse Transcription Kit (Takara, Tokyo, Japan) was utilized for reversely transcribing RNAs into cDNAs while 2−ΔΔCt method was used for RNA quantification via normalizing to GAPDH.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Over Expression, Plasmid Preparation

    FOXO1, which is directly targeted by miR-21 in addition to PTEN, controls Bim expression at a transcriptional level in DLBCLs A , The expression levels of miR-21, FOXO1, and PTEN were evaluated in GCB-DLBCL and ABC-DLBCL cell lines using qRT-PCR. The bar graph shows the relative expression levels (2 −ddCt ) of miR-21, FOXO1, and PTEN in each cell line compared with those of OCI-Ly1 cells. B , SU-DHL4 and SU-DHL5 cells were transfected with a miR-21 inhibitor or a negative (scramble) inhibitor using Lipofectamine 2000, and the cells were harvested at 24 hours after transfection. The expression levels of FOXO1 and PTEN in cells that were transfected with the miR-21 inhibitor were compared with those transfected with the negative inhibitor using qRT-PCR. C , OCI-Ly10 cells were transfected with miR-21 mimics or negative (scramble) mimics, and the differences in the expression levels of FOXO1 and PTEN between the negative- and miR-21 mimic-cells were assessed using qRT-PCR. D , The FOXO1 3′-untranslated region (UTR)-luciferase reporter construct containing many miR-21 binding motifs (5′-AGCUUAU-3′) was co-transfected into SU-DHL-4 and SU-DHL5 cells together with a miR-21 mimic, negative mimic, miR-21 inhibitor or negative inhibitor. At 24 hours post-transfection, the supernatant fractions of the lysed cells were recovered, and the luciferase activities were determined using a luminometer. E , At 24 hours after transfection of the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells, or miR-21 mimics or negative mimics into OCI-Ly10 cells, Western blotting was performed to determine FOXO1 level in the nucleus of tumor cells. F , Changes in the expression of FOXO1 target genes, including p27, p21, FasL and Bim, were evaluated using qRT-PCR after inhibiting the expression of miR-21 in SU-DHL4 and SU-DHL5 cells. G , A Bim promoter luciferase-reporter construct containing many copies of the FOXO1 binding motif (5′-GTAAACAA-3′) was co-transfected with either a miR-21 inhibitor or negative inhibitor into SU-DHL4 or SU-DHL5 cells. At 24 hours post-transfection, the supernatant fractions of the lysed cells were recovered, and the luciferase activities were measured using a luminometer. The values presented in the histogram are the mean values ± SD. Statistically significant differences are indicated by * and **, which signify P

    Journal: Oncotarget

    Article Title: MicroRNA-21 plays an oncogenic role by targeting FOXO1 and activating the PI3K/AKT pathway in diffuse large B-cell lymphoma

    doi:

    Figure Lengend Snippet: FOXO1, which is directly targeted by miR-21 in addition to PTEN, controls Bim expression at a transcriptional level in DLBCLs A , The expression levels of miR-21, FOXO1, and PTEN were evaluated in GCB-DLBCL and ABC-DLBCL cell lines using qRT-PCR. The bar graph shows the relative expression levels (2 −ddCt ) of miR-21, FOXO1, and PTEN in each cell line compared with those of OCI-Ly1 cells. B , SU-DHL4 and SU-DHL5 cells were transfected with a miR-21 inhibitor or a negative (scramble) inhibitor using Lipofectamine 2000, and the cells were harvested at 24 hours after transfection. The expression levels of FOXO1 and PTEN in cells that were transfected with the miR-21 inhibitor were compared with those transfected with the negative inhibitor using qRT-PCR. C , OCI-Ly10 cells were transfected with miR-21 mimics or negative (scramble) mimics, and the differences in the expression levels of FOXO1 and PTEN between the negative- and miR-21 mimic-cells were assessed using qRT-PCR. D , The FOXO1 3′-untranslated region (UTR)-luciferase reporter construct containing many miR-21 binding motifs (5′-AGCUUAU-3′) was co-transfected into SU-DHL-4 and SU-DHL5 cells together with a miR-21 mimic, negative mimic, miR-21 inhibitor or negative inhibitor. At 24 hours post-transfection, the supernatant fractions of the lysed cells were recovered, and the luciferase activities were determined using a luminometer. E , At 24 hours after transfection of the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells, or miR-21 mimics or negative mimics into OCI-Ly10 cells, Western blotting was performed to determine FOXO1 level in the nucleus of tumor cells. F , Changes in the expression of FOXO1 target genes, including p27, p21, FasL and Bim, were evaluated using qRT-PCR after inhibiting the expression of miR-21 in SU-DHL4 and SU-DHL5 cells. G , A Bim promoter luciferase-reporter construct containing many copies of the FOXO1 binding motif (5′-GTAAACAA-3′) was co-transfected with either a miR-21 inhibitor or negative inhibitor into SU-DHL4 or SU-DHL5 cells. At 24 hours post-transfection, the supernatant fractions of the lysed cells were recovered, and the luciferase activities were measured using a luminometer. The values presented in the histogram are the mean values ± SD. Statistically significant differences are indicated by * and **, which signify P

    Article Snippet: The miR-21 level was evaluated by qRT-PCR using Mir-X miRNA first strand synthesis and SYBR® qRT-PCR kits (Clontech Laboratories, Mountain View, CA, USA) with U6 snRNA as an internal control.

