real time quantitative polymerase chain reaction qrt pcr total rna  (TaKaRa)

 
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    Advantage RT for PCR Kit
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    TaKaRa real time quantitative polymerase chain reaction qrt pcr total rna
    LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. ( a ) The mRNA expression of PCNA was detected by <t>qRT-PCR</t> in rats′ PAs tissues. ( b , c ) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ## p

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    1) Product Images from "Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension"

    Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension

    Journal: Respiratory Research

    doi: 10.1186/s12931-019-1018-x

    LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. ( a ) The mRNA expression of PCNA was detected by qRT-PCR in rats′ PAs tissues. ( b , c ) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ## p
    Figure Legend Snippet: LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. ( a ) The mRNA expression of PCNA was detected by qRT-PCR in rats′ PAs tissues. ( b , c ) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ## p

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

    Up-regulation of lncRNA CASC2 inhibited proliferation in hypoxia-induced PASMCs. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in PASMCs. ( b ) Cell number of PASMCs was counted by CyQuant cell proliferation assay kit. ( c ) The mRNA expression of PCNA was detected by qRT-PCR in PASMCs. ( d ) The protein expression of PCNA was detected by western blot analysis in PASMCs. ( e , f ) Cell proliferation status of PASMCs was assessed by EdU staining assay. ### p
    Figure Legend Snippet: Up-regulation of lncRNA CASC2 inhibited proliferation in hypoxia-induced PASMCs. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in PASMCs. ( b ) Cell number of PASMCs was counted by CyQuant cell proliferation assay kit. ( c ) The mRNA expression of PCNA was detected by qRT-PCR in PASMCs. ( d ) The protein expression of PCNA was detected by western blot analysis in PASMCs. ( e , f ) Cell proliferation status of PASMCs was assessed by EdU staining assay. ### p

    Techniques Used: Expressing, Quantitative RT-PCR, CyQUANT Assay, Proliferation Assay, Western Blot, Staining

    LncRNA CASC2 was down-regulated in hypoxia-induced rats and reversed the increase of mPAP and progression of right ventricular hypertrophy caused by hypoxia. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in rat pulmonary arterial tissues. ( b ) Systemic blood pressure (SBP) was assessed in normoxia and hypoxia-induced rats. ( c ) Mean pulmonary arterial pressure (mPAP) was detected in normoxia and hypoxia-induced rats. ( d ) The right ventricular hypertrophy index (RVI) was calculated in normoxia and hypoxia-induced rats. # p
    Figure Legend Snippet: LncRNA CASC2 was down-regulated in hypoxia-induced rats and reversed the increase of mPAP and progression of right ventricular hypertrophy caused by hypoxia. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in rat pulmonary arterial tissues. ( b ) Systemic blood pressure (SBP) was assessed in normoxia and hypoxia-induced rats. ( c ) Mean pulmonary arterial pressure (mPAP) was detected in normoxia and hypoxia-induced rats. ( d ) The right ventricular hypertrophy index (RVI) was calculated in normoxia and hypoxia-induced rats. # p

    Techniques Used: Expressing, Quantitative RT-PCR

    2) Product Images from "RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study"

    Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study

    Journal: Forensic sciences research

    doi: 10.1080/20961790.2017.1349639

    RT-qPCR validation of 11 DEGs. (A) The transcript expression fold changes measured by RNA-seq and RT-qPCR are indicated by white and black columns, respectively. (A and B) The expression levels of nine up-regulated genes, Fosb, Atf3, IL6, Cxcl1, Zfp36, Jun, Rasd1, Fos and Apold1, two down-regulated genes, Sfrp2 and Fcna, were all consistent with the RNA-seq data. (B) The regression equation between RNA-seq ( x ) and RT-qPCR ( y ) data (Log 2 Ratio) is y = 0.860 7 x − 0.268 9, while the R 2 is 0.978 3. (C) Those 11 DEGs showed no difference between abdominal skins of the injury group (RT-qPCR-A) and control group (RT-qPCR-B).
    Figure Legend Snippet: RT-qPCR validation of 11 DEGs. (A) The transcript expression fold changes measured by RNA-seq and RT-qPCR are indicated by white and black columns, respectively. (A and B) The expression levels of nine up-regulated genes, Fosb, Atf3, IL6, Cxcl1, Zfp36, Jun, Rasd1, Fos and Apold1, two down-regulated genes, Sfrp2 and Fcna, were all consistent with the RNA-seq data. (B) The regression equation between RNA-seq ( x ) and RT-qPCR ( y ) data (Log 2 Ratio) is y = 0.860 7 x − 0.268 9, while the R 2 is 0.978 3. (C) Those 11 DEGs showed no difference between abdominal skins of the injury group (RT-qPCR-A) and control group (RT-qPCR-B).

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

    RT-qPCR results in postmortem human skin wounds. In line with animal experiments, Cxcl1, Jun, Fos and IL6 RNA expression levels were significantly up-regulated in postmortem human skin wounds than in intact skins, while Sfrp2 was down-regulated.
    Figure Legend Snippet: RT-qPCR results in postmortem human skin wounds. In line with animal experiments, Cxcl1, Jun, Fos and IL6 RNA expression levels were significantly up-regulated in postmortem human skin wounds than in intact skins, while Sfrp2 was down-regulated.

    Techniques Used: Quantitative RT-PCR, RNA Expression

    3) Product Images from "Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis"

    Article Title: Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074955

    Mice have increased myocardial fibrosis after CVB3 infection. ( A ) Groups of 8 mice were infected with 10 TCID 50 dose of CVB3, the myocardial inflammation was shown by H E staining 4 wks after infection. Representative heart sections were shown for each week (magnification: 200×). Arrows indicate inflammatory cell infiltrate. ( B ) At 1,2,3,4 wks after CVB3 infection, the mRNA expression levels of matrix proteins (collagen I, MMP-1, MMP-3, TIMP-1) were determined by quantitative real time-PCR. Data are from one representative experiment of three performed ones and represent as the mean±SEM. ( C ) Picrosirius-red stained heart sections of CVB3-infected mice revealed increased fibrosis (red) (magnification: 200×). Arrows indicate myocardial interstitial and perivascular fibrosis. The severity of fibrosis was scored by collagen volume fractions and represent as mean ± SEM of three separate experiments. *, P
    Figure Legend Snippet: Mice have increased myocardial fibrosis after CVB3 infection. ( A ) Groups of 8 mice were infected with 10 TCID 50 dose of CVB3, the myocardial inflammation was shown by H E staining 4 wks after infection. Representative heart sections were shown for each week (magnification: 200×). Arrows indicate inflammatory cell infiltrate. ( B ) At 1,2,3,4 wks after CVB3 infection, the mRNA expression levels of matrix proteins (collagen I, MMP-1, MMP-3, TIMP-1) were determined by quantitative real time-PCR. Data are from one representative experiment of three performed ones and represent as the mean±SEM. ( C ) Picrosirius-red stained heart sections of CVB3-infected mice revealed increased fibrosis (red) (magnification: 200×). Arrows indicate myocardial interstitial and perivascular fibrosis. The severity of fibrosis was scored by collagen volume fractions and represent as mean ± SEM of three separate experiments. *, P

    Techniques Used: Mouse Assay, Infection, Staining, Expressing, Real-time Polymerase Chain Reaction

    Adoptive transfer of Tregs ameliorated cardiac fibrosis in CVB3-infected mice. ( A ) Groups of 5 mice were transferred with 1×10 6 Tregs per mouse one day before CVB3 challenge via tail vein. The peripheral Treg frequency at wk 1 was determined. Graph representative of three independent experiments. Relative numbers of Treg frequency are represent as the mean ±SEM. ( B ) RT-PCR measurement of cardiac gene expression levels of collagen I and TIMP-1, TIMP-1/MMP-1 and TIMP-1/MMP-3 4 wks after CVB3 infection. Data are from one representative experiment of three performed ones and represent the mean ±SEM. ( C ) Picrosirius-red stained heart sections at week 4 (magnification: 200×) were shown and the collagen volume fraction (CVF) was evaluated. Arrows indicate myocardial interstitial and perivascular fibrosis. Individual experiments were performed three times with similar results. *, P
    Figure Legend Snippet: Adoptive transfer of Tregs ameliorated cardiac fibrosis in CVB3-infected mice. ( A ) Groups of 5 mice were transferred with 1×10 6 Tregs per mouse one day before CVB3 challenge via tail vein. The peripheral Treg frequency at wk 1 was determined. Graph representative of three independent experiments. Relative numbers of Treg frequency are represent as the mean ±SEM. ( B ) RT-PCR measurement of cardiac gene expression levels of collagen I and TIMP-1, TIMP-1/MMP-1 and TIMP-1/MMP-3 4 wks after CVB3 infection. Data are from one representative experiment of three performed ones and represent the mean ±SEM. ( C ) Picrosirius-red stained heart sections at week 4 (magnification: 200×) were shown and the collagen volume fraction (CVF) was evaluated. Arrows indicate myocardial interstitial and perivascular fibrosis. Individual experiments were performed three times with similar results. *, P

    Techniques Used: Adoptive Transfer Assay, Infection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining

    Mice Depleted of Tregs had aggravated cardiac fibrosis. ( A ) Groups of 5 mice were treated with 8 doses of 0.1 mg anti-CD25 or isotype mAb via intraperitoneal injection during the course of CVB3 infection to deplete Tregs. The peripheral Treg frequency at wk 1 was determined to confirm the depletion effect. Graph representative of three independent experiments. Relative numbers of Treg frequency are represent as the mean ±SEM. ( B ) 4 wks after CVB3 infection, mRNA levels of collagen I, TIMP-1 and relative expression of TIMP-1/MMP-1 as well as TIMP-1/MMP-3 in the heart tissues were detected. Data are from one representative experiment of three performed ones and represent as the mean ±SEM. ( C ) Representative Picrosirius-red stained heart sections (magnification: 200×) and the collagen volume fraction (CVF) at week 4. Data represent the mean ± SEM. Arrows indicate myocardial interstitial and perivascular fibrosis. Individual experiments were performed three times with similar results. *, P
    Figure Legend Snippet: Mice Depleted of Tregs had aggravated cardiac fibrosis. ( A ) Groups of 5 mice were treated with 8 doses of 0.1 mg anti-CD25 or isotype mAb via intraperitoneal injection during the course of CVB3 infection to deplete Tregs. The peripheral Treg frequency at wk 1 was determined to confirm the depletion effect. Graph representative of three independent experiments. Relative numbers of Treg frequency are represent as the mean ±SEM. ( B ) 4 wks after CVB3 infection, mRNA levels of collagen I, TIMP-1 and relative expression of TIMP-1/MMP-1 as well as TIMP-1/MMP-3 in the heart tissues were detected. Data are from one representative experiment of three performed ones and represent as the mean ±SEM. ( C ) Representative Picrosirius-red stained heart sections (magnification: 200×) and the collagen volume fraction (CVF) at week 4. Data represent the mean ± SEM. Arrows indicate myocardial interstitial and perivascular fibrosis. Individual experiments were performed three times with similar results. *, P

    Techniques Used: Mouse Assay, Injection, Infection, Expressing, Staining

    4) Product Images from "Identification and Characterization of MicroRNAs by High Through-Put Sequencing in Mesenchymal Stem Cells and Bone Tissue from Mice of Age-Related Osteoporosis"

    Article Title: Identification and Characterization of MicroRNAs by High Through-Put Sequencing in Mesenchymal Stem Cells and Bone Tissue from Mice of Age-Related Osteoporosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071895

    miRNA expression validated by qRT-PCR in MSCs and bone. qPCR results were normalized to U6 snRNA expression levels. Values showed that these miRNAs were significantly different between 2m and 25m samples.
    Figure Legend Snippet: miRNA expression validated by qRT-PCR in MSCs and bone. qPCR results were normalized to U6 snRNA expression levels. Values showed that these miRNAs were significantly different between 2m and 25m samples.

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

    5) Product Images from "Suppressor of Cytokine Signaling (SOCS) Genes Are Silenced by DNA Hypermethylation and Histone Deacetylation and Regulate Response to Radiotherapy in Cervical Cancer Cells"

    Article Title: Suppressor of Cytokine Signaling (SOCS) Genes Are Silenced by DNA Hypermethylation and Histone Deacetylation and Regulate Response to Radiotherapy in Cervical Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123133

    Effect of DNA methylation inhibitor and DNMT1 gene knockdown on SOCS gene expression. CaSki (A,D), HeLa (B,E), and ME-180 (C,F) cells were treated with 10 μM of 5-Azacytosine (5-Aza) for 72 h (A-C) or infected with control lentivirus (shSCR) or lentivirus expressing shRNA targeting DNMT1 (shDNMT1) (D-F). Knock-down of DNMT1 and SOCS gene expression was examined with qRT-PCR. Asterisk (*), statistically significant ( p
    Figure Legend Snippet: Effect of DNA methylation inhibitor and DNMT1 gene knockdown on SOCS gene expression. CaSki (A,D), HeLa (B,E), and ME-180 (C,F) cells were treated with 10 μM of 5-Azacytosine (5-Aza) for 72 h (A-C) or infected with control lentivirus (shSCR) or lentivirus expressing shRNA targeting DNMT1 (shDNMT1) (D-F). Knock-down of DNMT1 and SOCS gene expression was examined with qRT-PCR. Asterisk (*), statistically significant ( p

    Techniques Used: DNA Methylation Assay, Expressing, Infection, shRNA, Quantitative RT-PCR

    DNA methylation analysis of SOCS gene promoter. (A) Methyl-specific PCR analysis. Bisulfite-treated genomic DNA was amplified with unmethylated (U) or methylated (M) DNA specific primers. (B-D) Bisulfite sequencing of SOCS1 (B), SOCS3 (C), and SOCS5 (D). Unmethylated CpG site in amplified promoter region was showed as an open circle and methylated CpG as a closed circle.
    Figure Legend Snippet: DNA methylation analysis of SOCS gene promoter. (A) Methyl-specific PCR analysis. Bisulfite-treated genomic DNA was amplified with unmethylated (U) or methylated (M) DNA specific primers. (B-D) Bisulfite sequencing of SOCS1 (B), SOCS3 (C), and SOCS5 (D). Unmethylated CpG site in amplified promoter region was showed as an open circle and methylated CpG as a closed circle.

    Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Amplification, Methylation, Methylation Sequencing

    6) Product Images from "Contribution of Eukaryotic-Type Serine/Threonine Kinase to Stress Response and Virulence of Streptococcus suis"

    Article Title: Contribution of Eukaryotic-Type Serine/Threonine Kinase to Stress Response and Virulence of Streptococcus suis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0091971

    Virulence gene expression of different strains in vitro . Total RNA was extracted from SS2-1, Δ stk and C Δ stk grown in THY medium at an OD600 of 0.6–0.8 and used for qRT-PCR analysis. The mRNA level of each gene was normalized to that of 16S rRNA . Results are shown as relative expression ratios compared to expression in the parental strain SS2-1. Data from three independent assays are presented as the means±SE. Differences between SS2-1 and Δ stk was determined by two-way ANOVA. (**p
    Figure Legend Snippet: Virulence gene expression of different strains in vitro . Total RNA was extracted from SS2-1, Δ stk and C Δ stk grown in THY medium at an OD600 of 0.6–0.8 and used for qRT-PCR analysis. The mRNA level of each gene was normalized to that of 16S rRNA . Results are shown as relative expression ratios compared to expression in the parental strain SS2-1. Data from three independent assays are presented as the means±SE. Differences between SS2-1 and Δ stk was determined by two-way ANOVA. (**p

    Techniques Used: Expressing, In Vitro, Quantitative RT-PCR

    7) Product Images from "HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway"

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1214-5

    Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p
    Figure Legend Snippet: Japanese encephalitis virus (JEV)-induced IL-1β production by activated microglia is regulated by HSP60. Upper panel depicts the qRT-PCR data. a–c JEV infection increases HSP60 both at RNA level ( a , b ) and protein level ( d , e ) in N9 cells and mice brains respectively. Protein levels of HSP60 in the Western blot were normalized with β-actin levels while transcript expression of HSP60 was normalized with GAPDH expression. c Effect of JEV infection on the transcript level of HSP60 was also assessed in FFPE human brain sections infected with JEV and were compared with the control brains. f , g JEV infection increases IL-1β secretion both in vitro ( f ) and in vivo ( g ) which were analyzed using ELISA. h , i HSP60 knockdown leads to decrease in the IL-1β secretion as assessed by ELISA in N9 cells ( h ) and mice brain lysate ( i ). Both qRT-PCR and ELISA were performed in triplicates for each experiment. Data represented as mean ± SD of three independent experiments ( n = 3). * p

    Techniques Used: Quantitative RT-PCR, Infection, Mouse Assay, Western Blot, Expressing, Formalin-fixed Paraffin-Embedded, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay

    HSP60 critically regulates microglial IL-1β production both in vitro and in vivo . Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene ( a–e ) while right panel shows the IL-1β ELISA ( f–j ). IL-1β treatment increases its own expression in vitro ( a ) and induces its own secretion also ( f ). Similarly, IL-1β expression was checked through qRT-PCR ( d ) and ELISA ( i ) in vivo. b , g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β ( b ) and its secretion from microglia ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as secreted levels of IL-1β ( h, j ) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments ( n = 3). * p
    Figure Legend Snippet: HSP60 critically regulates microglial IL-1β production both in vitro and in vivo . Expression of IL-1β gene and its secretion by activated microglia was checked by qRT-PCR and ELISA respectively. Left panel depicts the qRT-PCR analysis of IL-1β gene ( a–e ) while right panel shows the IL-1β ELISA ( f–j ). IL-1β treatment increases its own expression in vitro ( a ) and induces its own secretion also ( f ). Similarly, IL-1β expression was checked through qRT-PCR ( d ) and ELISA ( i ) in vivo. b , g HSP60 overexpression in microglia leads to increase in transcript level of IL-1β ( b ) and its secretion from microglia ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as secreted levels of IL-1β ( h, j ) was also observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH. Both qRT-PCR analysis and ELISA were performed in triplicates for each experiment. Data shown is representative of three independent experiments ( n = 3). * p

    Techniques Used: In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Over Expression

    Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. ** p
    Figure Legend Snippet: Expression of IL-1β and HSP60 increase in various human brain diseases. The levels of IL-1β and HSP60 gene expression were checked by qRT-PCR in frontal cortex of different neurological conditions and were compared with age-matched controls. For glioma, qRT-PCR was done with tissue sample and the expression of IL-1β and HSP60 were compared with that of control tissue. The transcript levels of the genes were normalized with the levels of GAPDH. The graph depicts pooled analysis of fold change in the levels of IL-1β and HSP60 in different brain diseases as compared with control brain. Data represented as mean ± SD from two different sets of experiments. The graph represents the pooled analysis of qRT-PCR data. ** p

    Techniques Used: Expressing, Quantitative RT-PCR

    HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene ( a – e ) whereas the right panel shows the Western blot analysis ( f–j ). IL-1β treatment increased NLRP3 expression in vitro on both transcript level ( a ) and protein level ( f ). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR ( d ) and Western blotting ( i ). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level ( b ) and protein level ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as protein levels ( h, j ) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. * p
    Figure Legend Snippet: HSP60 regulates the expression of NLRP3 after IL-1β treatment. The left panel depicts the qRT-PCR analysis of NLRP3 gene ( a – e ) whereas the right panel shows the Western blot analysis ( f–j ). IL-1β treatment increased NLRP3 expression in vitro on both transcript level ( a ) and protein level ( f ). Similarly, NLRP3 expression was checked in vivo also through qRT-PCR ( d ) and Western blotting ( i ). HSP60 overexpression in microglial cells leads to increase in NLRP3 transcript level ( b ) and protein level ( g ). Effect of HSP60 knockdown on transcript levels ( c, e ) as well as protein levels ( h, j ) were observed in vitro and in vivo, respectively. Normalization of the transcript level was done with GAPDH while β-actin was used for the normalization of Western blots. For quantitative real-time PCR, each experiment was performed in triplicates. Representative blots of three independent experiments are shown here. Bar graphs below the blots represent the quantification of protein levels. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, In Vitro, In Vivo, Over Expression, Real-time Polymerase Chain Reaction

    8) Product Images from "PR-10, defensin and cold dehydrin genes are among those over expressed in Oxytropis (Fabaceae) species adapted to the arctic"

    Article Title: PR-10, defensin and cold dehydrin genes are among those over expressed in Oxytropis (Fabaceae) species adapted to the arctic

