quantitative real time polymerase chain reaction pcr  (Thermo Fisher)


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    Structured Review

    Thermo Fisher quantitative real time polymerase chain reaction pcr
    HDAC inhibitor TSA enhances basal Rgs10 expression and blocks LPS-stimulated suppression in microglia. (A) BV-2 cells were plated in 12-well plate and allowed to adhere overnight. Cells were treated with vehicle, 100, 250, and 500 nM TSA for 24 hours. Cells were harvested in TRIzol, and RNA was isolated. Rgs10 transcript was quantified using quantitative <t>RT-PCR</t> relative to actin. (B) BV-2 cells were treated with vehicle or 10 ng/mL LPS for 6 hours with or without 100 nM TSA, and Rgs10 transcript was quantified relative to the actin. (C) BV-2 cells were treated with vehicle or 1 µ g/mL LPS for 4 hours with or without 250 nM TSA, and Rgs10 . Cells were treated with vehicle or LPS (10 ng/mL) for 6 hours with or without TSA (100 nM). Rgs10 transcript expression was determined using quantitative RT-PCR relative to actin. (E) BV-2 cells were plated in six-well plate and allowed to adhere overnight. Cells were treated with either vehicle or LPS (10 ng/ml) for 48 hours with or without TSA (100 nM). Cells were harvested, and protein levels were assessed by Western blot analysis. Blot presented is a representative of three independent experiments. Densitometry values were normalized to GAPDH loading control and then to vehicle-treated condition (right panel). Data in each graph are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. ** P
    Quantitative Real Time Polymerase Chain Reaction Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity"

    Article Title: Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.116.106963

    HDAC inhibitor TSA enhances basal Rgs10 expression and blocks LPS-stimulated suppression in microglia. (A) BV-2 cells were plated in 12-well plate and allowed to adhere overnight. Cells were treated with vehicle, 100, 250, and 500 nM TSA for 24 hours. Cells were harvested in TRIzol, and RNA was isolated. Rgs10 transcript was quantified using quantitative RT-PCR relative to actin. (B) BV-2 cells were treated with vehicle or 10 ng/mL LPS for 6 hours with or without 100 nM TSA, and Rgs10 transcript was quantified relative to the actin. (C) BV-2 cells were treated with vehicle or 1 µ g/mL LPS for 4 hours with or without 250 nM TSA, and Rgs10 . Cells were treated with vehicle or LPS (10 ng/mL) for 6 hours with or without TSA (100 nM). Rgs10 transcript expression was determined using quantitative RT-PCR relative to actin. (E) BV-2 cells were plated in six-well plate and allowed to adhere overnight. Cells were treated with either vehicle or LPS (10 ng/ml) for 48 hours with or without TSA (100 nM). Cells were harvested, and protein levels were assessed by Western blot analysis. Blot presented is a representative of three independent experiments. Densitometry values were normalized to GAPDH loading control and then to vehicle-treated condition (right panel). Data in each graph are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. ** P
    Figure Legend Snippet: HDAC inhibitor TSA enhances basal Rgs10 expression and blocks LPS-stimulated suppression in microglia. (A) BV-2 cells were plated in 12-well plate and allowed to adhere overnight. Cells were treated with vehicle, 100, 250, and 500 nM TSA for 24 hours. Cells were harvested in TRIzol, and RNA was isolated. Rgs10 transcript was quantified using quantitative RT-PCR relative to actin. (B) BV-2 cells were treated with vehicle or 10 ng/mL LPS for 6 hours with or without 100 nM TSA, and Rgs10 transcript was quantified relative to the actin. (C) BV-2 cells were treated with vehicle or 1 µ g/mL LPS for 4 hours with or without 250 nM TSA, and Rgs10 . Cells were treated with vehicle or LPS (10 ng/mL) for 6 hours with or without TSA (100 nM). Rgs10 transcript expression was determined using quantitative RT-PCR relative to actin. (E) BV-2 cells were plated in six-well plate and allowed to adhere overnight. Cells were treated with either vehicle or LPS (10 ng/ml) for 48 hours with or without TSA (100 nM). Cells were harvested, and protein levels were assessed by Western blot analysis. Blot presented is a representative of three independent experiments. Densitometry values were normalized to GAPDH loading control and then to vehicle-treated condition (right panel). Data in each graph are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. ** P