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Luciferase, Construct, Binding Assay, Western Blot

    Expression of miR-21, the miR-17-92 cluster and miR-155 in tumor tissues of DLBCL patients and their prognostic implications A , The levels of expression of miR-21, the miR-17-92 cluster and miR-155 were evaluated in the tumor tissues of DLBCL patients (n = 200) and in normal tonsils as a control (n = 11) using qRT-PCR. Box-and-whisker plots demonstrate the levels of expression of miR-21, the miR-17-92 cluster, and miR-155 as dCt (= Ct (miRNA) - Ct (U6) ) values in the DLBCL tissues compared with those in normal tonsils. A lower dCt implies a higher relative level of miRNA. The differences in the miRNA levels of the DLBCL and normal group were evaluated using Student's t-test. B , The relative expression levels of the miRNAs were calculated as ddCt (= dCt (case) - mean dCt (control) ). The diagrams show the correlation of the relative expression levels of miR-21, the miR-17-92 cluster and miR-155 determined using Pearson's correlation analysis (r, correlation coefficient). Kaplan-Meier curves obtained using the log-rank test show C - E , the overall survival and F - H , the progression-free survival of patients with DLBCLs with high vs. low relative expression levels of miR-21, the miR-17-92 cluster, and miR-155.

    Journal: Oncotarget

    Article Title: MicroRNA-21 plays an oncogenic role by targeting FOXO1 and activating the PI3K/AKT pathway in diffuse large B-cell lymphoma

    doi:

    Figure Lengend Snippet: Expression of miR-21, the miR-17-92 cluster and miR-155 in tumor tissues of DLBCL patients and their prognostic implications A , The levels of expression of miR-21, the miR-17-92 cluster and miR-155 were evaluated in the tumor tissues of DLBCL patients (n = 200) and in normal tonsils as a control (n = 11) using qRT-PCR. Box-and-whisker plots demonstrate the levels of expression of miR-21, the miR-17-92 cluster, and miR-155 as dCt (= Ct (miRNA) - Ct (U6) ) values in the DLBCL tissues compared with those in normal tonsils. A lower dCt implies a higher relative level of miRNA. The differences in the miRNA levels of the DLBCL and normal group were evaluated using Student's t-test. B , The relative expression levels of the miRNAs were calculated as ddCt (= dCt (case) - mean dCt (control) ). The diagrams show the correlation of the relative expression levels of miR-21, the miR-17-92 cluster and miR-155 determined using Pearson's correlation analysis (r, correlation coefficient). Kaplan-Meier curves obtained using the log-rank test show C - E , the overall survival and F - H , the progression-free survival of patients with DLBCLs with high vs. low relative expression levels of miR-21, the miR-17-92 cluster, and miR-155.

    Article Snippet: The miR-21 level was evaluated by qRT-PCR using Mir-X miRNA first strand synthesis and SYBR® qRT-PCR kits (Clontech Laboratories, Mountain View, CA, USA) with U6 snRNA as an internal control.

    Techniques: Expressing, Quantitative RT-PCR, Whisker Assay

    MiR-21 regulates the PI3K/AKT/mTOR/FOXO1 pathway and involved in the drug resistance and proliferation of DLBCL cells At 24 hours after transfection of A , the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells or B , miR-21 mimics or negative mimics into OCI-Ly10 cells, Western blotting was performed to determine the levels of phospho-AKT, AKT, phospho-p70S6K, p70S6K, phospho-FOXO1, FOXO1 and Bim. C , SU-DHL4, SU-DHL5, and OCI-Ly10 cells were treated with increasing doses of LY294002, a PI3K inhibitor. At 24 hours after incubation, Western blotting was performed to determine the levels of FOXO1 and Bim. D , OCI-Ly10 cells were treated with increasing doses of AS1842856, a functional inhibitor of FOXO1, and 2 hours after incubation, Western blotting was performed to determine the levels of phospho-p70S6K and p70S6K. At 24 hours after transfection of E , the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells, or F , miR-21 mimics or negative mimics into OCI-Ly10 cells, the expression levels of ABCB1 (MDR1) and ABCG2 were evaluated using qRT-PCR. G , SU-DHL4 and SU-DHL5 cells were treated with the miR-21 inhibitor or negative inhibitor for 24 hours and co-incubated with the efflux-blocking drug, vinblastine, for the last 30 minutes. The cells were then stained with DiOC 2 , and their drug efflux activity was analyzed. A decrease in the percentage of DiOC 2 -staining cells determined using FACS represents an increase in the population of cells with drug efflux activity. H , The effect of miR-21 inhibition on the doxorubicin-induced cytotoxicity in SU-DHL4 and SU-DHL5 cells was evaluated using the CCK8 assay. I , The effect of miR-21 overexpression on the doxorubicin-induced cytotoxicity in OCI-Ly10 cells was assessed using the CCK8 assay. J , The relative rates of cell proliferation of OCI-Ly10 cells treated with DMSO (control) and doxorubicin and/or the FOXO1 inhibitor (AS1842856) were determined using the CCK8 assay. The values presented in the histogram are the mean values ± SD. Statistically significant differences are indicated by *, ** and ***, which signify P