    Journal: Functional & Integrative Genomics

    doi: 10.1007/s10142-011-0223-6

    Expression of selected genes compared in transcriptomes of two arctic Oxytropis species ( O. arctobia and O. maydelliana ) and two temperate species ( O. campestris subsp. johannensis and O. splendens ) under two climatic conditions. Real-time RT-PCR was used to measure gene expression in cDNA. Values on the Y -axis are the mean ratio of the selected gene relative to actin expression. Actin was used as a normalizing gene ±1 standard deviation of three biological replicates and two technical replicates. a Expression of arctic.contig47 (cold dehydrin); b arctic.contig61 (PR-10); c arctic.contig13/36 (PR-10); d temperate.contig98 (light harvesting protein I, lhcbI). Abbreviations: oa, O. arctobia ; om, O. maydelliana ; ocj, O. campestris subsp. johannensis ; os O. splendens . The white bars represent expression of plantlets from the arctic conditions, and the dark grey bars from the temperate conditions. Small letters a or b above bars denote values that are significantly different at the 0.05 level (one-way ANOVA-GLM with SNK)
    Figure Legend Snippet: Expression of selected genes compared in transcriptomes of two arctic Oxytropis species ( O. arctobia and O. maydelliana ) and two temperate species ( O. campestris subsp. johannensis and O. splendens ) under two climatic conditions. Real-time RT-PCR was used to measure gene expression in cDNA. Values on the Y -axis are the mean ratio of the selected gene relative to actin expression. Actin was used as a normalizing gene ±1 standard deviation of three biological replicates and two technical replicates. a Expression of arctic.contig47 (cold dehydrin); b arctic.contig61 (PR-10); c arctic.contig13/36 (PR-10); d temperate.contig98 (light harvesting protein I, lhcbI). Abbreviations: oa, O. arctobia ; om, O. maydelliana ; ocj, O. campestris subsp. johannensis ; os O. splendens . The white bars represent expression of plantlets from the arctic conditions, and the dark grey bars from the temperate conditions. Small letters a or b above bars denote values that are significantly different at the 0.05 level (one-way ANOVA-GLM with SNK)

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation

    9) Product Images from "Network-based approach to prediction and population-based validation of in silico drug repurposing"

    Article Title: Network-based approach to prediction and population-based validation of in silico drug repurposing

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05116-5

    Experimental validation of hydroxychloroquine’s likely mechanism-of-action in coronary artery disease (CAD). a A highlighted subnetwork shows the inferred mechanism-of-action for hydroxychloroquine’s protective effect in CAD by network analysis. A network analysis was designed to meet four criteria: (1) the shortest paths from the known drug targets (TLR7 and TLR9) in the human protein–protein interaction network; (2) the blood vessel-specific gene expression level based on RNA-seq data from Genotype-Tissue Expression database; (3) known CAD or cardiovascular disease (CVD) gene products (proteins); and (4) literature-reported in vitro and in vivo evidence. There are three proposed mechanisms: (i) ERK5 (encoded by MAPK7 ) activation prevents endothelial inflammation via inhibition of cell adhesion molecule expression (VCAM-1 and ICAM-1), (ii) suppression of pro-inflammatory cytokines (TNF-α and IL-1β), and (iii) improvement in endothelial dysfunction via enhanced nitric oxide production by endothelial nitric oxide synthase (NOS3). The node size scales show the blood vessel-specific expression level based on RNA-seq data from Genotype-Tissue Expression database (Methods section). b , d Endothelial cells were pretreated with various concentrations of hydroxychloroquine (HCQ, 10–50 µM) for 1 h prior to 24 h incubation with 5 ng/ml TNF-α. qRT-PCR was used to monitor gene expression of inflammatory genes ( b ) VCAM1 and IL1B ; and ( d ) NOS3 . Expression of the β-actin gene was used as an internal standard. VCAM1: no HCQ, no TNF, n = 8; TNF; n = 8; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 4; TNF+ 30 μM HCQ, n = 3; TNF+ 50 μM HCQ, n = 6. IL-1β and NOS3: no HCQ, no TNF, n = 9; TNF; n = 9; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 5; TNF+ 30 μM HCQ, n = 4; TNF+ 50 μM HCQ, n = 6. Error bars are standard deviations. *significantly different from TNF-α with no HCQ, p
    Figure Legend Snippet: Experimental validation of hydroxychloroquine’s likely mechanism-of-action in coronary artery disease (CAD). a A highlighted subnetwork shows the inferred mechanism-of-action for hydroxychloroquine’s protective effect in CAD by network analysis. A network analysis was designed to meet four criteria: (1) the shortest paths from the known drug targets (TLR7 and TLR9) in the human protein–protein interaction network; (2) the blood vessel-specific gene expression level based on RNA-seq data from Genotype-Tissue Expression database; (3) known CAD or cardiovascular disease (CVD) gene products (proteins); and (4) literature-reported in vitro and in vivo evidence. There are three proposed mechanisms: (i) ERK5 (encoded by MAPK7 ) activation prevents endothelial inflammation via inhibition of cell adhesion molecule expression (VCAM-1 and ICAM-1), (ii) suppression of pro-inflammatory cytokines (TNF-α and IL-1β), and (iii) improvement in endothelial dysfunction via enhanced nitric oxide production by endothelial nitric oxide synthase (NOS3). The node size scales show the blood vessel-specific expression level based on RNA-seq data from Genotype-Tissue Expression database (Methods section). b , d Endothelial cells were pretreated with various concentrations of hydroxychloroquine (HCQ, 10–50 µM) for 1 h prior to 24 h incubation with 5 ng/ml TNF-α. qRT-PCR was used to monitor gene expression of inflammatory genes ( b ) VCAM1 and IL1B ; and ( d ) NOS3 . Expression of the β-actin gene was used as an internal standard. VCAM1: no HCQ, no TNF, n = 8; TNF; n = 8; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 4; TNF+ 30 μM HCQ, n = 3; TNF+ 50 μM HCQ, n = 6. IL-1β and NOS3: no HCQ, no TNF, n = 9; TNF; n = 9; TNF+ 10 μM HCQ, n = 5; TNF+ 20 μM HCQ, n = 5; TNF+ 30 μM HCQ, n = 4; TNF+ 50 μM HCQ, n = 6. Error bars are standard deviations. *significantly different from TNF-α with no HCQ, p

    Techniques Used: Expressing, RNA Sequencing Assay, In Vitro, In Vivo, Activation Assay, Inhibition, Incubation, Quantitative RT-PCR

    10) Product Images from "Hypoxia-induced Suppression of c-Myc by HIF-2α in Human Pulmonary Endothelial Cells Attenuates TFAM Expression"

    Article Title: Hypoxia-induced Suppression of c-Myc by HIF-2α in Human Pulmonary Endothelial Cells Attenuates TFAM Expression

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2017.07.008

    Hypoxia suppresses TFAM expression. (A) Quantitative real-time PCR (qRT-PCR) with 0.5 μg RNA, using actin as an endogenous control, was performed to monitor TFAM (n=5) and PGC1β (n=3) gene expression after 24 h hypoxia. Shown are mean ± SD. (B) Western blot for TFAM and actin following 42 h hypoxia or normoxia. Graphs show the relative densitometries corrected for actin; mean ± SD (n=4) are plotted. (C) qRT-PCR with 12 μg RNA was used to monitor PGC1α gene expression in normoxia and hypoxia and to compare the Δ CT of PGC1α and PGC1β transcripts in normoxic conditions (normalized to actin) (n=4). *, significantly different (p
    Figure Legend Snippet: Hypoxia suppresses TFAM expression. (A) Quantitative real-time PCR (qRT-PCR) with 0.5 μg RNA, using actin as an endogenous control, was performed to monitor TFAM (n=5) and PGC1β (n=3) gene expression after 24 h hypoxia. Shown are mean ± SD. (B) Western blot for TFAM and actin following 42 h hypoxia or normoxia. Graphs show the relative densitometries corrected for actin; mean ± SD (n=4) are plotted. (C) qRT-PCR with 12 μg RNA was used to monitor PGC1α gene expression in normoxia and hypoxia and to compare the Δ CT of PGC1α and PGC1β transcripts in normoxic conditions (normalized to actin) (n=4). *, significantly different (p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    11) Product Images from "Molecular Characterization of Iron Binding Proteins from Glossina morsitans morsitans (Diptera: Glossinidae)"

    Article Title: Molecular Characterization of Iron Binding Proteins from Glossina morsitans morsitans (Diptera: Glossinidae)

    Journal: Insect biochemistry and molecular biology

    doi: 10.1016/j.ibmb.2006.09.003

    Transcript abundance of transferrin and ferritin during life stages and in isolated tissues of G. m. morsitans . ( A ). Northern blot analysis of G. m. morsitans RNA from early larva (EL), late larva (LL), early pupa (EP), late pupa (LP), and adult female fat body (A F ). ( B ). Tissue-specific expression of GmmTsf1, GmmFer1HCH , and GmmFer2LCH in adult male and female tsetse identified by semi-quantitative RT-PCR analysis. Amplification products were generated using cDNAs from midgut (MG), fat body (FB) and reproductive tissues (RT) as templates. ( C ). Northern blot analysis of GmmTsf1 transcript abundance in whole flies analyzed at 0, 6, 12, 24 and 48 hours after blood feeding. Flies were collected from points after their first blood meal post eclosion and acquisition of their second blood meal. The tsetse tubulin gene GmmTub was used in hybridizations as a loading control. ( D ). Quantification of GmmTsf1 expression levels. Transcript abundance for the Northern data in ( C ) was quantified and then normalized against the GmmTub loading control.
    Figure Legend Snippet: Transcript abundance of transferrin and ferritin during life stages and in isolated tissues of G. m. morsitans . ( A ). Northern blot analysis of G. m. morsitans RNA from early larva (EL), late larva (LL), early pupa (EP), late pupa (LP), and adult female fat body (A F ). ( B ). Tissue-specific expression of GmmTsf1, GmmFer1HCH , and GmmFer2LCH in adult male and female tsetse identified by semi-quantitative RT-PCR analysis. Amplification products were generated using cDNAs from midgut (MG), fat body (FB) and reproductive tissues (RT) as templates. ( C ). Northern blot analysis of GmmTsf1 transcript abundance in whole flies analyzed at 0, 6, 12, 24 and 48 hours after blood feeding. Flies were collected from points after their first blood meal post eclosion and acquisition of their second blood meal. The tsetse tubulin gene GmmTub was used in hybridizations as a loading control. ( D ). Quantification of GmmTsf1 expression levels. Transcript abundance for the Northern data in ( C ) was quantified and then normalized against the GmmTub loading control.

    Techniques Used: Isolation, Northern Blot, Expressing, Quantitative RT-PCR, Amplification, Generated

    12) Product Images from "Roles of N-Acetylglucosaminyltransferase III in Epithelial-to-Mesenchymal Transition Induced by Transforming Growth Factor ?1 (TGF-?1) in Epithelial Cell Lines *"

    Article Title: Roles of N-Acetylglucosaminyltransferase III in Epithelial-to-Mesenchymal Transition Induced by Transforming Growth Factor ?1 (TGF-?1) in Epithelial Cell Lines *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.262154

    Effects of TGF-β1 on cell morphology, E-cadherin expression, and changes in N -glycans in GE11 cells. GE11 cells were grown in 6-well (2 × 10 5 ) or bottom dishes (2 × 10 4 ) for 24 h and then replaced with fresh complete medium with or without TGF-β1 (5 ng/ml) for another 4 days of incubation. A, cell morphology of the indicated cells was photographed. Photographs were taken of living cells using a ×10 objective. Scale bar, 100 μm. B, E-cadherin expressed on the cell surface was stained with anti-E-cadherin primary antibody, followed by incubation with Alexa Fluor-conjugated secondary antibody. Scale bar, 25 μm. C, total expression levels of E-cadherin were analyzed using Western blotting. Cell lysates from those cells were immunoblotted with anti-E-cadherin antibody. D, equal amounts of cell lysate proteins (20 μg) were used as the enzymatic source for the examination of GnT-III activities. S, substrate; P, product. E, mRNA expression of GnT-III . Quantitative RT-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on an ABI StepOnePlus. The mean number of cycles to the threshold ( CT ) of fluorescence detection was calculated for each sample, and the results were normalized to the mean CT of GAPDH for each sample tested. The changes in N -glycans were detected by E4-PHA ( F ) and concanavalin A ( ConA ) ( G ) lectin blot. α-Tubulin was used as a load control. H, N -glycans of GE11 cells cultured under normal conditions and treated with ( bottom ) or without ( top ) TGF-β for 4 days were released with peptide: N -glycosidase F ( PNGaseF ), as described under “Experimental Procedures,” digested with sialidase, and subjected to reversed-phase HPLC. The elution times for those PA-bisected N -glycans were compared with standard PA- N -glycans.
    Figure Legend Snippet: Effects of TGF-β1 on cell morphology, E-cadherin expression, and changes in N -glycans in GE11 cells. GE11 cells were grown in 6-well (2 × 10 5 ) or bottom dishes (2 × 10 4 ) for 24 h and then replaced with fresh complete medium with or without TGF-β1 (5 ng/ml) for another 4 days of incubation. A, cell morphology of the indicated cells was photographed. Photographs were taken of living cells using a ×10 objective. Scale bar, 100 μm. B, E-cadherin expressed on the cell surface was stained with anti-E-cadherin primary antibody, followed by incubation with Alexa Fluor-conjugated secondary antibody. Scale bar, 25 μm. C, total expression levels of E-cadherin were analyzed using Western blotting. Cell lysates from those cells were immunoblotted with anti-E-cadherin antibody. D, equal amounts of cell lysate proteins (20 μg) were used as the enzymatic source for the examination of GnT-III activities. S, substrate; P, product. E, mRNA expression of GnT-III . Quantitative RT-PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on an ABI StepOnePlus. The mean number of cycles to the threshold ( CT ) of fluorescence detection was calculated for each sample, and the results were normalized to the mean CT of GAPDH for each sample tested. The changes in N -glycans were detected by E4-PHA ( F ) and concanavalin A ( ConA ) ( G ) lectin blot. α-Tubulin was used as a load control. H, N -glycans of GE11 cells cultured under normal conditions and treated with ( bottom ) or without ( top ) TGF-β for 4 days were released with peptide: N -glycosidase F ( PNGaseF ), as described under “Experimental Procedures,” digested with sialidase, and subjected to reversed-phase HPLC. The elution times for those PA-bisected N -glycans were compared with standard PA- N -glycans.

    Techniques Used: Expressing, Incubation, Staining, Western Blot, Quantitative RT-PCR, Fluorescence, SYBR Green Assay, Cell Culture, High Performance Liquid Chromatography

    13) Product Images from "Association Study of Genetic Variants in Autophagy Pathway and Risk of Non-syndromic Cleft Lip With or Without Cleft Palate"

    Article Title: Association Study of Genetic Variants in Autophagy Pathway and Risk of Non-syndromic Cleft Lip With or Without Cleft Palate

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.00576

    The rs2301104 alleles affect the enhancer activity and expression of HIF1A . (A) HEPM and (B) HOEC cells were transiently transfected with rs2301104 [G], rs2301104 [C], and HIF1A promoter with PGL3-basic luciferase reporter vector. pRL-SV40 was co-transfected into cells. The activity of luciferase was assayed 48h later. qRT-PCR analysis of HIF1A expression at 48 h after plasmids transfection in (C) HEPM and (D) HOEC cells. It was indicated that the expression of HIF1A was lower with C allele constructs, compared with G allele constructs. Results are shown as mean values with the standard deviation (SD) normalized to GAPDH. (E) HIF1A mRNA expression was detected by qRT-PCR in 68 NSCL/P lip tissue samples. The HIF1A expression was significantly lower in samples with GC/CC genotypes than those with GG genotypes. The results were normalized to GAPDH . The P -value was calculated with two-side t -test ( n = 6; * P
    Figure Legend Snippet: The rs2301104 alleles affect the enhancer activity and expression of HIF1A . (A) HEPM and (B) HOEC cells were transiently transfected with rs2301104 [G], rs2301104 [C], and HIF1A promoter with PGL3-basic luciferase reporter vector. pRL-SV40 was co-transfected into cells. The activity of luciferase was assayed 48h later. qRT-PCR analysis of HIF1A expression at 48 h after plasmids transfection in (C) HEPM and (D) HOEC cells. It was indicated that the expression of HIF1A was lower with C allele constructs, compared with G allele constructs. Results are shown as mean values with the standard deviation (SD) normalized to GAPDH. (E) HIF1A mRNA expression was detected by qRT-PCR in 68 NSCL/P lip tissue samples. The HIF1A expression was significantly lower in samples with GC/CC genotypes than those with GG genotypes. The results were normalized to GAPDH . The P -value was calculated with two-side t -test ( n = 6; * P

    Techniques Used: Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Quantitative RT-PCR, Construct, Standard Deviation

    14) Product Images from "Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice"

    Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014391

    Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative PCR analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic DNA (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.
    Figure Legend Snippet: Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative PCR analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic DNA (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.

    Techniques Used: Expressing, Transgenic Assay, Real-time Polymerase Chain Reaction, Isolation, Mouse Assay, Sequencing

    Identification of the tgε26 transgenic integration site. (A) Physical map of the genomic region between D17mit26-mit80-1 and D17mit 26-mit80-24. a–q: probes for Southern blot analysis. Details of these probes are listed in Supplementary 2 . 1–7: regions of quantitative genomic PCR analysis. S: SalI, E: EcoRI, A: AseI, K: KpnI, Hp: HpaI, Sc: SacI. (B–D) PFGE Southern blot analysis of SalI-digested WT and tgε26 +/+ genomic DNA using the indicated probes. (E) Sequence of Sstr5 -sequence-containing tgε26 +/+ genomic clones that also contained hCD3ε sequence (left). Physical map of the transgenic integration site at the Sstr5 locus (middle). H: HindIII. i: probe for Southern blot analysis. TS1F, TS1R, and CD3e9130F: primers for genomic PCR analysis. Genomic PCR analysis using TS1F/TS1R primers (lanes 1 and 2) and CDe9130F/TS1R primers (lanes 3 and 4) (right). (F, G) Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (H) Physical map of the transgenic integration site at the Metrn locus (left). N: NcoI. q: probe for Southern blot analysis. m7900, m8100, and CD3e23010F: primers for genomic PCR analysis. i-1R, i-1F, i-2R, and i-2F: primers for inverse and nested PCR amplification. Sequence of tgε26 +/+ genomic clones containing both Metrn and hCD3ε sequences (middle). Genomic PCR analysis using m7900/m8100 primers (lanes 1 and 2) and m7900/CD3e23010F primers (lanes 3 and 4) (right). (I, J) PFGE Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (K) Quantitative genomic PCR analysis of WT (open bars) and tgε26 +/+ (black bars) genomic DNA. Primers 1–7 are shown in (A). ct-1 and ct-2 are the genomic regions from chromosome 6 used as controls. Primer sequences are shown in Supplementary Table S3 . Data were normalized to ct-1 signals. Means and standard errors of 3 independent measurements are shown. (L) Configuration of the tgε26 allele.
    Figure Legend Snippet: Identification of the tgε26 transgenic integration site. (A) Physical map of the genomic region between D17mit26-mit80-1 and D17mit 26-mit80-24. a–q: probes for Southern blot analysis. Details of these probes are listed in Supplementary 2 . 1–7: regions of quantitative genomic PCR analysis. S: SalI, E: EcoRI, A: AseI, K: KpnI, Hp: HpaI, Sc: SacI. (B–D) PFGE Southern blot analysis of SalI-digested WT and tgε26 +/+ genomic DNA using the indicated probes. (E) Sequence of Sstr5 -sequence-containing tgε26 +/+ genomic clones that also contained hCD3ε sequence (left). Physical map of the transgenic integration site at the Sstr5 locus (middle). H: HindIII. i: probe for Southern blot analysis. TS1F, TS1R, and CD3e9130F: primers for genomic PCR analysis. Genomic PCR analysis using TS1F/TS1R primers (lanes 1 and 2) and CDe9130F/TS1R primers (lanes 3 and 4) (right). (F, G) Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (H) Physical map of the transgenic integration site at the Metrn locus (left). N: NcoI. q: probe for Southern blot analysis. m7900, m8100, and CD3e23010F: primers for genomic PCR analysis. i-1R, i-1F, i-2R, and i-2F: primers for inverse and nested PCR amplification. Sequence of tgε26 +/+ genomic clones containing both Metrn and hCD3ε sequences (middle). Genomic PCR analysis using m7900/m8100 primers (lanes 1 and 2) and m7900/CD3e23010F primers (lanes 3 and 4) (right). (I, J) PFGE Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (K) Quantitative genomic PCR analysis of WT (open bars) and tgε26 +/+ (black bars) genomic DNA. Primers 1–7 are shown in (A). ct-1 and ct-2 are the genomic regions from chromosome 6 used as controls. Primer sequences are shown in Supplementary Table S3 . Data were normalized to ct-1 signals. Means and standard errors of 3 independent measurements are shown. (L) Configuration of the tgε26 allele.