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

    Rgs10 transcript and RGS protein suppressed in BV-2 mouse microglial cells following LPS treatment in a dose-dependent fashion. (A) BV2 cells were treated with vehicle (serum-free media), 10 ng/ml, 100 ng/ml, 1 µ g/ml, and 10 µ g/ml LPS for 4 hours. Cells were harvested in TRIzol, and RNA was isolated. Rgs10 (top panel) and Tnf-α (bottom panel) transcripts were quantified using SYBR Green RT-PCR reagents and normalized to the housekeeping gene actin (2 −ΔΔCT ). (B) BV-2 cells were treated with vehicle, 10 ng/ml, 100 ng/ml, 1 µ g/ml, and 10 µ g/ml LPS for 24 hours, and total cell lysates were collected in SDS sample buffer and assessed using Western blotting with RGS10 and GAPDH antibodies. (C) BV-2 cells were treated with 100 ng/mL LPS for the times indicated, and Rgs10 transcript expression was determined using SYBR Green RT-PCR reagents and normalized to the housekeeping gene actin (2 −ΔΔCT ). Data are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. * P
    Figure Legend Snippet: Rgs10 transcript and RGS protein suppressed in BV-2 mouse microglial cells following LPS treatment in a dose-dependent fashion. (A) BV2 cells were treated with vehicle (serum-free media), 10 ng/ml, 100 ng/ml, 1 µ g/ml, and 10 µ g/ml LPS for 4 hours. Cells were harvested in TRIzol, and RNA was isolated. Rgs10 (top panel) and Tnf-α (bottom panel) transcripts were quantified using SYBR Green RT-PCR reagents and normalized to the housekeeping gene actin (2 −ΔΔCT ). (B) BV-2 cells were treated with vehicle, 10 ng/ml, 100 ng/ml, 1 µ g/ml, and 10 µ g/ml LPS for 24 hours, and total cell lysates were collected in SDS sample buffer and assessed using Western blotting with RGS10 and GAPDH antibodies. (C) BV-2 cells were treated with 100 ng/mL LPS for the times indicated, and Rgs10 transcript expression was determined using SYBR Green RT-PCR reagents and normalized to the housekeeping gene actin (2 −ΔΔCT ). Data are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. * P

    Techniques Used: Isolation, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

    The 5-Aza does not affect LPS-induced suppression of Rgs10 transcription. (A) BV-2 cells were plated in 12-well plate and allowed to adhere overnight. Cells were treated with vehicle (serum-free media) or LPS (10 ng/ml, for 6 hours) with or without 5-Aza (AZA, 10 µ M). Cells were harvested in TRIzol, and RNA was isolated. mRNA transcript levels were measured by quantitative real-time PCR. Transcripts were normalized to the housekeeping gene actin. (B) BV-2 cells were plated in 24-well plate and allowed to adhere overnight. Cells were treated with either vehicle (serum-free media) or LPS (10 ng/ml) for 48 hours or with 5-Aza (AZA, 10 µ M). Cells were harvested, and protein levels were assessed by Western blot analysis with RGS10 and GAPDH control antibodies. Data are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. ** P
    Figure Legend Snippet: The 5-Aza does not affect LPS-induced suppression of Rgs10 transcription. (A) BV-2 cells were plated in 12-well plate and allowed to adhere overnight. Cells were treated with vehicle (serum-free media) or LPS (10 ng/ml, for 6 hours) with or without 5-Aza (AZA, 10 µ M). Cells were harvested in TRIzol, and RNA was isolated. mRNA transcript levels were measured by quantitative real-time PCR. Transcripts were normalized to the housekeeping gene actin. (B) BV-2 cells were plated in 24-well plate and allowed to adhere overnight. Cells were treated with either vehicle (serum-free media) or LPS (10 ng/ml) for 48 hours or with 5-Aza (AZA, 10 µ M). Cells were harvested, and protein levels were assessed by Western blot analysis with RGS10 and GAPDH control antibodies. Data are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. ** P

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Western Blot

    2) Product Images from "Diabetes-Specific Regulation of Adipocyte Metabolism by the Adipose Tissue Extracellular Matrix"