    Journal: Oncotarget

    Article Title: MicroRNA-21 plays an oncogenic role by targeting FOXO1 and activating the PI3K/AKT pathway in diffuse large B-cell lymphoma

    doi:

    Figure Lengend Snippet: MiR-21 regulates the PI3K/AKT/mTOR/FOXO1 pathway and involved in the drug resistance and proliferation of DLBCL cells At 24 hours after transfection of A , the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells or B , miR-21 mimics or negative mimics into OCI-Ly10 cells, Western blotting was performed to determine the levels of phospho-AKT, AKT, phospho-p70S6K, p70S6K, phospho-FOXO1, FOXO1 and Bim. C , SU-DHL4, SU-DHL5, and OCI-Ly10 cells were treated with increasing doses of LY294002, a PI3K inhibitor. At 24 hours after incubation, Western blotting was performed to determine the levels of FOXO1 and Bim. D , OCI-Ly10 cells were treated with increasing doses of AS1842856, a functional inhibitor of FOXO1, and 2 hours after incubation, Western blotting was performed to determine the levels of phospho-p70S6K and p70S6K. At 24 hours after transfection of E , the miR-21 inhibitor or negative inhibitor into SU-DHL4 and SU-DHL5 cells, or F , miR-21 mimics or negative mimics into OCI-Ly10 cells, the expression levels of ABCB1 (MDR1) and ABCG2 were evaluated using qRT-PCR. G , SU-DHL4 and SU-DHL5 cells were treated with the miR-21 inhibitor or negative inhibitor for 24 hours and co-incubated with the efflux-blocking drug, vinblastine, for the last 30 minutes. The cells were then stained with DiOC 2 , and their drug efflux activity was analyzed. A decrease in the percentage of DiOC 2 -staining cells determined using FACS represents an increase in the population of cells with drug efflux activity. H , The effect of miR-21 inhibition on the doxorubicin-induced cytotoxicity in SU-DHL4 and SU-DHL5 cells was evaluated using the CCK8 assay. I , The effect of miR-21 overexpression on the doxorubicin-induced cytotoxicity in OCI-Ly10 cells was assessed using the CCK8 assay. J , The relative rates of cell proliferation of OCI-Ly10 cells treated with DMSO (control) and doxorubicin and/or the FOXO1 inhibitor (AS1842856) were determined using the CCK8 assay. The values presented in the histogram are the mean values ± SD. Statistically significant differences are indicated by *, ** and ***, which signify P

    Article Snippet: The miR-21 level was evaluated by qRT-PCR using Mir-X miRNA first strand synthesis and SYBR® qRT-PCR kits (Clontech Laboratories, Mountain View, CA, USA) with U6 snRNA as an internal control.

    Techniques: Transfection, Western Blot, Incubation, Functional Assay, Expressing, Quantitative RT-PCR, Blocking Assay, Staining, Activity Assay, FACS, Inhibition, CCK-8 Assay, Over Expression

    QRT-PCR validation of lipase , RNA helicase , PP2C , H + -ATPase , WRKY , and 14-3-3 genes. Panels A and B show gene expressions in blue light and dark, respectively. Expression changes are presented as normalized fold changes between the test tissues and reference tissue (WT/MS) in corresponding B and D. Positive and negative values indicate up and down regulations of the gene expression, respectively. Twofold threshold was considered as a cutoff value for significant changes in the expression. Error bars represent standard errors of three technical replicates based on DMNRT (p = 0.05).