    Techniques Used: Transgenic Assay, Southern Blot, Polymerase Chain Reaction, Sequencing, Clone Assay, Nested PCR, Amplification

    15) Product Images from "The positive feedback loop between ILF3 and lncRNA ILF3-AS1 promotes melanoma proliferation, migration, and invasion"

    Article Title: The positive feedback loop between ILF3 and lncRNA ILF3-AS1 promotes melanoma proliferation, migration, and invasion

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S186777

    ILF3-AS1 epigenetically activates ILF3 expression. Notes: ( A ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably overexpressed and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( B ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably depleted and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( C ) ILF3 mRNA levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by qRT-PCR. ( D ) ILF3 mRNA levels in ILF3-AS1 stably depleted and control A375 cells were determined by qRT-PCR. ( E ) ILF3 protein levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by Western blot. ( F ) ILF3 protein levels in ILF3-AS1 stably depleted and control A375 cells were determined by Western blot. Results are presented as mean ± SD based on at least three independent experiments. ** P
    Figure Legend Snippet: ILF3-AS1 epigenetically activates ILF3 expression. Notes: ( A ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably overexpressed and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( B ) The specific binding of EZH2 to ILF3 promoter and H3K27me3 levels at ILF3 promoter in ILF3-AS1 stably depleted and control A375 cells were determined by ChIP assays followed by qRT-PCR. ( C ) ILF3 mRNA levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by qRT-PCR. ( D ) ILF3 mRNA levels in ILF3-AS1 stably depleted and control A375 cells were determined by qRT-PCR. ( E ) ILF3 protein levels in ILF3-AS1 stably overexpressed and control A375 cells were determined by Western blot. ( F ) ILF3 protein levels in ILF3-AS1 stably depleted and control A375 cells were determined by Western blot. Results are presented as mean ± SD based on at least three independent experiments. ** P

    Techniques Used: Expressing, Binding Assay, Stable Transfection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Western Blot

    ILF3 is increased in melanoma and correlated with poor prognosis. Notes: ( A ) ILF3 expression levels in 37 benign nevi and 60 primary melanoma tissues were determined by qRT-PCR. Results are presented as median with IQR; P
    Figure Legend Snippet: ILF3 is increased in melanoma and correlated with poor prognosis. Notes: ( A ) ILF3 expression levels in 37 benign nevi and 60 primary melanoma tissues were determined by qRT-PCR. Results are presented as median with IQR; P

    Techniques Used: Expressing, Quantitative RT-PCR

    ILF3 physically binds and increases the stability of ILF3-AS1 transcript. Notes: ( A ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1 with ILF3-specific antibody compared with nonspecific IgG. β-Actin mRNA was used as a negative control. LincIN was used as a positive control. ( B ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, ILF3 protein levels were determined by Western blot. ( C ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, ILF3 protein levels were determined by Western blot. ( D ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, the cells were treated with 50 µM α-amanitin to block new RNA synthesis, and then, the stability of ILF3-AS1 transcript over time was determined by qRT-PCR. 18S rRNA, a product of RNA polymerase I that is unchanged by α-amanitin, was used as endogenous control. ( E ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, the cells were treated with 50 µM α-amanitin to block new RNA synthesis and, then, the stability of ILF3-AS1 transcript over time was determined by qRT-PCR. 18S rRNA, a product of RNA polymerase I that is unchanged by α-amanitin, was used as endogenous control. ( F ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, ILF3-AS1 transcript levels were determined by qRT-PCR. ( G ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, ILF3-AS1 transcript levels were determined by qRT-PCR. Results are presented as mean ± SD based on at least three independent experiments. ** P
    Figure Legend Snippet: ILF3 physically binds and increases the stability of ILF3-AS1 transcript. Notes: ( A ) RIP assays followed by qRT-PCR revealed the specific enrichment of ILF3-AS1 with ILF3-specific antibody compared with nonspecific IgG. β-Actin mRNA was used as a negative control. LincIN was used as a positive control. ( B ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, ILF3 protein levels were determined by Western blot. ( C ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, ILF3 protein levels were determined by Western blot. ( D ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, the cells were treated with 50 µM α-amanitin to block new RNA synthesis, and then, the stability of ILF3-AS1 transcript over time was determined by qRT-PCR. 18S rRNA, a product of RNA polymerase I that is unchanged by α-amanitin, was used as endogenous control. ( E ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, the cells were treated with 50 µM α-amanitin to block new RNA synthesis and, then, the stability of ILF3-AS1 transcript over time was determined by qRT-PCR. 18S rRNA, a product of RNA polymerase I that is unchanged by α-amanitin, was used as endogenous control. ( F ) Forty-eight hours after transiently transfecting ILF3 overexpression plasmid into SK-MEL-2 cells, ILF3-AS1 transcript levels were determined by qRT-PCR. ( G ) Forty-eight hours after transiently transfecting two independent ILF3-specific shRNAs into A375 cells, ILF3-AS1 transcript levels were determined by qRT-PCR. Results are presented as mean ± SD based on at least three independent experiments. ** P

    Techniques Used: Quantitative RT-PCR, Negative Control, Positive Control, Over Expression, Plasmid Preparation, Western Blot, Blocking Assay

    16) Product Images from "Interaction between Leptospiral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line, and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction"

    Article Title: Interaction between Leptospiral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line, and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction

    Journal: Asian-Australasian Journal of Animal Sciences

    doi: 10.5713/ajas.14.0440

    A relative mRNA expression ratio of TLR2 was quantified by qRT-PCR in LPS-induced PEFs_SV40 cells. (A) Cells (1×10 6 cells/well) were isolated after incubation with 0.1, 1.0, and 10.0 μg/mL concentrations of LPS at 3 h. (B) Cells were isolated at 3, 12, 24 h after incubation with 0.1 μg/mL LPS. Data shown is an average of n = 3 biological replicate±SD. Levels of expression and significance are relative to unstimulated cells (at each time point for time-dependent experiments). Asterisks indicate significant differences (* p
    Figure Legend Snippet: A relative mRNA expression ratio of TLR2 was quantified by qRT-PCR in LPS-induced PEFs_SV40 cells. (A) Cells (1×10 6 cells/well) were isolated after incubation with 0.1, 1.0, and 10.0 μg/mL concentrations of LPS at 3 h. (B) Cells were isolated at 3, 12, 24 h after incubation with 0.1 μg/mL LPS. Data shown is an average of n = 3 biological replicate±SD. Levels of expression and significance are relative to unstimulated cells (at each time point for time-dependent experiments). Asterisks indicate significant differences (* p

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Incubation

    A relative mRNA expression ratio of proinflammatory cytokines were quantified by qRT-PCR in LPS-induced PEFs_SV40 cells. (A) (B) IL-6 and IL-8 mRNA expression in PEFs_SV40 cells after incubation with 0.1, 1.0, and 10.0 μg/mL concentrations of LPS at 3 h. (C) (D) IL-6 and IL-8 mRNA expression in SV40 PEFs after incubation with 0.1 μg/mL LPS at 3, 12, and 24 h. Data shown is an average of n = 3 biological replicate±SD. Levels of expression and significance are relative to unstimulated cells (at each time point for time-dependent experiments). Asterisks indicate significant differences (* p
    Figure Legend Snippet: A relative mRNA expression ratio of proinflammatory cytokines were quantified by qRT-PCR in LPS-induced PEFs_SV40 cells. (A) (B) IL-6 and IL-8 mRNA expression in PEFs_SV40 cells after incubation with 0.1, 1.0, and 10.0 μg/mL concentrations of LPS at 3 h. (C) (D) IL-6 and IL-8 mRNA expression in SV40 PEFs after incubation with 0.1 μg/mL LPS at 3, 12, and 24 h. Data shown is an average of n = 3 biological replicate±SD. Levels of expression and significance are relative to unstimulated cells (at each time point for time-dependent experiments). Asterisks indicate significant differences (* p

    Techniques Used: Expressing, Quantitative RT-PCR, Incubation

    Detection of TLR4 mRNA expression on different kinds of pig cells by qRT-PCR analysis. Lane 1: pig peripheral blood mononuclear cells (PBMCs), lane 2: pig embryonic fibroblast cell line (PEFs_NCC1), lane 3: pig embryonic fibroblast cell line (PEFs_SV40), M: marker (100 bp DNA ladder). TLR4, toll-like receptor 4; qRT-PCR, quantitative real-time polymerase chain reaction.
    Figure Legend Snippet: Detection of TLR4 mRNA expression on different kinds of pig cells by qRT-PCR analysis. Lane 1: pig peripheral blood mononuclear cells (PBMCs), lane 2: pig embryonic fibroblast cell line (PEFs_NCC1), lane 3: pig embryonic fibroblast cell line (PEFs_SV40), M: marker (100 bp DNA ladder). TLR4, toll-like receptor 4; qRT-PCR, quantitative real-time polymerase chain reaction.

    Techniques Used: Expressing, Quantitative RT-PCR, Marker, Real-time Polymerase Chain Reaction

    17) Product Images from "Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling"

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    Journal: Bioscience Reports

    doi: 10.1042/BSR20170681

    The effect of SNHG20 knockdown on cell growth, apoptosis, and colony formation ( A ) The mRNA level of SNHG20 in ovarian cancer cells, which were transfected with siSNHG20, was detected, siNC acts as normal control. MTT assays showed SNHG20 knockdown inhibited cell proliferation of SKOV3 ( B ), OVCA429 ( C ), and OVCA433 ( D ) cells. ( E ) Flow cytometry analysis was performed for testing the effect of SNHG20 knockdown on the apoptosis of ovarian cancer cells. ( F ) Colony formation assay measuring colony formation of SNHG20 knockdown ovarian cancer cells. SNHG20 knockdown ovarian cancer cells were inoculated into nude mice and the tumor volume was detected every week. The growth curve of SKOV3 ( G ), OVCA429 ( H ), and OVCA433 ( I ) tumors were determined every week. Colony number was normalized to that obtained with cells transfected with siNC, which was set to 100%. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P
    Figure Legend Snippet: The effect of SNHG20 knockdown on cell growth, apoptosis, and colony formation ( A ) The mRNA level of SNHG20 in ovarian cancer cells, which were transfected with siSNHG20, was detected, siNC acts as normal control. MTT assays showed SNHG20 knockdown inhibited cell proliferation of SKOV3 ( B ), OVCA429 ( C ), and OVCA433 ( D ) cells. ( E ) Flow cytometry analysis was performed for testing the effect of SNHG20 knockdown on the apoptosis of ovarian cancer cells. ( F ) Colony formation assay measuring colony formation of SNHG20 knockdown ovarian cancer cells. SNHG20 knockdown ovarian cancer cells were inoculated into nude mice and the tumor volume was detected every week. The growth curve of SKOV3 ( G ), OVCA429 ( H ), and OVCA433 ( I ) tumors were determined every week. Colony number was normalized to that obtained with cells transfected with siNC, which was set to 100%. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Techniques Used: Transfection, MTT Assay, Flow Cytometry, Cytometry, Colony Assay, Mouse Assay, Quantitative RT-PCR

    LncRNA SNHG20 expression was increased in ovarian cancer ( A ) The expression of SNHG20 in 17 ovarian tumor samples and paired adjacent non-tumorous normal tissues was examined by q-PCR. ( B ) The expression in 13 metastatic ovarian tumor samples and paired primary tumor samples was also examined by q-PCR. ( C ) The expression of SNHG20 in HOSE and ovarian cancer cells was determined by q-PCR. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P
    Figure Legend Snippet: LncRNA SNHG20 expression was increased in ovarian cancer ( A ) The expression of SNHG20 in 17 ovarian tumor samples and paired adjacent non-tumorous normal tissues was examined by q-PCR. ( B ) The expression in 13 metastatic ovarian tumor samples and paired primary tumor samples was also examined by q-PCR. ( C ) The expression of SNHG20 in HOSE and ovarian cancer cells was determined by q-PCR. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Techniques Used: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

    The effect of SNHG20 overexpression on normal ovarian epithelial cell growth, apoptosis, and colony formation ( A ) HOSE cells were infected with lentivirus carrying SNHG20 and then the mRNA level was examined by q-PCR. MTT assays showed the effects of SNHG20 overexpression on HOSE cell growth ( B ), cell apoptosis ( C ), and colony formation ( D ). Colony number was normalized to that obtained from cells transfected with siNC, which was set to 100%. ( E ) SNHG20 overexpressing HOSE cells were inoculated into nude mice and the tumor volumes were evaluated every week. ( F ) The tumor weight was detected at the end point after we killed all groups of mice. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P
    Figure Legend Snippet: The effect of SNHG20 overexpression on normal ovarian epithelial cell growth, apoptosis, and colony formation ( A ) HOSE cells were infected with lentivirus carrying SNHG20 and then the mRNA level was examined by q-PCR. MTT assays showed the effects of SNHG20 overexpression on HOSE cell growth ( B ), cell apoptosis ( C ), and colony formation ( D ). Colony number was normalized to that obtained from cells transfected with siNC, which was set to 100%. ( E ) SNHG20 overexpressing HOSE cells were inoculated into nude mice and the tumor volumes were evaluated every week. ( F ) The tumor weight was detected at the end point after we killed all groups of mice. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Techniques Used: Over Expression, Infection, Polymerase Chain Reaction, MTT Assay, Transfection, Mouse Assay, Quantitative RT-PCR

    The effect of SNHG20 knockdown on Wnt/β-catenin signaling pathway in ovarian cancer cells Ovarian cancer cells were transfected with siSNHG20 or siNC, then these cells were subjected to q-PCR ( A ) and Western blot ( B ) for testing the mRNA and protein level of E-cadherin. ( C–E ) The mRNA level and protein level of β-catenin, GSK-3β, p-GSK-3β and several targets of Wnt/β-catenin signaling were examined by q-PCR and western blot in SKOV3 and OVCA429 cells. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P
    Figure Legend Snippet: The effect of SNHG20 knockdown on Wnt/β-catenin signaling pathway in ovarian cancer cells Ovarian cancer cells were transfected with siSNHG20 or siNC, then these cells were subjected to q-PCR ( A ) and Western blot ( B ) for testing the mRNA and protein level of E-cadherin. ( C–E ) The mRNA level and protein level of β-catenin, GSK-3β, p-GSK-3β and several targets of Wnt/β-catenin signaling were examined by q-PCR and western blot in SKOV3 and OVCA429 cells. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Techniques Used: Transfection, Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR

    18) Product Images from "Activation of NF-?B by the RANKL/RANK system up-regulates snail and twist expressions and induces epithelial-to-mesenchymal transition in mammary tumor cell lines"

    Article Title: Activation of NF-?B by the RANKL/RANK system up-regulates snail and twist expressions and induces epithelial-to-mesenchymal transition in mammary tumor cell lines

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-32-62

    RANKL enhances Snail and Twist expression and induces changes in the morphology of cells. (A) Analysis of cell morphology after cell treatment of with 100 ng/mL RANKL. RANKL induces changes in the epithelial morphology of 4T1, MCF-7, and NMuMG cells (×40 magnification). (B-D) Total RNA was extracted, and the mRNA expression levels of vimentin, E-cadherin, N-cadherin, Snail, Slug, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p
    Figure Legend Snippet: RANKL enhances Snail and Twist expression and induces changes in the morphology of cells. (A) Analysis of cell morphology after cell treatment of with 100 ng/mL RANKL. RANKL induces changes in the epithelial morphology of 4T1, MCF-7, and NMuMG cells (×40 magnification). (B-D) Total RNA was extracted, and the mRNA expression levels of vimentin, E-cadherin, N-cadherin, Snail, Slug, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effects of DMF on RANKL-induced EMT and EMT-related mRNA expression. (A) Analysis of 4T1 cell morphology after cell treatment of with 100 ng/mL RANKL or 100 μM DMF (× 40 magnification). (B) Total RNA was extracted, and the mRNA levels of vimentin, E-cadherin, N-cadherin, Snail, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p
    Figure Legend Snippet: Effects of DMF on RANKL-induced EMT and EMT-related mRNA expression. (A) Analysis of 4T1 cell morphology after cell treatment of with 100 ng/mL RANKL or 100 μM DMF (× 40 magnification). (B) Total RNA was extracted, and the mRNA levels of vimentin, E-cadherin, N-cadherin, Snail, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    19) Product Images from "Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo"

    Article Title: Lentivirus-mediated LIGHT overexpression inhibits human colorectal carcinoma cell growth in vitro and in vivo

    Journal: Oncology Letters

    doi: 10.3892/ol.2013.1505

    Effects of overexpressing LIGHT on the mRNA level of LIGHT. (A) Electrophoresis and semiquantitative analysis of PCR products. The mRNA level of LIGHT was higher in the HCT116/LIGHT cells compared with the HCT116/GFP and HCT116 cells. Lanes 1 and 4, HCT116; lanes 2 and 5, HCT116/GFP; lanes 3 and 6, HCT116/LIGHT; M, DL1000 DNA marker. (B) Statistical analysis of the mRNA level of LIGHT relative to that of GAPDH. LIGHT, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells.
    Figure Legend Snippet: Effects of overexpressing LIGHT on the mRNA level of LIGHT. (A) Electrophoresis and semiquantitative analysis of PCR products. The mRNA level of LIGHT was higher in the HCT116/LIGHT cells compared with the HCT116/GFP and HCT116 cells. Lanes 1 and 4, HCT116; lanes 2 and 5, HCT116/GFP; lanes 3 and 6, HCT116/LIGHT; M, DL1000 DNA marker. (B) Statistical analysis of the mRNA level of LIGHT relative to that of GAPDH. LIGHT, lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells.

    Techniques Used: Electrophoresis, Polymerase Chain Reaction, Marker, Binding Assay

    20) Product Images from "Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses"

    Article Title: Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-42400-w

    EXA1 homologs in tomato and rice complement the role of EXA1 in infection by PlAMV and PVX. ( a ) Expression analysis of SlEXA1 and OsEXA1 using RT-PCR in tomato and rice plants. Total RNA was extracted from tomato and rice leaves. DW: distilled water. ( b ) The accumulation of NbEXA1a , SlEXA1 , and OsEXA1 transcripts in N . benthamiana leaves inoculated with Agrobacterium carrying a plasmid encoding the genomic sequence of each EXA1 homolog and GUS . Nb18S was used as the internal control. ( c ) Functional complementation analysis of PlAMV-GFP (upper) and PVX-GFP (bottom) infection by the transient expression of NbEXA1a , SlEXA1 , or OsEXA1 in NbEXA1 -silenced (right) and control (left) plants. Accumulation of PlAMV-GFP or PVX-GFP co-expressed with each EXA1 homolog and GUS was observed under UV light at 4 dpi. ( d ) Quantification of the accumulation of PlAMV-GFP and PVX-GFP RNA in NbEXA1 -silenced and control plants using qRT-PCR. Total RNA extracted from the inoculated leaves at 4 dpi. Error bars represent SD of six (PlAMV-GFP) and three (PVX-GFP) samples. The mean level of viral RNA in control plants expressed with GUS was used as the standard (1.0), and scores for other conditions are shown above the bars. * P
    Figure Legend Snippet: EXA1 homologs in tomato and rice complement the role of EXA1 in infection by PlAMV and PVX. ( a ) Expression analysis of SlEXA1 and OsEXA1 using RT-PCR in tomato and rice plants. Total RNA was extracted from tomato and rice leaves. DW: distilled water. ( b ) The accumulation of NbEXA1a , SlEXA1 , and OsEXA1 transcripts in N . benthamiana leaves inoculated with Agrobacterium carrying a plasmid encoding the genomic sequence of each EXA1 homolog and GUS . Nb18S was used as the internal control. ( c ) Functional complementation analysis of PlAMV-GFP (upper) and PVX-GFP (bottom) infection by the transient expression of NbEXA1a , SlEXA1 , or OsEXA1 in NbEXA1 -silenced (right) and control (left) plants. Accumulation of PlAMV-GFP or PVX-GFP co-expressed with each EXA1 homolog and GUS was observed under UV light at 4 dpi. ( d ) Quantification of the accumulation of PlAMV-GFP and PVX-GFP RNA in NbEXA1 -silenced and control plants using qRT-PCR. Total RNA extracted from the inoculated leaves at 4 dpi. Error bars represent SD of six (PlAMV-GFP) and three (PVX-GFP) samples. The mean level of viral RNA in control plants expressed with GUS was used as the standard (1.0), and scores for other conditions are shown above the bars. * P

    Techniques Used: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Functional Assay, Quantitative RT-PCR

    Downregulation of SlEXA1 expression inhibits PepMV RNA accumulation in tomato. ( a ) Morphological phenotypes of control and SlEXA1 -silenced plants. Tomato seedlings were inoculated with TRV (control) and TRV- SlEXA1 ( SlEXA1 -silenced) by agroinfiltration. Photographs were taken from the top (upper) and side (bottom) of plants at 4 weeks post-inoculation. Un-inoculated healthy plants served as visual controls. ( b ) Quantification of the accumulation of PepMV RNA and SlEXA1 mRNA in control and SlEXA1 -silenced plants using qRT-PCR. Six control plants and six SlEXA1 -silenced plants were mechanically inoculated with PepMV. Total RNA extracted from PepMV-inoculated leaves at 5 dpi served as templates for qRT-PCR analysis of PepMV RNA and SlEXA1 mRNA. Error bars represent the standard error (SE) of six samples. The mean level of viral RNA or SlEXA1 mRNA in control plants was used as the standard (1.0), and scores for other conditions are shown above the bars. * P
    Figure Legend Snippet: Downregulation of SlEXA1 expression inhibits PepMV RNA accumulation in tomato. ( a ) Morphological phenotypes of control and SlEXA1 -silenced plants. Tomato seedlings were inoculated with TRV (control) and TRV- SlEXA1 ( SlEXA1 -silenced) by agroinfiltration. Photographs were taken from the top (upper) and side (bottom) of plants at 4 weeks post-inoculation. Un-inoculated healthy plants served as visual controls. ( b ) Quantification of the accumulation of PepMV RNA and SlEXA1 mRNA in control and SlEXA1 -silenced plants using qRT-PCR. Six control plants and six SlEXA1 -silenced plants were mechanically inoculated with PepMV. Total RNA extracted from PepMV-inoculated leaves at 5 dpi served as templates for qRT-PCR analysis of PepMV RNA and SlEXA1 mRNA. Error bars represent the standard error (SE) of six samples. The mean level of viral RNA or SlEXA1 mRNA in control plants was used as the standard (1.0), and scores for other conditions are shown above the bars. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P
    Figure Legend Snippet: Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P

    Techniques Used: Clone Assay, Functional Assay, Infection, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    Evaluation of the accumulation of viral RNA in NbEXA1 -silenced or control plants using qRT-PCR. Plants were mechanically inoculated with ( a ) potexviruses, ( b ) a lolavirus, ( c ) a carlavirus, and ( d ) a tobamovirus. Total RNA was extracted from inoculated leaves at 4 dpi for AltMV, CymMV, HdRSV, PepMV, PVM, and YoMV; 5 dpi for LoLV; and 8 dpi for WClMV. The mean level of viral RNA in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bars. Error bars represent SD of eight samples, except for PepMV (four samples). * P
    Figure Legend Snippet: Evaluation of the accumulation of viral RNA in NbEXA1 -silenced or control plants using qRT-PCR. Plants were mechanically inoculated with ( a ) potexviruses, ( b ) a lolavirus, ( c ) a carlavirus, and ( d ) a tobamovirus. Total RNA was extracted from inoculated leaves at 4 dpi for AltMV, CymMV, HdRSV, PepMV, PVM, and YoMV; 5 dpi for LoLV; and 8 dpi for WClMV. The mean level of viral RNA in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bars. Error bars represent SD of eight samples, except for PepMV (four samples). * P

    Techniques Used: Quantitative RT-PCR

    21) Product Images from "CircPTPRA acts as a tumor suppressor in bladder cancer by sponging miR-636 and upregulating KLF9"

    Article Title: CircPTPRA acts as a tumor suppressor in bladder cancer by sponging miR-636 and upregulating KLF9

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102530

    CircPTPRA acts as a sponge of miR-636. ( A ) Predicted miRNA target of circPTPRA identified on circInteractome and circBank. ( B , C ) Expression of circPTPRA detected by qRT-PCR analysis and gel electrophoresis of samples obtained by RNA pull-down assay. ( D , E ) Analysis of miRNAs bound to circPTPRA and control probes, measured by qRT-PCR. ( F ) Luciferase reporter assay results demonstrating the interaction between circPTPRA and miR-636. ( G ) Enrichment of circPTPRA captured by biotin-coupled miR-636 mimic or its mutant variant, as detected by qRT-PCR. ( H ) Co-localization of circPTPRA and miR-636 determined by FISH assay. Scale bar=50μm. Data are presented as the mean ± SD of three experiments. * P
    Figure Legend Snippet: CircPTPRA acts as a sponge of miR-636. ( A ) Predicted miRNA target of circPTPRA identified on circInteractome and circBank. ( B , C ) Expression of circPTPRA detected by qRT-PCR analysis and gel electrophoresis of samples obtained by RNA pull-down assay. ( D , E ) Analysis of miRNAs bound to circPTPRA and control probes, measured by qRT-PCR. ( F ) Luciferase reporter assay results demonstrating the interaction between circPTPRA and miR-636. ( G ) Enrichment of circPTPRA captured by biotin-coupled miR-636 mimic or its mutant variant, as detected by qRT-PCR. ( H ) Co-localization of circPTPRA and miR-636 determined by FISH assay. Scale bar=50μm. Data are presented as the mean ± SD of three experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Nucleic Acid Electrophoresis, Pull Down Assay, Luciferase, Reporter Assay, Mutagenesis, Variant Assay, Fluorescence In Situ Hybridization

    Characterization of circPTPRA in BC cell lines. ( A ) Expression of circPTPRA in normal SV-HUC-1 cells and two BC cell lines (T24 and UM-UC-3). ( B ) Gel electrophoresis of qRT-PCR products resulting from divergent and convergent primers. GAPDH was used as internal control. ( C ) Schematic diagram depicting the circPTPRA’s origin from exons 8 and 9 of the PTPRA gene. Sanger sequencing confirmed the back-splicing junction site (blue arrow). ( D , E ) Analysis of PTPRA mRNA and circPTPRA by qRT-PCR in BC cell lines after actinomycin D treatment. ( F ) PTPRA mRNA and circPTPRA levels measured by qRT-PCR after RNase R treatment in BC cell lines. ( G ) Cellular localization of circPTPRA in BC cell lines, as assessed by cytoplasmic and nuclear fractionation assay. ( H ) FISH assay of indicating the cellular distribution of circPTPRA in UM-UC-3 cells. Scale bar=50μm. Data are presented as mean ± SD. * P
    Figure Legend Snippet: Characterization of circPTPRA in BC cell lines. ( A ) Expression of circPTPRA in normal SV-HUC-1 cells and two BC cell lines (T24 and UM-UC-3). ( B ) Gel electrophoresis of qRT-PCR products resulting from divergent and convergent primers. GAPDH was used as internal control. ( C ) Schematic diagram depicting the circPTPRA’s origin from exons 8 and 9 of the PTPRA gene. Sanger sequencing confirmed the back-splicing junction site (blue arrow). ( D , E ) Analysis of PTPRA mRNA and circPTPRA by qRT-PCR in BC cell lines after actinomycin D treatment. ( F ) PTPRA mRNA and circPTPRA levels measured by qRT-PCR after RNase R treatment in BC cell lines. ( G ) Cellular localization of circPTPRA in BC cell lines, as assessed by cytoplasmic and nuclear fractionation assay. ( H ) FISH assay of indicating the cellular distribution of circPTPRA in UM-UC-3 cells. Scale bar=50μm. Data are presented as mean ± SD. * P

    Techniques Used: Expressing, Nucleic Acid Electrophoresis, Quantitative RT-PCR, Sequencing, Fractionation, Fluorescence In Situ Hybridization

    22) Product Images from "Transcription factor NF-Y is involved in differentiation of R7 photoreceptor cell in Drosophila"

    Article Title: Transcription factor NF-Y is involved in differentiation of R7 photoreceptor cell in Drosophila

    Journal: Biology Open

    doi: 10.1242/bio.2011013

    Knockdown of dNF-YA reduces sev mRNA levels in third instar larvae. dNF-YA mRNA and sev mRNA levels were measured by quantitative RT-PCR. mRNA for Rp49 was used as a negative control. Fold differences against the amplification with RNA samples from Canton S are shown with standard deviations from three independent preparations of RNA.
    Figure Legend Snippet: Knockdown of dNF-YA reduces sev mRNA levels in third instar larvae. dNF-YA mRNA and sev mRNA levels were measured by quantitative RT-PCR. mRNA for Rp49 was used as a negative control. Fold differences against the amplification with RNA samples from Canton S are shown with standard deviations from three independent preparations of RNA.

    Techniques Used: Quantitative RT-PCR, Negative Control, Amplification

    23) Product Images from "Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms"

    Article Title: Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007534

    siRNA (-2752-21) targets tomato SlLNR1 . (A) Schematic illustration of SlLNR1 structure and its associated siRNAs. The sense SlLNR1 , SlLNR1 (+), from the susceptible cultivar is an 1132-nt lncRNA, which is validated by RACE. Its anti-sense, SlLNR1 (-), is generated by sequencing a segmental RT-PCR (the 54 th bp of the 3’ end to the 1008 th ). The full sequence of SlLNR1 was shown in S4 Fig . The vertical bar indicate the target site of siRNA(-2752-21). The data derived from small RNA sequencing of TYLCV infected plants or mock was aligned to SlLNR1 and the number indicates the aligned siRNA reads. (B) Negative correlation of expression of siRNA(-2752-21) and SlLNR1 . SlLNR1 (+) or SlLNR1 (-) was expressed with siRNA(-2752-21) in N . benthamiana . The RNA sample was extracted at 48 h after agroinfiltration. The EF1a gene of N . benthamiana was used as a refernce. (C) SlLNR1 was down regulated in the pTRV2:IR inoculated susceptible plants but not in the resistant plants. The RNA sample was extracted at 15 days after pTRV2:IR and EV inoculated plants. The relative expression of SlLNR1 was measured by qRT-PCR and calculated in relation to EV inoculated plants according to the ΔΔ Ct method. The tomato actin gene was set as reference gene. Error bars represented SE of three biological replicates and significant differences by Student’s t test (*, p
    Figure Legend Snippet: siRNA (-2752-21) targets tomato SlLNR1 . (A) Schematic illustration of SlLNR1 structure and its associated siRNAs. The sense SlLNR1 , SlLNR1 (+), from the susceptible cultivar is an 1132-nt lncRNA, which is validated by RACE. Its anti-sense, SlLNR1 (-), is generated by sequencing a segmental RT-PCR (the 54 th bp of the 3’ end to the 1008 th ). The full sequence of SlLNR1 was shown in S4 Fig . The vertical bar indicate the target site of siRNA(-2752-21). The data derived from small RNA sequencing of TYLCV infected plants or mock was aligned to SlLNR1 and the number indicates the aligned siRNA reads. (B) Negative correlation of expression of siRNA(-2752-21) and SlLNR1 . SlLNR1 (+) or SlLNR1 (-) was expressed with siRNA(-2752-21) in N . benthamiana . The RNA sample was extracted at 48 h after agroinfiltration. The EF1a gene of N . benthamiana was used as a refernce. (C) SlLNR1 was down regulated in the pTRV2:IR inoculated susceptible plants but not in the resistant plants. The RNA sample was extracted at 15 days after pTRV2:IR and EV inoculated plants. The relative expression of SlLNR1 was measured by qRT-PCR and calculated in relation to EV inoculated plants according to the ΔΔ Ct method. The tomato actin gene was set as reference gene. Error bars represented SE of three biological replicates and significant differences by Student’s t test (*, p

    Techniques Used: Generated, Sequencing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, RNA Sequencing Assay, Infection, Expressing, Quantitative RT-PCR

    Identification of a 25-nt segment and a vsRNA that induce stunt and curled leaves in tomato. (A) VsRNAs generated by the IR and sequence alignment of vsRNAs and SlLNR1 . Location and frequency of TYCLV-derived siRNAs (vsRNAs) were mapped to the IR in sense- (above the x-axis) or antisense- (below the x-axis) orientation. Genome organization of the IR was shown at the top in which the inverted repeat was symbolized as a stem loop. Numbers indicate the first (2616) and last (147) nucleotides of the IR sequence. The histogram of location, frequency and size distribution of vsRNAs corresponding to the 25-nt-fragment (2730–2754) were shown at the medium panel. The fragment was highly complemented with SlLNR1 (-). The scissor means the cleavage site determined by 5’-RACE analysis. (B) Phenotypes of tomato inoculated with pTRV2:4TR. TYLCV-susceptible tomato plants were inoculated with pTRV2 containing 4×25-nt-fragment (2730–2754). The photos were taken at 15 dpi. (C) Validation of siRNA(-2752-21) in the tomato plants by siRNA Northern blot. The leaves of susceptible tomato plants inoculated by TYLCV infectious clone, natural infection by viruliferous whiteflies, agroinfiltrated with pTRV2:4TR and pTRV2:IR were used for total RNA extraction and analyzed at 24 dpi. U6 gene was set as the internal control. (D) Validation of siRNA(-2752-21) presence and downregulation of SlLNR1 in the overexpressed plants. Two individual transgenic lines (pCAMBIA2301:siRNA-1/2) with overexpression of siRNA(-2752-21) were used for total RNA extraction and analyzed. EV indicates the transgenic plant with the EV. The lower panel shows the relative expressi o n of SlLNR1 that was measured by qRT-PCR and calculated in relation to the transgenic plants according to the ΔΔ Ct method using tomat o actin gene as the reference. Error bars represented SE of three biological replicates and significant differences by Student’ s t test (*, p
    Figure Legend Snippet: Identification of a 25-nt segment and a vsRNA that induce stunt and curled leaves in tomato. (A) VsRNAs generated by the IR and sequence alignment of vsRNAs and SlLNR1 . Location and frequency of TYCLV-derived siRNAs (vsRNAs) were mapped to the IR in sense- (above the x-axis) or antisense- (below the x-axis) orientation. Genome organization of the IR was shown at the top in which the inverted repeat was symbolized as a stem loop. Numbers indicate the first (2616) and last (147) nucleotides of the IR sequence. The histogram of location, frequency and size distribution of vsRNAs corresponding to the 25-nt-fragment (2730–2754) were shown at the medium panel. The fragment was highly complemented with SlLNR1 (-). The scissor means the cleavage site determined by 5’-RACE analysis. (B) Phenotypes of tomato inoculated with pTRV2:4TR. TYLCV-susceptible tomato plants were inoculated with pTRV2 containing 4×25-nt-fragment (2730–2754). The photos were taken at 15 dpi. (C) Validation of siRNA(-2752-21) in the tomato plants by siRNA Northern blot. The leaves of susceptible tomato plants inoculated by TYLCV infectious clone, natural infection by viruliferous whiteflies, agroinfiltrated with pTRV2:4TR and pTRV2:IR were used for total RNA extraction and analyzed at 24 dpi. U6 gene was set as the internal control. (D) Validation of siRNA(-2752-21) presence and downregulation of SlLNR1 in the overexpressed plants. Two individual transgenic lines (pCAMBIA2301:siRNA-1/2) with overexpression of siRNA(-2752-21) were used for total RNA extraction and analyzed. EV indicates the transgenic plant with the EV. The lower panel shows the relative expressi o n of SlLNR1 that was measured by qRT-PCR and calculated in relation to the transgenic plants according to the ΔΔ Ct method using tomat o actin gene as the reference. Error bars represented SE of three biological replicates and significant differences by Student’ s t test (*, p

    Techniques Used: Generated, Sequencing, Derivative Assay, Northern Blot, Infection, RNA Extraction, Transgenic Assay, Over Expression, Quantitative RT-PCR

    24) Product Images from "NIK Stabilization in Osteoclasts Results in Osteoporosis and Enhanced Inflammatory Osteolysis"

    Article Title: NIK Stabilization in Osteoclasts Results in Osteoporosis and Enhanced Inflammatory Osteolysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015383

    Expression of NIKΔT3 induced by both catK-Cre and lysM-Cre. A . Diagram of NT3 locus. NIKΔT3 (NT3) cDNA was knocked into the ROSA26 locus, flanked upstream by a loxP- Neo R .STOP-loxP cassette. Cre-mediated loxP recombination removes the Neo R .STOP sequence, putting NT3 under control of the ROSA 26 promoter. Black arrows indicate the pair of primers used to amplify across the loxP. B–C . Genomic DNA amplification across the loxP sites using NT3.catK (B) and NT3.lysM (C) BMMs cultured with 20 ng/ml RANKL for up to 4 days. ∼3kb STOP and ∼300bp lox bands are generated before and after Cre-mediated deletion, respectively. The left most lanes (-) are negative controls with no genomic DNA in PCR reactions. D–E . Western blot analysis of NT3 expression in total cell lysates, using BMMs cultured as in B. NIK KO BMMs serve as a negative control. F–G . p100/p52 processing was assessed by Western blot in total cell lysates from Ctl and NT3.catK BMMs cultured in RANKL for the indicated times, or in unstimulated NT3.lysM BMMs. H . Evaluation of RelB and p65 levels in nuclear extracts of NT3.lysM BMMs. I . Western blot analysis of IκBα phosphorylation in NT3.lysM BMM total lysates.
    Figure Legend Snippet: Expression of NIKΔT3 induced by both catK-Cre and lysM-Cre. A . Diagram of NT3 locus. NIKΔT3 (NT3) cDNA was knocked into the ROSA26 locus, flanked upstream by a loxP- Neo R .STOP-loxP cassette. Cre-mediated loxP recombination removes the Neo R .STOP sequence, putting NT3 under control of the ROSA 26 promoter. Black arrows indicate the pair of primers used to amplify across the loxP. B–C . Genomic DNA amplification across the loxP sites using NT3.catK (B) and NT3.lysM (C) BMMs cultured with 20 ng/ml RANKL for up to 4 days. ∼3kb STOP and ∼300bp lox bands are generated before and after Cre-mediated deletion, respectively. The left most lanes (-) are negative controls with no genomic DNA in PCR reactions. D–E . Western blot analysis of NT3 expression in total cell lysates, using BMMs cultured as in B. NIK KO BMMs serve as a negative control. F–G . p100/p52 processing was assessed by Western blot in total cell lysates from Ctl and NT3.catK BMMs cultured in RANKL for the indicated times, or in unstimulated NT3.lysM BMMs. H . Evaluation of RelB and p65 levels in nuclear extracts of NT3.lysM BMMs. I . Western blot analysis of IκBα phosphorylation in NT3.lysM BMM total lysates.