    Journal: PLoS ONE

    Article Title: DNA Methylation and Transcriptomic Changes in Response to Different Lights and Stresses in 7B-1 Male-Sterile Tomato

    doi: 10.1371/journal.pone.0121864

    Figure Lengend Snippet: QRT-PCR validation of lipase , RNA helicase , PP2C , H + -ATPase , WRKY , and 14-3-3 genes. Panels A and B show gene expressions in blue light and dark, respectively. Expression changes are presented as normalized fold changes between the test tissues and reference tissue (WT/MS) in corresponding B and D. Positive and negative values indicate up and down regulations of the gene expression, respectively. Twofold threshold was considered as a cutoff value for significant changes in the expression. Error bars represent standard errors of three technical replicates based on DMNRT (p = 0.05).

    Article Snippet: PCR conditions were set at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, and annealing/extension at 60°C for 20 s. Expressions of miRNAs and tasiRNAs were validated using the Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR kit (Clontech).

    Techniques: Quantitative RT-PCR, Expressing, Mass Spectrometry

    QRT-PCR validation of CRY1 , CRY2 , PHOT1 , PHOT2 , and HY5 genes in 7B-1 and WT seedlings in response to 5-azaC. Expression changes are presented as normalized fold changes between the test tissues and reference tissue (untreated WT). Positive and negative values indicate up and down regulations of the gene expression, respectively. Twofold threshold was considered as a cutoff value for significant changes in the expression. Error bars represent standard errors of three technical replicates based on DMNRT (p = 0.05).

    Journal: PLoS ONE

    Article Title: DNA Methylation and Transcriptomic Changes in Response to Different Lights and Stresses in 7B-1 Male-Sterile Tomato

    doi: 10.1371/journal.pone.0121864

    Figure Lengend Snippet: QRT-PCR validation of CRY1 , CRY2 , PHOT1 , PHOT2 , and HY5 genes in 7B-1 and WT seedlings in response to 5-azaC. Expression changes are presented as normalized fold changes between the test tissues and reference tissue (untreated WT). Positive and negative values indicate up and down regulations of the gene expression, respectively. Twofold threshold was considered as a cutoff value for significant changes in the expression. Error bars represent standard errors of three technical replicates based on DMNRT (p = 0.05).

    Article Snippet: PCR conditions were set at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, and annealing/extension at 60°C for 20 s. Expressions of miRNAs and tasiRNAs were validated using the Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR kit (Clontech).

    Techniques: Quantitative RT-PCR, Expressing

    QRT-PCR validation of miRNAs, tasiRNAs, and ARF s in 5-azaC-treated 7B-1 seedlings. Expression changes are presented as normalized fold changes between the test tissues and reference tissue (untreated 7B-1 ). Positive and negative values indicate up and down regulations of the gene expression, respectively. Twofold threshold was considered as a cutoff value for significant changes in the expression. Error bars represent standard errors of three technical replicates based on DMNRT (p = 0.05).

    Journal: PLoS ONE

    Article Title: DNA Methylation and Transcriptomic Changes in Response to Different Lights and Stresses in 7B-1 Male-Sterile Tomato

    doi: 10.1371/journal.pone.0121864

    Figure Lengend Snippet: QRT-PCR validation of miRNAs, tasiRNAs, and ARF s in 5-azaC-treated 7B-1 seedlings. Expression changes are presented as normalized fold changes between the test tissues and reference tissue (untreated 7B-1 ). Positive and negative values indicate up and down regulations of the gene expression, respectively. Twofold threshold was considered as a cutoff value for significant changes in the expression. Error bars represent standard errors of three technical replicates based on DMNRT (p = 0.05).

    Article Snippet: PCR conditions were set at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, and annealing/extension at 60°C for 20 s. Expressions of miRNAs and tasiRNAs were validated using the Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR kit (Clontech).

    Techniques: Quantitative RT-PCR, Expressing

    QRT-PCR validation of the differentially regulated genes in response to 5-azaC treatment. Expression changes are presented as normalized fold changes between the test tissues and reference tissue (untreated WT). Positive and negative values indicate up and down regulations of the gene expression, respectively. Twofold threshold was considered as a cutoff value for significant changes in the expression. Error bars represent standard errors of three technical replicates based on DMNRT (p = 0.05).

    Journal: PLoS ONE

    Article Title: DNA Methylation and Transcriptomic Changes in Response to Different Lights and Stresses in 7B-1 Male-Sterile Tomato

    doi: 10.1371/journal.pone.0121864

    Figure Lengend Snippet: QRT-PCR validation of the differentially regulated genes in response to 5-azaC treatment. Expression changes are presented as normalized fold changes between the test tissues and reference tissue (untreated WT). Positive and negative values indicate up and down regulations of the gene expression, respectively. Twofold threshold was considered as a cutoff value for significant changes in the expression. Error bars represent standard errors of three technical replicates based on DMNRT (p = 0.05).

    Article Snippet: PCR conditions were set at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, and annealing/extension at 60°C for 20 s. Expressions of miRNAs and tasiRNAs were validated using the Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR kit (Clontech).

    Techniques: Quantitative RT-PCR, Expressing