    Techniques Used: Expressing, Sequencing, Amplification, Cell Culture, Generated, Polymerase Chain Reaction, Western Blot, Negative Control, CTL Assay

    25) Product Images from "Injury factors alter miRNAs profiles of exosomes derived from islets and circulation"

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

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101689

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    26) Product Images from "Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics"

    Article Title: Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics

    Journal: Translational Vision Science & Technology

    doi: 10.1167/tvst.8.6.28

    Rational optimization tests of the 725 hhRz. (A) In vitro co-synthesis with cleavage assay comparing the 725 hhRz with 4-bp stem II in the pPrislei versus pUC-VAL VAI RNA scaffolds against the short RHO target. Linearized plasmids in defined molar ratio (6:1 ribozyme:target) were transcribed by T7 RNA polymerase and cleavage emerged during the 3-hour incubation prior to analysis by PAGE-urea gel electrophoresis and staining with RNA dye (SYBR Gold). Substrate short RHO is labeled (green arrow) and the mobility of the different scaffold-hhRz RNAs is labeled (blue arrow). Note that the extent of cleavage products (P1, P2) (red arrows) with the 725 hhRz within the Prislei VAI scaffold is greater than with the same 725 hhRz within the pUC-VAL VAI scaffold. There is no cleavage without the hhRz within the scaffold (not shown for pPrislei) and inactivating mutations in the catalytic core of the 725 hhRz (G12C) obviates cleavage (data not shown for pPrislei). Eight percent (wt/vol) PEG 8000 was used to simulate the viscosity of the cytoplasm. (B) In cellula cotransfection of plasmids that express the 4-bp stem II hhRz within the pUC-VAL and pPrislei scaffolds with plasmid that expresses full-length fix-5′UT human RHO mRNA. Additional comparisons are the inactivated 725 hhRz, 725 hhRzs with upstream tertiary accessory elements (RzB, RzC, RzD). Human RHO mRNA was quantified by real time RT/PCR with hRHO cDNA standardized data comparison. For the pUC-VAL scaffold one-way ANOVA showed significant differences between samples (P = 0.042). There was a 49.4% reduction of hRHO mRNA by the active 725 hhRz relative to control (P = 0.03375). Catalytic core mutation caused 14.2% reduction of hRHO mRNA but this was not statistically different relative to control or to the active 725 hhRz (P > 0.05). In the pUC-VAL scaffold the remaining constructs (RzB, RzC, RzD) did not show significant knockdown of hRHO mRNA relative to control. For the pPrislei scaffold one-way ANOVA showed significant differences among all the samples (P = 1.92 E−5 ). The active 725 hhRz showed 68.4% knockdown of hRHO mRNA relative to control (scaffold alone; P = 1.36 E−6 ). The inactivated 725 hhRz exerted 23.8% knockdown but this was not significantly different from control (P = 0.17586). However, the active and inactivated 725 hhRzs showed significantly different knockdown of hRHO mRNA by t-test (P = 4.91 E−5 ). All other hhRzs within pPrislei with TAE elements did not achieve significant knockdown of hRHO (P > 0.05). (C) Verifying accessibility in the predicted large platform (nts: 653–763) of accessibility in hRHO mRNA (green arrow). Various hhRzs (blue arrow) within the pPrislei scaffold targeted sites both inside (725, 689, 700, 707) and outside (785) the predicted accessibility platform were tested for capacity to cleave hRHO mRNA. All targeting sites were GUC↓ cleavage motifs. Reaction conditions used were as in Figure 8A. Cleavage products (red arrows) are only present when attacking predicted accessible target sites. Cleavage sites vary in size depending upon the location of the cleavage motif. Inactivating the core of the hhRz prevents cleavage. The noncleaving 785 site is just outside the accessible region but is in one of the most stable regions of the target hRHO mRNA fold.
    Figure Legend Snippet: Rational optimization tests of the 725 hhRz. (A) In vitro co-synthesis with cleavage assay comparing the 725 hhRz with 4-bp stem II in the pPrislei versus pUC-VAL VAI RNA scaffolds against the short RHO target. Linearized plasmids in defined molar ratio (6:1 ribozyme:target) were transcribed by T7 RNA polymerase and cleavage emerged during the 3-hour incubation prior to analysis by PAGE-urea gel electrophoresis and staining with RNA dye (SYBR Gold). Substrate short RHO is labeled (green arrow) and the mobility of the different scaffold-hhRz RNAs is labeled (blue arrow). Note that the extent of cleavage products (P1, P2) (red arrows) with the 725 hhRz within the Prislei VAI scaffold is greater than with the same 725 hhRz within the pUC-VAL VAI scaffold. There is no cleavage without the hhRz within the scaffold (not shown for pPrislei) and inactivating mutations in the catalytic core of the 725 hhRz (G12C) obviates cleavage (data not shown for pPrislei). Eight percent (wt/vol) PEG 8000 was used to simulate the viscosity of the cytoplasm. (B) In cellula cotransfection of plasmids that express the 4-bp stem II hhRz within the pUC-VAL and pPrislei scaffolds with plasmid that expresses full-length fix-5′UT human RHO mRNA. Additional comparisons are the inactivated 725 hhRz, 725 hhRzs with upstream tertiary accessory elements (RzB, RzC, RzD). Human RHO mRNA was quantified by real time RT/PCR with hRHO cDNA standardized data comparison. For the pUC-VAL scaffold one-way ANOVA showed significant differences between samples (P = 0.042). There was a 49.4% reduction of hRHO mRNA by the active 725 hhRz relative to control (P = 0.03375). Catalytic core mutation caused 14.2% reduction of hRHO mRNA but this was not statistically different relative to control or to the active 725 hhRz (P > 0.05). In the pUC-VAL scaffold the remaining constructs (RzB, RzC, RzD) did not show significant knockdown of hRHO mRNA relative to control. For the pPrislei scaffold one-way ANOVA showed significant differences among all the samples (P = 1.92 E−5 ). The active 725 hhRz showed 68.4% knockdown of hRHO mRNA relative to control (scaffold alone; P = 1.36 E−6 ). The inactivated 725 hhRz exerted 23.8% knockdown but this was not significantly different from control (P = 0.17586). However, the active and inactivated 725 hhRzs showed significantly different knockdown of hRHO mRNA by t-test (P = 4.91 E−5 ). All other hhRzs within pPrislei with TAE elements did not achieve significant knockdown of hRHO (P > 0.05). (C) Verifying accessibility in the predicted large platform (nts: 653–763) of accessibility in hRHO mRNA (green arrow). Various hhRzs (blue arrow) within the pPrislei scaffold targeted sites both inside (725, 689, 700, 707) and outside (785) the predicted accessibility platform were tested for capacity to cleave hRHO mRNA. All targeting sites were GUC↓ cleavage motifs. Reaction conditions used were as in Figure 8A. Cleavage products (red arrows) are only present when attacking predicted accessible target sites. Cleavage sites vary in size depending upon the location of the cleavage motif. Inactivating the core of the hhRz prevents cleavage. The noncleaving 785 site is just outside the accessible region but is in one of the most stable regions of the target hRHO mRNA fold.

    Techniques Used: In Vitro, Cleavage Assay, Incubation, Polyacrylamide Gel Electrophoresis, Nucleic Acid Electrophoresis, Staining, Labeling, Cotransfection, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis, Construct

    In vitro and in cellula 725 ribozyme cleavage assays-stem-II optimization. (A) In vitro transcription/cleavage reactions were performed and run on a 5% denaturing PAGE gel with 8 M urea relative to RNA size markers (lane 1). The mobility of the scaffold and active and inactive (G12C) ribozymes (lanes 2–4) and the short RHO substrate (511 nt; lane 5) are shown. Cleavage products of 371 nt (red arrow) and 140 nt (blue arrow) are only identified with a catalytically active 725 hhRz 16 is embedded in the scaffold (lane 7) but not from the pUC-VAL scaffold itself (lane 6) or with a 725 hhRz 16 that had a catalytic core inactivating mutation (G12C; lane 8). (B) In vitro analysis of short hRHO (511 nt) and full-length (1532 nt) hRHO mRNAs, transcribed in vitro but analyzed on nondenaturing PAGE gels relative to RNA size mobility markers. For the short hRHO (left panel) there are two prominent bands. For the full-length hRHO (right panel) there appear to be at least three well-populated conformational states. (C) In cellula assay to assess target hRHO mRNA knockdown in the pUC-VAI scaffold. HEK293S cells were transiently co-transfected with pNEB-VAI-hhRz-1 and pRHO-fix5UT. Total RNA was extracted from transfected cells 48 hours posttransfection and analyzed for relative hRHO mRNA levels using qRT-PCR. Mean percent control vector transfection hRHO mRNA levels are shown ± SEM (one-way ANOVA P = 3.54, P = 0.03). Asterisks denote significant (P
    Figure Legend Snippet: In vitro and in cellula 725 ribozyme cleavage assays-stem-II optimization. (A) In vitro transcription/cleavage reactions were performed and run on a 5% denaturing PAGE gel with 8 M urea relative to RNA size markers (lane 1). The mobility of the scaffold and active and inactive (G12C) ribozymes (lanes 2–4) and the short RHO substrate (511 nt; lane 5) are shown. Cleavage products of 371 nt (red arrow) and 140 nt (blue arrow) are only identified with a catalytically active 725 hhRz 16 is embedded in the scaffold (lane 7) but not from the pUC-VAL scaffold itself (lane 6) or with a 725 hhRz 16 that had a catalytic core inactivating mutation (G12C; lane 8). (B) In vitro analysis of short hRHO (511 nt) and full-length (1532 nt) hRHO mRNAs, transcribed in vitro but analyzed on nondenaturing PAGE gels relative to RNA size mobility markers. For the short hRHO (left panel) there are two prominent bands. For the full-length hRHO (right panel) there appear to be at least three well-populated conformational states. (C) In cellula assay to assess target hRHO mRNA knockdown in the pUC-VAI scaffold. HEK293S cells were transiently co-transfected with pNEB-VAI-hhRz-1 and pRHO-fix5UT. Total RNA was extracted from transfected cells 48 hours posttransfection and analyzed for relative hRHO mRNA levels using qRT-PCR. Mean percent control vector transfection hRHO mRNA levels are shown ± SEM (one-way ANOVA P = 3.54, P = 0.03). Asterisks denote significant (P

    Techniques Used: In Vitro, Polyacrylamide Gel Electrophoresis, Mutagenesis, Transfection, Quantitative RT-PCR, Plasmid Preparation

    Toward the development of RNA agents for use in preclinical trial of for adRP. HEK293S cells were transiently co-transfected with plasmids encoding PTGS agents and WT target or hardened hRHO targets containing either a single silent mutation at the RHO 725-hhRz cleavage site (RHO-725-HARD) or a double silent mutation at the RHO 725 and RHO 731-hhRz cleavage sites (RHO-725-731-HARD). Total RNA was extracted from transfected cells 48 hours posttransfection and analyzed for relative hRHO mRNA levels using qRT-PCR. Cytoplasmic protein was extracted from transfected cells 72 hours posttransfection and analyzed for relative RHO protein levels by western blotting. Mean percent control (scrambled shRNA or VAI control) vector transfection RHO mRNA or protein levels are shown ± SEM in bar graphs. Asterisks indicate significant (P
    Figure Legend Snippet: Toward the development of RNA agents for use in preclinical trial of for adRP. HEK293S cells were transiently co-transfected with plasmids encoding PTGS agents and WT target or hardened hRHO targets containing either a single silent mutation at the RHO 725-hhRz cleavage site (RHO-725-HARD) or a double silent mutation at the RHO 725 and RHO 731-hhRz cleavage sites (RHO-725-731-HARD). Total RNA was extracted from transfected cells 48 hours posttransfection and analyzed for relative hRHO mRNA levels using qRT-PCR. Cytoplasmic protein was extracted from transfected cells 72 hours posttransfection and analyzed for relative RHO protein levels by western blotting. Mean percent control (scrambled shRNA or VAI control) vector transfection RHO mRNA or protein levels are shown ± SEM in bar graphs. Asterisks indicate significant (P

    Techniques Used: Transfection, Mutagenesis, Quantitative RT-PCR, Western Blot, shRNA, Plasmid Preparation

    Impact of shRNAs and hhRzs targeting accessible regions of full-length hRHO mRNA target in HEK293S cells and HER224 cells. (A) HEK293S cells. shRNA agents were designed targeting RHO at the 310 region (RHOi2), the 725 region (RHOi-725), and the 266 region (RHOi-266). The pSUPER expression system was used, and HEK293S cells were transiently co-transfected with pSUPER plasmids along with pRHO-fix5UT. Total RNA was extracted from transfected cells 48 hours posttransfection and analyzed for relative hRHO mRNA levels using qRT-PCR. Mean percent of control vector (scrambled shRNA) transfection hRHO mRNA levels are shown ± SEM (one-way ANOVA F = 219.98, P = 9.44 E−11 ). Asterisks indicate significant (P
    Figure Legend Snippet: Impact of shRNAs and hhRzs targeting accessible regions of full-length hRHO mRNA target in HEK293S cells and HER224 cells. (A) HEK293S cells. shRNA agents were designed targeting RHO at the 310 region (RHOi2), the 725 region (RHOi-725), and the 266 region (RHOi-266). The pSUPER expression system was used, and HEK293S cells were transiently co-transfected with pSUPER plasmids along with pRHO-fix5UT. Total RNA was extracted from transfected cells 48 hours posttransfection and analyzed for relative hRHO mRNA levels using qRT-PCR. Mean percent of control vector (scrambled shRNA) transfection hRHO mRNA levels are shown ± SEM (one-way ANOVA F = 219.98, P = 9.44 E−11 ). Asterisks indicate significant (P

    Techniques Used: shRNA, Expressing, Transfection, Quantitative RT-PCR, Plasmid Preparation

    27) Product Images from "Long Non-Coding RNA LINC01783 Promotes the Progression of Cervical Cancer by Sponging miR-199b-5p to Mediate GBP1 Expression"

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

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S230171

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    28) Product Images from "The tRNA-Derived Small RNAs Regulate Gene Expression through Triggering Sequence-Specific Degradation of Target Transcripts in the Oomycete Pathogen Phytophthora sojae"

    Article Title: The tRNA-Derived Small RNAs Regulate Gene Expression through Triggering Sequence-Specific Degradation of Target Transcripts in the Oomycete Pathogen Phytophthora sojae

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2016.01938

    The negative correlation between tsRNA abundance and target transcript accumulation in P. sojae . (A) Heatmap showing the digital gene expression profile of tsRNA target genes. Column samples MY, SP, CY, GC, and IF, extracted from the Phytophthora Transcriptome Database, correspond to the P. sojae developmental stages of M, S, C, G, and I as described in this study, respectively. Each row represents one predicted target gene. Dashed vertical line indicates the median of expression levels of all predicted target genes at each of the five developmental stages; vertical trace line inside each column indicates expression level of the corresponding gene. The color-key on the top indicates the relative expression levels of the target genes, from low (cyan) to high (magenta). Dendrogram on the left shows the distance of each two target genes based on their transcript levels. The colored column on the left represent the four clusters of expression patterns of putative tsRNA targets; the cluster in orange color are genes that in general show a negative expression correlation with tsRNA accumulation. (B) Quantitative RT-PCR analysis of 10 selected tsRNA putative targets. Error bars indicate the standard deviation of three technical replicates. The bottom text of each barplot indicates the corresponding tsRNA. O, oospores; M, mycelia; S, sporangia; C, cysts; G, germinated cysts; I, soybean infection at 48 hpi. The RNA level in M was set to 1.
    Figure Legend Snippet: The negative correlation between tsRNA abundance and target transcript accumulation in P. sojae . (A) Heatmap showing the digital gene expression profile of tsRNA target genes. Column samples MY, SP, CY, GC, and IF, extracted from the Phytophthora Transcriptome Database, correspond to the P. sojae developmental stages of M, S, C, G, and I as described in this study, respectively. Each row represents one predicted target gene. Dashed vertical line indicates the median of expression levels of all predicted target genes at each of the five developmental stages; vertical trace line inside each column indicates expression level of the corresponding gene. The color-key on the top indicates the relative expression levels of the target genes, from low (cyan) to high (magenta). Dendrogram on the left shows the distance of each two target genes based on their transcript levels. The colored column on the left represent the four clusters of expression patterns of putative tsRNA targets; the cluster in orange color are genes that in general show a negative expression correlation with tsRNA accumulation. (B) Quantitative RT-PCR analysis of 10 selected tsRNA putative targets. Error bars indicate the standard deviation of three technical replicates. The bottom text of each barplot indicates the corresponding tsRNA. O, oospores; M, mycelia; S, sporangia; C, cysts; G, germinated cysts; I, soybean infection at 48 hpi. The RNA level in M was set to 1.

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation, Infection

    Identification and verification of P. sojae tsRNAs. (A) Alignment of tsRNA-AlaAGC reads to its cognate tRNA. The first two lines indicate the position of tRNA. The third line is the tRNA sequence. The RNA secondary structure is shown in the fourth line. The Base Deep Index (BDI, a number to indicate the relative coverage of each base) of each nucleotide is in the fifth line. The rest lines are the tsRNA reads and alignments. Numbers on the right indicate the number of tsRNA reads. (B) RT-PCR amplification of adaptor-ligated P. sojae sRNAs. The vertical line indicates the amplified products. Lanes A and B are two technical repeats. (C) Clover-leaf view of cloned tsRNAs derived from tRNA-IleAAT. The candidate tsRNA region is highlighted in red. (D) Northern analysis of Phytophthora tsRNAs. The lower bands (30–38 nt) are the tsRNAs, while the upper bands (larger than 38 nt) are the cognate tRNAs. Ps and Pp are P. sojae and P. parasitica RNAs pooled from an equal amount of total RNAs of oospores, mycelia, sporangia, cysts and germinated cysts, respectively. (E) Processing patterns of tsRNAs. tRNA-SerTGA, tRNA-ArgACG and tRNA-GlnCTG produce 5′, 3′ and both 5′ and 3′ tsRNAs, respectively. The x-axis shows the position of each base in the tRNA.
    Figure Legend Snippet: Identification and verification of P. sojae tsRNAs. (A) Alignment of tsRNA-AlaAGC reads to its cognate tRNA. The first two lines indicate the position of tRNA. The third line is the tRNA sequence. The RNA secondary structure is shown in the fourth line. The Base Deep Index (BDI, a number to indicate the relative coverage of each base) of each nucleotide is in the fifth line. The rest lines are the tsRNA reads and alignments. Numbers on the right indicate the number of tsRNA reads. (B) RT-PCR amplification of adaptor-ligated P. sojae sRNAs. The vertical line indicates the amplified products. Lanes A and B are two technical repeats. (C) Clover-leaf view of cloned tsRNAs derived from tRNA-IleAAT. The candidate tsRNA region is highlighted in red. (D) Northern analysis of Phytophthora tsRNAs. The lower bands (30–38 nt) are the tsRNAs, while the upper bands (larger than 38 nt) are the cognate tRNAs. Ps and Pp are P. sojae and P. parasitica RNAs pooled from an equal amount of total RNAs of oospores, mycelia, sporangia, cysts and germinated cysts, respectively. (E) Processing patterns of tsRNAs. tRNA-SerTGA, tRNA-ArgACG and tRNA-GlnCTG produce 5′, 3′ and both 5′ and 3′ tsRNAs, respectively. The x-axis shows the position of each base in the tRNA.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Clone Assay, Derivative Assay, Northern Blot

    29) Product Images from "Expression of miR-27a-3p is an independent predictive factor for recurrence in clear cell renal cell carcinoma"

    Article Title: Expression of miR-27a-3p is an independent predictive factor for recurrence in clear cell renal cell carcinoma

    Journal: Oncotarget

    doi:

    miR-27a-3p inhibitor significantly reduced the cell growth, migration and invasion ability in Caki 1 cells A. Expression of miR-27a-3p in ccRCC cell lines was examined by quantitative real-time PCR. B. The proliferation of Caki-1 cells transfected with the miR-27a-3p inhibitor or negative control miRNA inhibitor for 48, 72, 96 and 120 h were examined by MTS assay. Values are means ± S.D. of 6 independent experiments. * p
    Figure Legend Snippet: miR-27a-3p inhibitor significantly reduced the cell growth, migration and invasion ability in Caki 1 cells A. Expression of miR-27a-3p in ccRCC cell lines was examined by quantitative real-time PCR. B. The proliferation of Caki-1 cells transfected with the miR-27a-3p inhibitor or negative control miRNA inhibitor for 48, 72, 96 and 120 h were examined by MTS assay. Values are means ± S.D. of 6 independent experiments. * p

    Techniques Used: Migration, Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control, MTS Assay

    The association of miRNAs expression levels with TNM staging, cancer-specific survival and progression-free survival Levels of miR-21-3p A. and miR193b-5p B. differed significantly based on tumor staging (Wilcoxon test). However, there was no significant correlation associated with miR-27a-3p expression C. We divided the samples into two groups, high or low levels, based on real time PCR results. High expression of miR-21-3p D. was significantly associated with cancer-specific survival (Kaplan-Meier, log rank test). High levels of miR-27a-3p were positively associated with recurrence E. and cancer-specific survival F. in ccRCC (Kaplan-Meier log rank test).
    Figure Legend Snippet: The association of miRNAs expression levels with TNM staging, cancer-specific survival and progression-free survival Levels of miR-21-3p A. and miR193b-5p B. differed significantly based on tumor staging (Wilcoxon test). However, there was no significant correlation associated with miR-27a-3p expression C. We divided the samples into two groups, high or low levels, based on real time PCR results. High expression of miR-21-3p D. was significantly associated with cancer-specific survival (Kaplan-Meier, log rank test). High levels of miR-27a-3p were positively associated with recurrence E. and cancer-specific survival F. in ccRCC (Kaplan-Meier log rank test).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    30) Product Images from "LINC00978 predicts poor prognosis in breast cancer patients"

    Article Title: LINC00978 predicts poor prognosis in breast cancer patients

    Journal: Scientific Reports

    doi: 10.1038/srep37936

    LINC00978 is highly expressed in breast cancer cell lines and tissues. ( a ) Comparison of LINC00978 expression between 12 cancer cell lines and the normal cell line 10 A by qRT-PCR. GAPDH was used as an internal control. ( b ) Comparison of LINC00978 expression in 36 pairs of breast cancer tissues and adjacent tissues. Abbreviations: 453, MDA-MB-453; 436, MDA-MB-436; 468, MDA-MB-468; 231, MDA-MB-231; 231HM, highly metastatic lung cancer cell line MDA-MB-231. * p
    Figure Legend Snippet: LINC00978 is highly expressed in breast cancer cell lines and tissues. ( a ) Comparison of LINC00978 expression between 12 cancer cell lines and the normal cell line 10 A by qRT-PCR. GAPDH was used as an internal control. ( b ) Comparison of LINC00978 expression in 36 pairs of breast cancer tissues and adjacent tissues. Abbreviations: 453, MDA-MB-453; 436, MDA-MB-436; 468, MDA-MB-468; 231, MDA-MB-231; 231HM, highly metastatic lung cancer cell line MDA-MB-231. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification

    31) Product Images from "MicroRNA-638 inhibits cell proliferation by targeting phospholipase D1 in human gastric carcinoma"

    Article Title: MicroRNA-638 inhibits cell proliferation by targeting phospholipase D1 in human gastric carcinoma

    Journal: Protein & Cell

    doi: 10.1007/s13238-015-0187-8

    Identification of PLD1 as the target of miR-638 . (A) Validation of the microarray results in both MKN-45 and SGC-7901 cells using qRT-PCR. A panel of 12 genes were indeed down-regulated by miR-638. (B) Proliferation assays performed on MKN-45 and SGC-7901 cells transfected with si-PLD1 or si-DEF6. Depleted PLD1 expression showed the most obvious growth repression effect. (C) Schematic of the wild-type (WT) or mutant-type (MT) 3′UTRs of the PLD1 plasmids. The complementary site of the seed region of miR-638 was selected for mutation. The free energy of hybrids between miR-638 and PLD1 was −21.1 kcal/mol. (D) Relative luciferase activity assays of luciferase reporter plasmids containing PLD1WT or MT 3′UTR were performed in cells (HEK-293T, MKN-45, and SGC-7901). Luciferase activity was determined 48 h after transfection and normalized to the Renilla luciferase activity. (E) The protein levels of PLD1 were determined by Western blotting in MKN-45 and SGC-7901 cells transfected with miR-638 mimic, miR-638 inhibitor or the corresponding NC. Beta-actin served as an internal control
    Figure Legend Snippet: Identification of PLD1 as the target of miR-638 . (A) Validation of the microarray results in both MKN-45 and SGC-7901 cells using qRT-PCR. A panel of 12 genes were indeed down-regulated by miR-638. (B) Proliferation assays performed on MKN-45 and SGC-7901 cells transfected with si-PLD1 or si-DEF6. Depleted PLD1 expression showed the most obvious growth repression effect. (C) Schematic of the wild-type (WT) or mutant-type (MT) 3′UTRs of the PLD1 plasmids. The complementary site of the seed region of miR-638 was selected for mutation. The free energy of hybrids between miR-638 and PLD1 was −21.1 kcal/mol. (D) Relative luciferase activity assays of luciferase reporter plasmids containing PLD1WT or MT 3′UTR were performed in cells (HEK-293T, MKN-45, and SGC-7901). Luciferase activity was determined 48 h after transfection and normalized to the Renilla luciferase activity. (E) The protein levels of PLD1 were determined by Western blotting in MKN-45 and SGC-7901 cells transfected with miR-638 mimic, miR-638 inhibitor or the corresponding NC. Beta-actin served as an internal control

    Techniques Used: Microarray, Quantitative RT-PCR, Transfection, Expressing, Mutagenesis, Luciferase, Activity Assay, Western Blot

    The expression of miR-638 was down-regulated in GC . (A) The expression of miR-638 was tested by qRT-PCR in 64 paired GC and adjacent noncancerous tissues (NCTs). (B) The levels of miR-638 were obviously down-regulated in 56.25% tumor tissues. (C) The DNA copy number of miR-638 was checked by qPCR in 24 paired GC and NCTs. (D) Kaplan-Meier analysis of the effect of the miR-638 expression on overall survival of 64 GC patients
    Figure Legend Snippet: The expression of miR-638 was down-regulated in GC . (A) The expression of miR-638 was tested by qRT-PCR in 64 paired GC and adjacent noncancerous tissues (NCTs). (B) The levels of miR-638 were obviously down-regulated in 56.25% tumor tissues. (C) The DNA copy number of miR-638 was checked by qPCR in 24 paired GC and NCTs. (D) Kaplan-Meier analysis of the effect of the miR-638 expression on overall survival of 64 GC patients

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

    32) Product Images from "Regulation of cell proliferation and metastasis by microRNA-593-5p in human gastric cancer"

    Article Title: Regulation of cell proliferation and metastasis by microRNA-593-5p in human gastric cancer

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S178151

    miR-593-5p is lowly expressed in gastric cancer (GC) tissues and cell lines. Notes: ( A ) miR-593-5p expression was assessed by quantitative RT-PCR (qRT-PCR) in 73 GC tumor specimens as compared to normal adjacent gastric tissue. ( B ) miR-593-5p expression was assessed by qRT-PCR in GC cells (HGC-27, AGS, SGC-7901, MGC-803) and normal gastric epithelial cells (GES). U6 was assessed as an internal control. * P
    Figure Legend Snippet: miR-593-5p is lowly expressed in gastric cancer (GC) tissues and cell lines. Notes: ( A ) miR-593-5p expression was assessed by quantitative RT-PCR (qRT-PCR) in 73 GC tumor specimens as compared to normal adjacent gastric tissue. ( B ) miR-593-5p expression was assessed by qRT-PCR in GC cells (HGC-27, AGS, SGC-7901, MGC-803) and normal gastric epithelial cells (GES). U6 was assessed as an internal control. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    33) Product Images from "The general PTS component HPr determines the preference for glucose over mannitol"

    Article Title: The general PTS component HPr determines the preference for glucose over mannitol

    Journal: Scientific Reports

    doi: 10.1038/srep43431

    Inactivation of mtlR completely abolishes the glucose-dependent repression of the mtl operon and glucose preference over mannitol. ( A , B ) Total RNA was isolated from wild type ( A ) and an mtlR deletion mutant ( B ) of E. coli MG1655 grown to early exponential phase in LB medium or LB medium containing the indicated sugars (0.2% each when added alone, or 0.1% each when added in combination), and the expression level of the mtl operon was then quantified by qRT-PCR as described in the Materials and Methods section. Representative data (mean ± SD) from three independent experiments (n = 3 each) are shown. Statistical significance was determined by Student’s t -test (***P
    Figure Legend Snippet: Inactivation of mtlR completely abolishes the glucose-dependent repression of the mtl operon and glucose preference over mannitol. ( A , B ) Total RNA was isolated from wild type ( A ) and an mtlR deletion mutant ( B ) of E. coli MG1655 grown to early exponential phase in LB medium or LB medium containing the indicated sugars (0.2% each when added alone, or 0.1% each when added in combination), and the expression level of the mtl operon was then quantified by qRT-PCR as described in the Materials and Methods section. Representative data (mean ± SD) from three independent experiments (n = 3 each) are shown. Statistical significance was determined by Student’s t -test (***P

    Techniques Used: Isolation, Mutagenesis, Expressing, Quantitative RT-PCR

    Dephosphorylated HPr inhibits derepression of the mtl operon by mannitol. ( A ) E. coli MG1655 was grown in M9 minimal medium containing the indicated sugars (0.04% each). The phosphorylation state of HPr was determined when OD 600 reached 0.15 as described in the Materials and Methods section. Purified HPr (2 ng) was used as a positive control. Band intensities were analyzed using Multi Gauge version 3.0 software. The table below the gel shows the percentage of dephospho-HPr over total HPr with means and standard deviations from three independent experiments. ( B ) The phosphorylation state of HPr was determined in E. coli MG1655 cells grown in M9 minimal medium containing 0.04% glucose and 0.04% mannitol at OD 600 0.15 and 0.6. Purified HPr (2 ng) was used as a positive control. ( C ) The wild-type E. coli MG1655 was cultivated as in ( A ), and harvested at indicated OD 600 . The expression level of mtlA was then quantified by qRT-PCR. Representative data (mean ± SD) from three independent experiments (n = 3 each) are shown relative to that observed in glycerol (Gly)-grown cells, and statistical significance was determined by Student’s t -test (***P
    Figure Legend Snippet: Dephosphorylated HPr inhibits derepression of the mtl operon by mannitol. ( A ) E. coli MG1655 was grown in M9 minimal medium containing the indicated sugars (0.04% each). The phosphorylation state of HPr was determined when OD 600 reached 0.15 as described in the Materials and Methods section. Purified HPr (2 ng) was used as a positive control. Band intensities were analyzed using Multi Gauge version 3.0 software. The table below the gel shows the percentage of dephospho-HPr over total HPr with means and standard deviations from three independent experiments. ( B ) The phosphorylation state of HPr was determined in E. coli MG1655 cells grown in M9 minimal medium containing 0.04% glucose and 0.04% mannitol at OD 600 0.15 and 0.6. Purified HPr (2 ng) was used as a positive control. ( C ) The wild-type E. coli MG1655 was cultivated as in ( A ), and harvested at indicated OD 600 . The expression level of mtlA was then quantified by qRT-PCR. Representative data (mean ± SD) from three independent experiments (n = 3 each) are shown relative to that observed in glycerol (Gly)-grown cells, and statistical significance was determined by Student’s t -test (***P

    Techniques Used: Purification, Positive Control, Software, Expressing, Quantitative RT-PCR

    The interaction between HPr and MtlR is sufficient to confer glucose preference over mannitol. ( A , B ) Growth of a ptsH deletion mutant harboring a pACYC184-derived expression vector for wild-type HPr (pACYC-HPr, closed diamonds), pACYC-HPr(K27E) (closed triangles) or pACYC-HPr(L47A/F48A) (open squares) in M9 medium containing 0.2% glucose ( A ) or 0.2% mannitol ( B ). Growth of the ptsH deletion mutant (open circles) is shown as a negative control. The inset in panel ( B ) shows the expression level of mtlA measured by qRT-PCR in the ptsH deletion mutant harboring pACYC-HPr(K27E) relative to that in the strain harboring pACYC-HPr. ( C , D ) The ptsH deletion strain harboring pACYC-HPr ( C ) or pACYC-HPr(K27E) ( D ) was grown in M9 minimal medium supplemented with 0.04% glucose and 0.04% mannitol. Growth rates (optical density at 600 nm, gray lines with circles) and the concentrations of sugars (open squares for glucose and closed squares for mannitol) remaining in the medium were measured as a function of incubation time. Representative data from three independent and reproducible measurements are shown.
    Figure Legend Snippet: The interaction between HPr and MtlR is sufficient to confer glucose preference over mannitol. ( A , B ) Growth of a ptsH deletion mutant harboring a pACYC184-derived expression vector for wild-type HPr (pACYC-HPr, closed diamonds), pACYC-HPr(K27E) (closed triangles) or pACYC-HPr(L47A/F48A) (open squares) in M9 medium containing 0.2% glucose ( A ) or 0.2% mannitol ( B ). Growth of the ptsH deletion mutant (open circles) is shown as a negative control. The inset in panel ( B ) shows the expression level of mtlA measured by qRT-PCR in the ptsH deletion mutant harboring pACYC-HPr(K27E) relative to that in the strain harboring pACYC-HPr. ( C , D ) The ptsH deletion strain harboring pACYC-HPr ( C ) or pACYC-HPr(K27E) ( D ) was grown in M9 minimal medium supplemented with 0.04% glucose and 0.04% mannitol. Growth rates (optical density at 600 nm, gray lines with circles) and the concentrations of sugars (open squares for glucose and closed squares for mannitol) remaining in the medium were measured as a function of incubation time. Representative data from three independent and reproducible measurements are shown.

    Techniques Used: Mutagenesis, Derivative Assay, Expressing, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Incubation

    34) Product Images from "PDL1 And LDHA act as ceRNAs in triple negative breast cancer by regulating miR-34a"

    Article Title: PDL1 And LDHA act as ceRNAs in triple negative breast cancer by regulating miR-34a

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0593-2

    PDL1 is a target of miR-34a, and its functions could be inhibited by miR-34a. a The expression level of miR-34a was determined by qRT-PCR in the above cell lines. U6 snRNA was used as an internal control. b HCC38 and MDA-MB-231 cells were transfected with miR-34a mimic or scrambled oligonucleotide, and qRT-PCR analysis demonstrated that the transfection was successful. c HCC38 and MDA-MB-231 cells were transfected as described, and the mRNA and protein expression of PDL1 was suppressed by miR-34a. d Histogram presenting cell viability based on MTS assays for HCC38 and MDA-MB-231 cells 48 h after transfection. e Transwell invasion assays demonstrated that the PDL1 3’UTR promoted cell invasion. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. f The expression levels of PDL1 were determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. g The impact of miR-34a on immune cell populations in the tumor microenvironment. Flow cytometry revealed that miR-34a increased the number of CD8+ cells and CD4+ cells and reduced the number of macrophages and Tregs. All the data are shown as the mean ± s.e.m. * P
    Figure Legend Snippet: PDL1 is a target of miR-34a, and its functions could be inhibited by miR-34a. a The expression level of miR-34a was determined by qRT-PCR in the above cell lines. U6 snRNA was used as an internal control. b HCC38 and MDA-MB-231 cells were transfected with miR-34a mimic or scrambled oligonucleotide, and qRT-PCR analysis demonstrated that the transfection was successful. c HCC38 and MDA-MB-231 cells were transfected as described, and the mRNA and protein expression of PDL1 was suppressed by miR-34a. d Histogram presenting cell viability based on MTS assays for HCC38 and MDA-MB-231 cells 48 h after transfection. e Transwell invasion assays demonstrated that the PDL1 3’UTR promoted cell invasion. Representative images of invaded cells are shown in the left panel, and the results are summarized in the right panel. f The expression levels of PDL1 were determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. g The impact of miR-34a on immune cell populations in the tumor microenvironment. Flow cytometry revealed that miR-34a increased the number of CD8+ cells and CD4+ cells and reduced the number of macrophages and Tregs. All the data are shown as the mean ± s.e.m. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Transfection, Western Blot, Flow Cytometry, Cytometry

    LDHA is highly expressed in TNBC and correlated with a poor outcome. a The expression level of LDHA was determined by qRT-PCR and Western blotting in the above cell lines. β-Actin was used as an internal control. b The expression levels of LDHA in 20 pairs of TNBC tissues and their matched normal adjacent tissues. c OS (left) and DFS (right) curves for breast cancer patients with positive or negative LDHA expression. d OS (left) and DFS (right) curves for TNBC patients with positive or negative LDHA expression. All the data are shown as the mean ± s.e.m. * P
    Figure Legend Snippet: LDHA is highly expressed in TNBC and correlated with a poor outcome. a The expression level of LDHA was determined by qRT-PCR and Western blotting in the above cell lines. β-Actin was used as an internal control. b The expression levels of LDHA in 20 pairs of TNBC tissues and their matched normal adjacent tissues. c OS (left) and DFS (right) curves for breast cancer patients with positive or negative LDHA expression. d OS (left) and DFS (right) curves for TNBC patients with positive or negative LDHA expression. All the data are shown as the mean ± s.e.m. * P

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

    PDL1 is highly expressed in TNBC and correlated with a poor outcome. a The expression level of PDL1 was determined by qRT-PCR and Western blotting in seven different mammary cell lines, including one HME cell line (MCF-10A) and six TNBC cell lines. PDL1 expression was normalized using β-actin expression. b The expression level of PDL1 in 20 pairs of TNBC tissues and their matched normal adjacent tissues. c OS (left) and DFS (right) curves for breast cancer patients with positive or negative PDL1 expression. d OS (left) and DFS (right) curves for TNBC patients with positive or negative PDL1 expression. All the data are shown as the mean ± s.e.m. * P
    Figure Legend Snippet: PDL1 is highly expressed in TNBC and correlated with a poor outcome. a The expression level of PDL1 was determined by qRT-PCR and Western blotting in seven different mammary cell lines, including one HME cell line (MCF-10A) and six TNBC cell lines. PDL1 expression was normalized using β-actin expression. b The expression level of PDL1 in 20 pairs of TNBC tissues and their matched normal adjacent tissues. c OS (left) and DFS (right) curves for breast cancer patients with positive or negative PDL1 expression. d OS (left) and DFS (right) curves for TNBC patients with positive or negative PDL1 expression. All the data are shown as the mean ± s.e.m. * P

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

    PDL1 and LDHA act as ceRNAs in TNBC by regulating miR-34a. a The expression level of miR-34a was determined by qRT-PCR in stable cell lines expressing the PDL1 3’UTR or LDHA 3’UTR. U6 snRNA was used as an internal control. b The expression level of PDL1 was determined by qRT-PCR and Western blotting. β-Actin was used as an internal control. c The expression level of LDHA was determined by qRT-PCR and Western blotting. β-Actin was used as an internal control. d The expression level of LDHA was determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. e The expression level of PDL1 was determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. f Lactate production and glucose consumption was evaluated by measuring the lactate and glucose levels in cell medium. g The impact of the LDHA 3’UTR on immune cell populations in the tumor microenvironment. Flow cytometry revealed that the LDHA 3’UTR increased the number of macrophages and Tregs and reduced the number of CD8+ cells and CD4+ cells. All of the data are shown as the mean ± s.e.m. ** P
    Figure Legend Snippet: PDL1 and LDHA act as ceRNAs in TNBC by regulating miR-34a. a The expression level of miR-34a was determined by qRT-PCR in stable cell lines expressing the PDL1 3’UTR or LDHA 3’UTR. U6 snRNA was used as an internal control. b The expression level of PDL1 was determined by qRT-PCR and Western blotting. β-Actin was used as an internal control. c The expression level of LDHA was determined by qRT-PCR and Western blotting. β-Actin was used as an internal control. d The expression level of LDHA was determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. e The expression level of PDL1 was determined by Western blotting in xenograft tumors (six in each group). β-Actin was used as an internal control. f Lactate production and glucose consumption was evaluated by measuring the lactate and glucose levels in cell medium. g The impact of the LDHA 3’UTR on immune cell populations in the tumor microenvironment. Flow cytometry revealed that the LDHA 3’UTR increased the number of macrophages and Tregs and reduced the number of CD8+ cells and CD4+ cells. All of the data are shown as the mean ± s.e.m. ** P

    Techniques Used: Activated Clotting Time Assay, Expressing, Quantitative RT-PCR, Stable Transfection, Western Blot, Flow Cytometry, Cytometry

    35) Product Images from "LincHOTAIR epigenetically silences miR34a by binding to PRC2 to promote the epithelial-to-mesenchymal transition in human gastric cancer"

    Article Title: LincHOTAIR epigenetically silences miR34a by binding to PRC2 to promote the epithelial-to-mesenchymal transition in human gastric cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.150

    Correlation between the expression of HOTAIR and the miR34a . ( a and b ) MiR34a expression was detected in BGC-823 and SGC-7901 cells with si-HOTAIR by qRT-PCR. All experiments were performed in triplicate with three technical replicates. ( c ) The expression level of miR34a in 61 paired tumors and peritumoral gastric cancers was detected by qRT-PCR. ( d and e ) Correlation analysis between the expression of HOTAIR and miR34a was examined in diffuse- and intestinal-type gastric cancer tissues. * P
    Figure Legend Snippet: Correlation between the expression of HOTAIR and the miR34a . ( a and b ) MiR34a expression was detected in BGC-823 and SGC-7901 cells with si-HOTAIR by qRT-PCR. All experiments were performed in triplicate with three technical replicates. ( c ) The expression level of miR34a in 61 paired tumors and peritumoral gastric cancers was detected by qRT-PCR. ( d and e ) Correlation analysis between the expression of HOTAIR and miR34a was examined in diffuse- and intestinal-type gastric cancer tissues. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    The association of HOTAIR with the PRC2 protein complex is critical for its regulation of miR34a . ( a ) Genome browser and analyzed H3K27me3 enrichment peaks in the miR34a promoter region. ( b ) RIP experiments were performed using the EZH2 antibodies for immunoprecipitation. Specific primers for HOTAIR were used to detect HOTAIR . ( c and d ) Expression of miR34a in BGC-823, SGC-7901 and MGC-803 cells transfected with si-EZH2, si-SUZ12 was detected by qRT-PCR. ( e and f ) ChIP analyses in SGC-7901 transfected with Si-HOTAIR and Si-EZH2 cells were performed on the miR34a promoter regions using anti-H3K27me3 and EZH2 antibodies. Enrichment was determined relative to the input controls. All experiments were performed in triplicate with three technical replicates. * P
    Figure Legend Snippet: The association of HOTAIR with the PRC2 protein complex is critical for its regulation of miR34a . ( a ) Genome browser and analyzed H3K27me3 enrichment peaks in the miR34a promoter region. ( b ) RIP experiments were performed using the EZH2 antibodies for immunoprecipitation. Specific primers for HOTAIR were used to detect HOTAIR . ( c and d ) Expression of miR34a in BGC-823, SGC-7901 and MGC-803 cells transfected with si-EZH2, si-SUZ12 was detected by qRT-PCR. ( e and f ) ChIP analyses in SGC-7901 transfected with Si-HOTAIR and Si-EZH2 cells were performed on the miR34a promoter regions using anti-H3K27me3 and EZH2 antibodies. Enrichment was determined relative to the input controls. All experiments were performed in triplicate with three technical replicates. * P

    Techniques Used: Immunoprecipitation, Expressing, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation

    HOTAIR induces EMT by silencing miR34a in gastric cancer. ( a ) qRT-PCR was used to detect miR34a expression of BGC-823 and SGC-7901 cells with miR34a mimics. ( b ) Transwell assays were used to investigate the changes in the migratory and invasive abilities of gastric cancer cells with miR34a mimics. ( c ) Western blot assays were performed to detect the protein of C-Met, Snail and EMT markers in BGC-823 and SGC-7901 cells with miR34a mimics. GAPDH was used as a control. ( d and e ) Western blot assay and qRT-PCR were performed for analysis of E-cadherin, N-cadherin and vimentin; C-Met and Snail in BGC-823 and SGC-7901 cells. All experiments were performed in triplicate with three technical replicates. * P
    Figure Legend Snippet: HOTAIR induces EMT by silencing miR34a in gastric cancer. ( a ) qRT-PCR was used to detect miR34a expression of BGC-823 and SGC-7901 cells with miR34a mimics. ( b ) Transwell assays were used to investigate the changes in the migratory and invasive abilities of gastric cancer cells with miR34a mimics. ( c ) Western blot assays were performed to detect the protein of C-Met, Snail and EMT markers in BGC-823 and SGC-7901 cells with miR34a mimics. GAPDH was used as a control. ( d and e ) Western blot assay and qRT-PCR were performed for analysis of E-cadherin, N-cadherin and vimentin; C-Met and Snail in BGC-823 and SGC-7901 cells. All experiments were performed in triplicate with three technical replicates. * P

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

    Relative HOTAIR expression in gastric cancer tissues and its clinical significance. ( a ) Relative expression of HOTAIR in the intestinal type of gastric cancer ( n =35) compared to diffuse-type gastric cancer ( n =26). HOTAIR expression was evaluated by qRT-PCR and normalized to GAPDH expression. Final results were presented as fold change in tumor tissues relative to normal tissues. Fold change is ≥2.0 for high expression and
    Figure Legend Snippet: Relative HOTAIR expression in gastric cancer tissues and its clinical significance. ( a ) Relative expression of HOTAIR in the intestinal type of gastric cancer ( n =35) compared to diffuse-type gastric cancer ( n =26). HOTAIR expression was evaluated by qRT-PCR and normalized to GAPDH expression. Final results were presented as fold change in tumor tissues relative to normal tissues. Fold change is ≥2.0 for high expression and

    Techniques Used: Expressing, Quantitative RT-PCR

    HOTAIR -promoted gastric cancer cell invasion, metastasis and EMT. ( a ) qRT-PCR was used to detect HOTAIR expression of BGC-823 and SGC-7901 cells with si-HOTAIRs. ( b ) Transwell assays were used to investigate the changes in migratory and invasive abilities of GC cells. ( c ) The lungs from mice in each experimental group with the numbers of tumor nodules on lung surfaces and weight were shown. ( d ) Visualization of the entire lung and HE-stained lung sections. ( e and f ) Western blotting and qRT-PCR were performed for analysis of E-cadherin, N-cadherin and vimentin in BGC-823 and SGC-7901 cells with si-HOTAIR. All experiments were performed in triplicate with three technical replicates. * P
    Figure Legend Snippet: HOTAIR -promoted gastric cancer cell invasion, metastasis and EMT. ( a ) qRT-PCR was used to detect HOTAIR expression of BGC-823 and SGC-7901 cells with si-HOTAIRs. ( b ) Transwell assays were used to investigate the changes in migratory and invasive abilities of GC cells. ( c ) The lungs from mice in each experimental group with the numbers of tumor nodules on lung surfaces and weight were shown. ( d ) Visualization of the entire lung and HE-stained lung sections. ( e and f ) Western blotting and qRT-PCR were performed for analysis of E-cadherin, N-cadherin and vimentin in BGC-823 and SGC-7901 cells with si-HOTAIR. All experiments were performed in triplicate with three technical replicates. * P

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

    36) Product Images from "Bufalin Inhibits Proliferation and Induces Apoptosis in Osteosarcoma Cells by Downregulating MicroRNA-221"

    Article Title: Bufalin Inhibits Proliferation and Induces Apoptosis in Osteosarcoma Cells by Downregulating MicroRNA-221

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2016/7319464

    Bufalin downregulates miR-221 in U-2OS and Saos-2 cells. (a) Microarray analysis was used to compare the expression profiles of miRNAs in Saos-2 cells that were untreated or treated with bufalin at its IC50. miR-221, one of the most markedly downregulated miRNAs, was labeled with a red box. (b) Detected by qRT-PCR, miR-221 level dramatically decreased 2–5-fold after being treated with bufalin in both U-2OS and Saos-2 cells ( ∗ P
    Figure Legend Snippet: Bufalin downregulates miR-221 in U-2OS and Saos-2 cells. (a) Microarray analysis was used to compare the expression profiles of miRNAs in Saos-2 cells that were untreated or treated with bufalin at its IC50. miR-221, one of the most markedly downregulated miRNAs, was labeled with a red box. (b) Detected by qRT-PCR, miR-221 level dramatically decreased 2–5-fold after being treated with bufalin in both U-2OS and Saos-2 cells ( ∗ P

    Techniques Used: Microarray, Expressing, Labeling, Quantitative RT-PCR

    37) Product Images from "Overexpression of zinc-α2-glycoprotein suppressed seizures and seizure-related neuroflammation in pentylenetetrazol-kindled rats"

    Article Title: Overexpression of zinc-α2-glycoprotein suppressed seizures and seizure-related neuroflammation in pentylenetetrazol-kindled rats

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1132-6

    Expression of GFP and AZGP 1 after injection of AAV vectors. a Fluorescent images showing GFP expression in the hippocampus of rats 3 and 9 weeks after AAV injection. The scale bar = 500 μm. b qrt-PCR showed increased AZGP1 mRNA level in the hippocampus of rats in AAV– AZGP1 group compared to AAV–GFP group 3 and 9 weeks after AAV injection. c Western blots showed increased ZAG protein level in the hippocampus of rats in AAV– AZGP1 group compared to AAV–GFP group 3 and 9 weeks after AAV injection. Optic density was normalized by GAPDH. n = 5 for each group, * p
    Figure Legend Snippet: Expression of GFP and AZGP 1 after injection of AAV vectors. a Fluorescent images showing GFP expression in the hippocampus of rats 3 and 9 weeks after AAV injection. The scale bar = 500 μm. b qrt-PCR showed increased AZGP1 mRNA level in the hippocampus of rats in AAV– AZGP1 group compared to AAV–GFP group 3 and 9 weeks after AAV injection. c Western blots showed increased ZAG protein level in the hippocampus of rats in AAV– AZGP1 group compared to AAV–GFP group 3 and 9 weeks after AAV injection. Optic density was normalized by GAPDH. n = 5 for each group, * p

    Techniques Used: Expressing, Injection, Quantitative RT-PCR, Western Blot

    38) Product Images from "Sirtuin 6 protects cardiomyocytes from hypertrophy in vitro via inhibition of NF-?B-dependent transcriptional activity"

    Article Title: Sirtuin 6 protects cardiomyocytes from hypertrophy in vitro via inhibition of NF-?B-dependent transcriptional activity

    Journal: British Journal of Pharmacology

    doi: 10.1111/j.1476-5381.2012.01903.x

    SIRT6 mRNA and protein levels were upregulated in the in vitro model of cardiomyocyte hypertrophy. (A) Primary neonatal rat cardiomyocytes were treated with 100 nM of AngII for 24 h, and quantitative RT-PCR was performed to screen the mRNA changes of all seven sirtuins. (B and C) Cardiomyocytes were treated with indicated concentrations of AngII for 24 h (left panel) or with 100 nM of AngII for indicated time (right panel). The levels of mRNA (B) and protein (C) of SIRT6 were measured and normalized (mRNA by GAPDH and protein by α-tubulin), and then presented as the fold of control levels. Data are presented as means ± SE. * P
    Figure Legend Snippet: SIRT6 mRNA and protein levels were upregulated in the in vitro model of cardiomyocyte hypertrophy. (A) Primary neonatal rat cardiomyocytes were treated with 100 nM of AngII for 24 h, and quantitative RT-PCR was performed to screen the mRNA changes of all seven sirtuins. (B and C) Cardiomyocytes were treated with indicated concentrations of AngII for 24 h (left panel) or with 100 nM of AngII for indicated time (right panel). The levels of mRNA (B) and protein (C) of SIRT6 were measured and normalized (mRNA by GAPDH and protein by α-tubulin), and then presented as the fold of control levels. Data are presented as means ± SE. * P

    Techniques Used: In Vitro, Quantitative RT-PCR

    SIRT6 physically interacted with the NF-κB catalytic subunit p65 and repressed NF-κB-dependent transcriptional activity. (A) After immunoprecipitation of cardiomyocyte nuclear extracts with anti-SIRT6 or control IgG, samples of precipitated proteins (IP), of lysate (Before IP) and supernatant (After IP), were separated by 10% SDS-PAGE gels and immunoblotted with anti-SIRT6 and anti-p65 antibodies. (B) Cardiomyocytes were treated with 100 mM AngII for 60 min, and then the co-localization of SIRT6 (green) and p65 (red) were examined by confocal immunofluorescence microscopy. DAPI was used to mark the position of nuclei. (C) Cardiomyocytes were treated with 100 nM AngII for indicated time. Co-IP assay was performed to show the effect of AngII on SIRT6-p65 interaction. The samples of lysate before co-immunoprecipitation are lebelled as ‘Input’. Images representative of three independent experiments are shown. (D) SIRT6 depleted cardiomyocytes (siSIRT6) were treated with or without PDTC, parthenolide or transfected with sip65. The mRNA levels of hypertrophic biomarkers were measured by quantitative RT-PCR. Data are presented as means ± SE. * P
    Figure Legend Snippet: SIRT6 physically interacted with the NF-κB catalytic subunit p65 and repressed NF-κB-dependent transcriptional activity. (A) After immunoprecipitation of cardiomyocyte nuclear extracts with anti-SIRT6 or control IgG, samples of precipitated proteins (IP), of lysate (Before IP) and supernatant (After IP), were separated by 10% SDS-PAGE gels and immunoblotted with anti-SIRT6 and anti-p65 antibodies. (B) Cardiomyocytes were treated with 100 mM AngII for 60 min, and then the co-localization of SIRT6 (green) and p65 (red) were examined by confocal immunofluorescence microscopy. DAPI was used to mark the position of nuclei. (C) Cardiomyocytes were treated with 100 nM AngII for indicated time. Co-IP assay was performed to show the effect of AngII on SIRT6-p65 interaction. The samples of lysate before co-immunoprecipitation are lebelled as ‘Input’. Images representative of three independent experiments are shown. (D) SIRT6 depleted cardiomyocytes (siSIRT6) were treated with or without PDTC, parthenolide or transfected with sip65. The mRNA levels of hypertrophic biomarkers were measured by quantitative RT-PCR. Data are presented as means ± SE. * P

    Techniques Used: Activity Assay, Immunoprecipitation, SDS Page, Immunofluorescence, Microscopy, Co-Immunoprecipitation Assay, Transfection, Quantitative RT-PCR

    SIRT6 knockdown mimicked the hypertrophic responses in primary neonatal rat cardiomyocytes. (A and B) Three independent siRNAs (marked as S1, S2 and S3), as well as the negative control (Neg), were transfected into cardiomyocytes. The mRNA and protein expression levels of SIRT6 were measured by quantitative RT-PCR (A) and Western blotting (B). (C) Cardiomyocytes were treated with AngII (100 nM, for 24 h) or subjected to SIRT6 knockdown (siSIRT6). Cell surface area was measured to demonstrate the hypertrophic responses in cardiomyocytes. (D) The mRNA levels of hypertrophic biomarkers ANF, BNP, and β-MHC were detected by quantitative RT-PCR. Data are presented as means ± SE. * P
    Figure Legend Snippet: SIRT6 knockdown mimicked the hypertrophic responses in primary neonatal rat cardiomyocytes. (A and B) Three independent siRNAs (marked as S1, S2 and S3), as well as the negative control (Neg), were transfected into cardiomyocytes. The mRNA and protein expression levels of SIRT6 were measured by quantitative RT-PCR (A) and Western blotting (B). (C) Cardiomyocytes were treated with AngII (100 nM, for 24 h) or subjected to SIRT6 knockdown (siSIRT6). Cell surface area was measured to demonstrate the hypertrophic responses in cardiomyocytes. (D) The mRNA levels of hypertrophic biomarkers ANF, BNP, and β-MHC were detected by quantitative RT-PCR. Data are presented as means ± SE. * P

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

    39) Product Images from "Mitochondrial NDUFA4L2 protein promotes the vitality of lung cancer cells by repressing oxidative stress"

    Article Title: Mitochondrial NDUFA4L2 protein promotes the vitality of lung cancer cells by repressing oxidative stress

    Journal: Thoracic Cancer

    doi: 10.1111/1759-7714.12984

    NDUFA4L2 is involved in the survival and migration of non‐small cell lung cancer (NSCLC) in hypoxia. ( a ) NDUFA4L2 messenger RNA (mRNA) of human NSCLC tissue samples was measured by quantitative PCR. ( b ) Four pairs of tissue samples were randomly extracted and measured by Western blotting. ( c ) The results of quantitative analysis of NDUFA4L2 were significant. ( d ) The expression of NDUFA4L2 and HIF‐1α was measured by immunohistochemical staining. A high percentage of tumor cells and high staining intensity indicate high expression. Pneumocytes were the normal cells. ( e ) HIF‐1α and NDUFA4L2 protein levels in NSCLC cell lines were measured by Western blotting assays. ( f–i ) The results of quantitative analysis of NDUFA4L2 and HIF‐1α were significant. ( ) Normoxia (NX), and ( ) Hypoxia (HY). ( j ) Wound healing assays in NSCLC cell lines cultured in normoxia and hypoxia are shown. All data were analyzed as the mean ± standard deviation from at least three independent experiments. * P
    Figure Legend Snippet: NDUFA4L2 is involved in the survival and migration of non‐small cell lung cancer (NSCLC) in hypoxia. ( a ) NDUFA4L2 messenger RNA (mRNA) of human NSCLC tissue samples was measured by quantitative PCR. ( b ) Four pairs of tissue samples were randomly extracted and measured by Western blotting. ( c ) The results of quantitative analysis of NDUFA4L2 were significant. ( d ) The expression of NDUFA4L2 and HIF‐1α was measured by immunohistochemical staining. A high percentage of tumor cells and high staining intensity indicate high expression. Pneumocytes were the normal cells. ( e ) HIF‐1α and NDUFA4L2 protein levels in NSCLC cell lines were measured by Western blotting assays. ( f–i ) The results of quantitative analysis of NDUFA4L2 and HIF‐1α were significant. ( ) Normoxia (NX), and ( ) Hypoxia (HY). ( j ) Wound healing assays in NSCLC cell lines cultured in normoxia and hypoxia are shown. All data were analyzed as the mean ± standard deviation from at least three independent experiments. * P

    Techniques Used: Migration, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemistry, Staining, Cell Culture, Standard Deviation

    40) Product Images from "microRNA-103 regulates the growth and invasion of endometrial cancer cells through the downregulation of tissue inhibitor of metalloproteinase 3"

    Article Title: microRNA-103 regulates the growth and invasion of endometrial cancer cells through the downregulation of tissue inhibitor of metalloproteinase 3

    Journal: Oncology Letters

    doi: 10.3892/ol.2012.638

    TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. * P
    Figure Legend Snippet: TIMP-3 3’-UTR is a direct target for miR-103. (A) Suppression of miR-103 by anti-miR-103 in Ishikawa cells is shown. The cells were frst transfected with anti-miR-103 or negative control oligonucleotide. The cells were harvested 2 days later and total RNA was extracted using TRIzol reagent. Mature miR-103 was detected by SYBR qRT-PCR, using U6 RNA for normalization. (B) Suppression of miR-103 by anti-miR-103 in HEC-1B cells is shown. Following transfection with anti-miR-103 (Anti-miR) or negative control oligonucleotide (Neg Control), mature miR-103 was detected as in (A). (C) An evolutionarily conserved target sequence for miR-103 is detected in the 3’-UTR of TIMP-3. (D and E) The luciferase assay was performed using Ishikawa and HEC-1B cells, respectively. The effect of miR-103 on the luciferase activity of pGL3-TIMP-3-wt and pGL3-TIMP-3-mut was measured as described in Materials and methods. A statistically significant upregulation in luciferase activity was found when the cells were transfected with pGL3-TIMP-3-wt together with anti-miR-103. pGL3-TIMP-3-wt, vector with wild-type TIMP-3 3’-UTR; pGL3-TIMP-3-mut, vector with mutated TIMP-3 3’-UTR. Each bar is the mean ± SEM from three independent experiments. * P

    Techniques Used: Transfection, Negative Control, Quantitative RT-PCR, Sequencing, Luciferase, Activity Assay, Plasmid Preparation

    Related Articles

    Amplification:

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    Article Snippet: .. Total RNA was extracted by using Atlas Pure Total RNA Labeling Kit (CLONTECH). cDNA was synthesized from 1 μg total RNA, by using Advantage RT-for-PCR Kit (CLONTECH). cDNA samples were subjected to PCR amplification with DNA primers selective for the human genes. .. For each gene, the DNA primers were derived from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA.

    Article Title: Isolation and characterization of multipotent stem cells from human cruciate ligaments
    Article Snippet: .. Complementary DNA (cDNA) was obtained by RT of 1 µg total RNA using Advantage RT‐for‐PCR (Clontech, Palo Alto, CA, USA) per manufacturer's instructions. cDNA was amplified using ABI GeneAmp PCR system 2400 (PerkinElmer Applied Biosystems, Boston, MA, USA) at 94 °C for 40 s, 56 °C for 50 s, and 72 °C for 60 s for 30 cycles, after initial denaturation at 94 °C for 5 min. Primers used for amplification are listed in . .. Briefly, cells were treated with 0.06 µg/ml colcemid (Invitrogen) for 2–4 h, trypsinized, incubated in 0.075 m of KCl for 10 min, and fixed in Carnoy's fixative.

    Synthesized:

    Article Title: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells
    Article Snippet: .. Total RNA was extracted by using Atlas Pure Total RNA Labeling Kit (CLONTECH). cDNA was synthesized from 1 μg total RNA, by using Advantage RT-for-PCR Kit (CLONTECH). cDNA samples were subjected to PCR amplification with DNA primers selective for the human genes. .. For each gene, the DNA primers were derived from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA.

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway
    Article Snippet: .. The RNA isolation was performed from human FFPE sections, human glioma brain tissue, N9 cells, and mouse brains using Tri reagent (Sigma-Aldrich), and cDNA was synthesized using an Advantage RT-for-PCR kit (Clontech Laboratories) as per manufacturer’s protocol. qRT-PCR was carried out as described previously [ ] from 500 ng RNA, using primers specific for mouse IL-1β, HSP60, and NLRP3 genes. ..

    Article Title: Network-based approach to prediction and population-based validation of in silico drug repurposing
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    Isolation:

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    Article Title: Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression
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    Incubation:

    Article Title: Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC
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    Labeling:

    Article Title: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells
    Article Snippet: .. Total RNA was extracted by using Atlas Pure Total RNA Labeling Kit (CLONTECH). cDNA was synthesized from 1 μg total RNA, by using Advantage RT-for-PCR Kit (CLONTECH). cDNA samples were subjected to PCR amplification with DNA primers selective for the human genes. .. For each gene, the DNA primers were derived from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA.

    Produced:

    Article Title: Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC
    Article Snippet: .. Total cDNA was produced by using the Advantage RT-for-PCR kit (Clontech), following the manufacturer's instructions, with oligo(dT) primer (GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTV), and incubated for 2 min at 70°C, then 1 h at 42°C, and then 5 min at 94°C. .. The coding region sequence of CD1.2 was from a clone with no 5′UTR and 51 bp of 3′UTR, derived using 0.5 μl (25 ng) of cDNA from various CB tissues and 20 pmol each primer [primer nos.

    Polymerase Chain Reaction:

    Article Title: Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells
    Article Snippet: .. Total RNA was extracted by using Atlas Pure Total RNA Labeling Kit (CLONTECH). cDNA was synthesized from 1 μg total RNA, by using Advantage RT-for-PCR Kit (CLONTECH). cDNA samples were subjected to PCR amplification with DNA primers selective for the human genes. .. For each gene, the DNA primers were derived from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA.

    Article Title: Isolation and characterization of multipotent stem cells from human cruciate ligaments
    Article Snippet: .. Complementary DNA (cDNA) was obtained by RT of 1 µg total RNA using Advantage RT‐for‐PCR (Clontech, Palo Alto, CA, USA) per manufacturer's instructions. cDNA was amplified using ABI GeneAmp PCR system 2400 (PerkinElmer Applied Biosystems, Boston, MA, USA) at 94 °C for 40 s, 56 °C for 50 s, and 72 °C for 60 s for 30 cycles, after initial denaturation at 94 °C for 5 min. Primers used for amplification are listed in . .. Briefly, cells were treated with 0.06 µg/ml colcemid (Invitrogen) for 2–4 h, trypsinized, incubated in 0.075 m of KCl for 10 min, and fixed in Carnoy's fixative.

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    Generated:

    Article Title: Expression of Gab1 Lacking the Pleckstrin Homology Domain Is Associated with Neoplastic Progression
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    Formalin-fixed Paraffin-Embedded:

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway
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    Quantitative RT-PCR:

    Article Title: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway
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    Reverse Transcription Polymerase Chain Reaction:

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    TaKaRa real time quantitative polymerase chain reaction qrt pcr total rna
    LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. ( a ) The mRNA expression of PCNA was detected by <t>qRT-PCR</t> in rats′ PAs tissues. ( b , c ) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ## p
    Real Time Quantitative Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Real-time quantitative <t>PCR</t> analysis of IL-4, IL-13, IL-6, IL-12p35, IL-12p40, and IFNγ mRNA levels in splenocytes isolated from OVA-immunized mice that were subsequently treated with 10 μg/mL OVA + 3.0 μM ODN 1612 , ODN 1555 , A151, H154, or iSG3. Mouse splenocytes were pre-incubated in medium for 3 h prior to exposure to OVA and ODNs for 72 h. Expression of IL-4 (A), IL-13 (B), IL-6 (C), IL-12p35 (D), IL-12p40 (E), and IFNγ (F) mRNAs were determined with real-time quantitative PCR. Results are shown as the ratio of <t>cytokine</t> mRNA levels (normalized to β-actin; see Section 2 ) in stimulated versus non-treated cells, as well as the level in the medium control. Values represent means, and error bars indicate the SD of three independent experiments. Values with different letters ( i.e., a, b, c, d, and e) were significantly different ( P
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    <t>qRT-PCR</t> analysis of the expression of defense signaling genes in transgenic eggplants. Quantitative reverse-transcription PCR (qPCR) was performed using a SYBR Premix Ex Taq kit (TaKaRa, Dalian, China), following the manufacturer’s protocols. Triplicate qPCR reactions were performed for each sample and the relative gene expression data was analyzed using the 2 −ΔΔ Ct method. ( a ) qRT-PCR analysis of defense signaling genes in SmNAC oveexpressing plants. CK represents non-transgenic plants from the E-31 line, whereas 1–3 show the SmNAC overexpressing transgenic T 0 plants EGT 0–87 , EGT 0–145 , and EGT 0–204 , and 4–6 show the SmNAC overexpressing transgenic T 1 plants EGT 1–87 , EGT 1–145 , and EGT 1–204 . ( b ) qRT-PCR analysis of defense signaling genes in RNAi- SmNAC plants. CK represents non-transgenic plants from line E-32. 1–5 show the RNAi- SmNAC transgenic plants (T0) RNAi-1, RNAi-2, RNAi-3, RNAi-4, and RNAi-5.
    Quantitative Reverse Transcription Qrt Pcr Analysis Quantitative Reverse Transcription Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa real time quantitative pcr analysis qrt pcr total rna
    LncRNA DBH-AS1 is inactivated by p53 A. The potential p53-binding site upstream of DBH-AS1 predicted by JASPAR database. B. Western blot analysis showed the reduced levels of p53 protein in HepG2 cells and LO2 cells transfected with siRNAs. C. Reduced p53 mRNA expression by siRNAs in HepG2 cells and LO2 cells was shown by <t>qRT-PCR.</t> D. Expression of DBH-AS1 transcripts was quantified by qRT-PCR. Data shown are the mean ± SD of three independent experiments. * P
    Real Time Quantitative Pcr Analysis Qrt Pcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time quantitative pcr analysis qrt pcr total rna/product/TaKaRa
    Average 88 stars, based on 1 article reviews
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    Image Search Results


    LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. ( a ) The mRNA expression of PCNA was detected by qRT-PCR in rats′ PAs tissues. ( b , c ) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ## p

    Journal: Respiratory Research

    Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension

    doi: 10.1186/s12931-019-1018-x

    Figure Lengend Snippet: LncRNA CASC2 inhibited hypoxia-induced cell proliferation in rat pulmonary arterial tissues. ( a ) The mRNA expression of PCNA was detected by qRT-PCR in rats′ PAs tissues. ( b , c ) The protein expression of PCNA was detected by western blot analysis in rat pulmonary arterial tissues. ## p

    Article Snippet: Real time-quantitative polymerase chain reaction (qRT-PCR) Total RNA from PASMCs or rat pulmonary arterial tissues was extracted using the Trizol reagent and reversely transcribed into cDNA using Prime-Script RT-PCR kit (Takara, Dalian, China) following the manufacturer′s instructions.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Up-regulation of lncRNA CASC2 inhibited proliferation in hypoxia-induced PASMCs. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in PASMCs. ( b ) Cell number of PASMCs was counted by CyQuant cell proliferation assay kit. ( c ) The mRNA expression of PCNA was detected by qRT-PCR in PASMCs. ( d ) The protein expression of PCNA was detected by western blot analysis in PASMCs. ( e , f ) Cell proliferation status of PASMCs was assessed by EdU staining assay. ### p

    Journal: Respiratory Research

    Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension

    doi: 10.1186/s12931-019-1018-x

    Figure Lengend Snippet: Up-regulation of lncRNA CASC2 inhibited proliferation in hypoxia-induced PASMCs. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in PASMCs. ( b ) Cell number of PASMCs was counted by CyQuant cell proliferation assay kit. ( c ) The mRNA expression of PCNA was detected by qRT-PCR in PASMCs. ( d ) The protein expression of PCNA was detected by western blot analysis in PASMCs. ( e , f ) Cell proliferation status of PASMCs was assessed by EdU staining assay. ### p

    Article Snippet: Real time-quantitative polymerase chain reaction (qRT-PCR) Total RNA from PASMCs or rat pulmonary arterial tissues was extracted using the Trizol reagent and reversely transcribed into cDNA using Prime-Script RT-PCR kit (Takara, Dalian, China) following the manufacturer′s instructions.

    Techniques: Expressing, Quantitative RT-PCR, CyQUANT Assay, Proliferation Assay, Western Blot, Staining

    LncRNA CASC2 was down-regulated in hypoxia-induced rats and reversed the increase of mPAP and progression of right ventricular hypertrophy caused by hypoxia. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in rat pulmonary arterial tissues. ( b ) Systemic blood pressure (SBP) was assessed in normoxia and hypoxia-induced rats. ( c ) Mean pulmonary arterial pressure (mPAP) was detected in normoxia and hypoxia-induced rats. ( d ) The right ventricular hypertrophy index (RVI) was calculated in normoxia and hypoxia-induced rats. # p

    Journal: Respiratory Research

    Article Title: Long non-coding RNA CASC2 suppresses pulmonary artery smooth muscle cell proliferation and phenotypic switch in hypoxia-induced pulmonary hypertension

    doi: 10.1186/s12931-019-1018-x

    Figure Lengend Snippet: LncRNA CASC2 was down-regulated in hypoxia-induced rats and reversed the increase of mPAP and progression of right ventricular hypertrophy caused by hypoxia. ( a ) The expression of lncRNA CASC2 was detected by qRT-PCR in rat pulmonary arterial tissues. ( b ) Systemic blood pressure (SBP) was assessed in normoxia and hypoxia-induced rats. ( c ) Mean pulmonary arterial pressure (mPAP) was detected in normoxia and hypoxia-induced rats. ( d ) The right ventricular hypertrophy index (RVI) was calculated in normoxia and hypoxia-induced rats. # p

    Article Snippet: Real time-quantitative polymerase chain reaction (qRT-PCR) Total RNA from PASMCs or rat pulmonary arterial tissues was extracted using the Trizol reagent and reversely transcribed into cDNA using Prime-Script RT-PCR kit (Takara, Dalian, China) following the manufacturer′s instructions.

    Techniques: Expressing, Quantitative RT-PCR

    Real-time quantitative PCR analysis of IL-4, IL-13, IL-6, IL-12p35, IL-12p40, and IFNγ mRNA levels in splenocytes isolated from OVA-immunized mice that were subsequently treated with 10 μg/mL OVA + 3.0 μM ODN 1612 , ODN 1555 , A151, H154, or iSG3. Mouse splenocytes were pre-incubated in medium for 3 h prior to exposure to OVA and ODNs for 72 h. Expression of IL-4 (A), IL-13 (B), IL-6 (C), IL-12p35 (D), IL-12p40 (E), and IFNγ (F) mRNAs were determined with real-time quantitative PCR. Results are shown as the ratio of cytokine mRNA levels (normalized to β-actin; see Section 2 ) in stimulated versus non-treated cells, as well as the level in the medium control. Values represent means, and error bars indicate the SD of three independent experiments. Values with different letters ( i.e., a, b, c, d, and e) were significantly different ( P

    Journal: FEBS Open Bio

    Article Title: Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression

    doi: 10.1016/j.fob.2012.11.002

    Figure Lengend Snippet: Real-time quantitative PCR analysis of IL-4, IL-13, IL-6, IL-12p35, IL-12p40, and IFNγ mRNA levels in splenocytes isolated from OVA-immunized mice that were subsequently treated with 10 μg/mL OVA + 3.0 μM ODN 1612 , ODN 1555 , A151, H154, or iSG3. Mouse splenocytes were pre-incubated in medium for 3 h prior to exposure to OVA and ODNs for 72 h. Expression of IL-4 (A), IL-13 (B), IL-6 (C), IL-12p35 (D), IL-12p40 (E), and IFNγ (F) mRNAs were determined with real-time quantitative PCR. Results are shown as the ratio of cytokine mRNA levels (normalized to β-actin; see Section 2 ) in stimulated versus non-treated cells, as well as the level in the medium control. Values represent means, and error bars indicate the SD of three independent experiments. Values with different letters ( i.e., a, b, c, d, and e) were significantly different ( P

    Article Snippet: An equivalent volume of cDNA was used for quantification of various cytokine cDNAs with real-time quantitative PCR using a Thermal Cycler Dice® Real Time System (TaKaRa Bio Inc., Tokyo, Japan).

    Techniques: Real-time Polymerase Chain Reaction, Isolation, Mouse Assay, Incubation, Expressing

    qRT-PCR analysis of the expression of defense signaling genes in transgenic eggplants. Quantitative reverse-transcription PCR (qPCR) was performed using a SYBR Premix Ex Taq kit (TaKaRa, Dalian, China), following the manufacturer’s protocols. Triplicate qPCR reactions were performed for each sample and the relative gene expression data was analyzed using the 2 −ΔΔ Ct method. ( a ) qRT-PCR analysis of defense signaling genes in SmNAC oveexpressing plants. CK represents non-transgenic plants from the E-31 line, whereas 1–3 show the SmNAC overexpressing transgenic T 0 plants EGT 0–87 , EGT 0–145 , and EGT 0–204 , and 4–6 show the SmNAC overexpressing transgenic T 1 plants EGT 1–87 , EGT 1–145 , and EGT 1–204 . ( b ) qRT-PCR analysis of defense signaling genes in RNAi- SmNAC plants. CK represents non-transgenic plants from line E-32. 1–5 show the RNAi- SmNAC transgenic plants (T0) RNAi-1, RNAi-2, RNAi-3, RNAi-4, and RNAi-5.

    Journal: Scientific Reports

    Article Title: Overexpression of the Eggplant (Solanum melongena) NAC Family Transcription Factor SmNAC Suppresses Resistance to Bacterial Wilt

    doi: 10.1038/srep31568

    Figure Lengend Snippet: qRT-PCR analysis of the expression of defense signaling genes in transgenic eggplants. Quantitative reverse-transcription PCR (qPCR) was performed using a SYBR Premix Ex Taq kit (TaKaRa, Dalian, China), following the manufacturer’s protocols. Triplicate qPCR reactions were performed for each sample and the relative gene expression data was analyzed using the 2 −ΔΔ Ct method. ( a ) qRT-PCR analysis of defense signaling genes in SmNAC oveexpressing plants. CK represents non-transgenic plants from the E-31 line, whereas 1–3 show the SmNAC overexpressing transgenic T 0 plants EGT 0–87 , EGT 0–145 , and EGT 0–204 , and 4–6 show the SmNAC overexpressing transgenic T 1 plants EGT 1–87 , EGT 1–145 , and EGT 1–204 . ( b ) qRT-PCR analysis of defense signaling genes in RNAi- SmNAC plants. CK represents non-transgenic plants from line E-32. 1–5 show the RNAi- SmNAC transgenic plants (T0) RNAi-1, RNAi-2, RNAi-3, RNAi-4, and RNAi-5.

    Article Snippet: Quantitative reverse-transcription (qRT-PCR) analysis Quantitative reverse-transcription PCR (qPCR) was performed using gene-specific primers ( ) and a SYBR Premix Ex Taq kit (TaKaRa, Dalian, China), following the manufacturer’s protocols.

    Techniques: Quantitative RT-PCR, Expressing, Transgenic Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    LncRNA DBH-AS1 is inactivated by p53 A. The potential p53-binding site upstream of DBH-AS1 predicted by JASPAR database. B. Western blot analysis showed the reduced levels of p53 protein in HepG2 cells and LO2 cells transfected with siRNAs. C. Reduced p53 mRNA expression by siRNAs in HepG2 cells and LO2 cells was shown by qRT-PCR. D. Expression of DBH-AS1 transcripts was quantified by qRT-PCR. Data shown are the mean ± SD of three independent experiments. * P

    Journal: Oncotarget

    Article Title: HBx-related long non-coding RNA DBH-AS1 promotes cell proliferation and survival by activating MAPK signaling in hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: LncRNA DBH-AS1 is inactivated by p53 A. The potential p53-binding site upstream of DBH-AS1 predicted by JASPAR database. B. Western blot analysis showed the reduced levels of p53 protein in HepG2 cells and LO2 cells transfected with siRNAs. C. Reduced p53 mRNA expression by siRNAs in HepG2 cells and LO2 cells was shown by qRT-PCR. D. Expression of DBH-AS1 transcripts was quantified by qRT-PCR. Data shown are the mean ± SD of three independent experiments. * P

    Article Snippet: RNA extraction and real-time quantitative PCR analysis (qRT-PCR) Total RNA was extracted from cultured cells or tissues using TRIzol Reagent (Takara, Dalian, China).

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

    LncRNA DBH-AS1 induces cell-cycle progression in HCC cells A. HepG2 and SMMC-7721 cells with elevated DBH-AS1 expression were seeded on 96-well plates, and cell proliferation was examined by EdU immunofluorescence staining. Effect of DBH-AS1 knockdown on Hep3B and SK-Hep1 cell proliferation was also measured by EdU immunofluorescence staining. The graph on the right shows the percentage of EdU-positive nuclei. B. Cell-cycle analysis of HepG2 and SMMC-7721 cells overexpressing DBH-AS1 and Hep3B and SK-Hep1 cells with stably silenced DBH-AS1 expression. C. Proportion of cells in various phases of the cell cycle. D. - E. The relative expression levels of cell cycle associated genes, including CDK6, CCND1, CCNE1, P16, P21 and P27, were detected in HepG2 cells overexpressing DBH-AS1 and Hep3B cells with stably down-regulated DBH-AS1 expression by qRT-PCR D. and western blot with quantitative analysis E. . The results show the means ± SD from at least 3 separate experiments. * P

    Journal: Oncotarget

    Article Title: HBx-related long non-coding RNA DBH-AS1 promotes cell proliferation and survival by activating MAPK signaling in hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: LncRNA DBH-AS1 induces cell-cycle progression in HCC cells A. HepG2 and SMMC-7721 cells with elevated DBH-AS1 expression were seeded on 96-well plates, and cell proliferation was examined by EdU immunofluorescence staining. Effect of DBH-AS1 knockdown on Hep3B and SK-Hep1 cell proliferation was also measured by EdU immunofluorescence staining. The graph on the right shows the percentage of EdU-positive nuclei. B. Cell-cycle analysis of HepG2 and SMMC-7721 cells overexpressing DBH-AS1 and Hep3B and SK-Hep1 cells with stably silenced DBH-AS1 expression. C. Proportion of cells in various phases of the cell cycle. D. - E. The relative expression levels of cell cycle associated genes, including CDK6, CCND1, CCNE1, P16, P21 and P27, were detected in HepG2 cells overexpressing DBH-AS1 and Hep3B cells with stably down-regulated DBH-AS1 expression by qRT-PCR D. and western blot with quantitative analysis E. . The results show the means ± SD from at least 3 separate experiments. * P

    Article Snippet: RNA extraction and real-time quantitative PCR analysis (qRT-PCR) Total RNA was extracted from cultured cells or tissues using TRIzol Reagent (Takara, Dalian, China).

    Techniques: Expressing, Immunofluorescence, Staining, Cell Cycle Assay, Stable Transfection, Quantitative RT-PCR, Western Blot

    HBx induces the expression of lncRNA DBH-AS1 A. Ectopic re-expression of HBx was detected in Lv-HBx-transfected HepG2 and LO2 cells by qRT-PCR and western blot. β-actin was used as a loading control. B. The relative expression of lncRNA DBH-AS1 in HepG2 and LO2 cells re-expressing HBx compared with controls by qRT-PCR. Data are shown as the mean±SD based on at least three independent experiments. C. Comparison of levels of DBH-AS1 in HCC patients with and without HBV infection (independent t test). D. The correlation between DBH-AS1 transcript level and HBx mRNA level in 31 HCC tissues. The ΔCt values were subjected to Pearson correlation analysis. * P

    Journal: Oncotarget

    Article Title: HBx-related long non-coding RNA DBH-AS1 promotes cell proliferation and survival by activating MAPK signaling in hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: HBx induces the expression of lncRNA DBH-AS1 A. Ectopic re-expression of HBx was detected in Lv-HBx-transfected HepG2 and LO2 cells by qRT-PCR and western blot. β-actin was used as a loading control. B. The relative expression of lncRNA DBH-AS1 in HepG2 and LO2 cells re-expressing HBx compared with controls by qRT-PCR. Data are shown as the mean±SD based on at least three independent experiments. C. Comparison of levels of DBH-AS1 in HCC patients with and without HBV infection (independent t test). D. The correlation between DBH-AS1 transcript level and HBx mRNA level in 31 HCC tissues. The ΔCt values were subjected to Pearson correlation analysis. * P

    Article Snippet: RNA extraction and real-time quantitative PCR analysis (qRT-PCR) Total RNA was extracted from cultured cells or tissues using TRIzol Reagent (Takara, Dalian, China).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Infection

    LncRNA DBH-AS1 promotes HCC cell proliferation in vitro A. HepG2 and SMMC-7721 cells were infected with lentivirus carrying the DBH-AS1 gene, and HepG2 and SMMC-7721 cells stably overexpressing DBH-AS1 were screened by qRT-PCR. B. Short hairpin RNA against DBH-AS1 stably decreased the expression of DBH-AS1 in sh-DBH-AS1 Hep3B and SK-Hep1 cells compared with sh-control cells by qRT-PCR. C. After overexpression of DBH-AS1 in HepG2 and SMMC-7721 cells, the cell viability was assessed by CCK-8 assays daily for 3 days. D. Cell viability was assessed by CCK-8 assays daily for 3 days in Hep3B and SK-Hep1 cells with silenced DBH-AS1 expression. E. Colony formation assays were performed on HepG2 and SMMC-7721 cells stably overexpressing DBH-AS1 for 2 weeks. F. In vitro proliferative ability of Hep3B and SK-Hep1 cells was significantly decreased in DBH-AS1-suppressed cells compared to sh-control cells by colony formation assays. Data are presented as mean ± SD for at least three independent experiments, * P

    Journal: Oncotarget

    Article Title: HBx-related long non-coding RNA DBH-AS1 promotes cell proliferation and survival by activating MAPK signaling in hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: LncRNA DBH-AS1 promotes HCC cell proliferation in vitro A. HepG2 and SMMC-7721 cells were infected with lentivirus carrying the DBH-AS1 gene, and HepG2 and SMMC-7721 cells stably overexpressing DBH-AS1 were screened by qRT-PCR. B. Short hairpin RNA against DBH-AS1 stably decreased the expression of DBH-AS1 in sh-DBH-AS1 Hep3B and SK-Hep1 cells compared with sh-control cells by qRT-PCR. C. After overexpression of DBH-AS1 in HepG2 and SMMC-7721 cells, the cell viability was assessed by CCK-8 assays daily for 3 days. D. Cell viability was assessed by CCK-8 assays daily for 3 days in Hep3B and SK-Hep1 cells with silenced DBH-AS1 expression. E. Colony formation assays were performed on HepG2 and SMMC-7721 cells stably overexpressing DBH-AS1 for 2 weeks. F. In vitro proliferative ability of Hep3B and SK-Hep1 cells was significantly decreased in DBH-AS1-suppressed cells compared to sh-control cells by colony formation assays. Data are presented as mean ± SD for at least three independent experiments, * P

    Article Snippet: RNA extraction and real-time quantitative PCR analysis (qRT-PCR) Total RNA was extracted from cultured cells or tissues using TRIzol Reagent (Takara, Dalian, China).

    Techniques: In Vitro, Infection, Stable Transfection, Quantitative RT-PCR, shRNA, Expressing, Over Expression, CCK-8 Assay