    Article Title: Diabetes-Specific Regulation of Adipocyte Metabolism by the Adipose Tissue Extracellular Matrix

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi: 10.1210/jc.2016-2915

    Adipocyte 2D culture. (A) Human adipocytes in in vitro 2D culture. Representative photomicrographs of in vitro differentiated human VAT and SAT adipocytes (top, magnification ×20; bottom, magnification ×40). (B) Adipogenic gene expression in human adipocytes. Quantitative real-time PCR data comparing adipogenic gene transcript levels in RNA from human adipocytes differentiated for 14 days in vitro with undifferentiated preadipocytes (PA) cultured for 72 hours in nonadipogenic media. Ordinate: fold difference in transcript level in mature adipocytes relative to undifferentiated preadipocyte referent = 1. * P
    Figure Legend Snippet: Adipocyte 2D culture. (A) Human adipocytes in in vitro 2D culture. Representative photomicrographs of in vitro differentiated human VAT and SAT adipocytes (top, magnification ×20; bottom, magnification ×40). (B) Adipogenic gene expression in human adipocytes. Quantitative real-time PCR data comparing adipogenic gene transcript levels in RNA from human adipocytes differentiated for 14 days in vitro with undifferentiated preadipocytes (PA) cultured for 72 hours in nonadipogenic media. Ordinate: fold difference in transcript level in mature adipocytes relative to undifferentiated preadipocyte referent = 1. * P

    Techniques Used: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Cell Culture

    3D-ECM adipocyte culture. (A) ECM-AD model. Adipose tissue was decellularized and then repopulated with preadipocytes that are subsequently differentiated within the ECM. (B) Decellularized adipose tissue ECM maintains collagen 1 microarchitecture and supports adipocyte differentiation. Top: Collagen 1 immunohistochemistry of whole human VAT before and after decellularization demonstrating maintenance of microarchitecture. Middle: 3D confocal photomicrographs of live human adipocytes within ECM; intact decellularized human VAT stained with Oil Red-O before reseeding with preadipocytes and 4 days and 14 days after seeding with preadipocytes followed by adipogenic differentiation. Blue: DAPI staining of cell nuclei; red: Oil Red-O staining of intracellular lipid. Bottom: Formalin-fixed, paraffin-embedded, 5- μ M–sectioned, Oil Red- O –stained human VAT prior to decellularization, immediately after decellularization, and after decellularization, preadipocyte-seeding, and 14 days of adipogenic differentiation, demonstrating cytoplasmic lipid accumulation in adipocytes within ECM. (C) Adipocytes in ECM upregulate adipogenic gene expression. Quantitative real-time PCR data comparing adipogenic gene transcript levels in RNA from human adipocytes differentiated in ECM for 14 days to undifferentiated preadipocytes (PA) in ECM cultured for 72 hours in nonadipogenic media. Ordinate: Fold difference in transcript level in mature adipocytes relative to undifferentiated preadipocyte referent = 1; all fold differences were significant ( P
    Figure Legend Snippet: 3D-ECM adipocyte culture. (A) ECM-AD model. Adipose tissue was decellularized and then repopulated with preadipocytes that are subsequently differentiated within the ECM. (B) Decellularized adipose tissue ECM maintains collagen 1 microarchitecture and supports adipocyte differentiation. Top: Collagen 1 immunohistochemistry of whole human VAT before and after decellularization demonstrating maintenance of microarchitecture. Middle: 3D confocal photomicrographs of live human adipocytes within ECM; intact decellularized human VAT stained with Oil Red-O before reseeding with preadipocytes and 4 days and 14 days after seeding with preadipocytes followed by adipogenic differentiation. Blue: DAPI staining of cell nuclei; red: Oil Red-O staining of intracellular lipid. Bottom: Formalin-fixed, paraffin-embedded, 5- μ M–sectioned, Oil Red- O –stained human VAT prior to decellularization, immediately after decellularization, and after decellularization, preadipocyte-seeding, and 14 days of adipogenic differentiation, demonstrating cytoplasmic lipid accumulation in adipocytes within ECM. (C) Adipocytes in ECM upregulate adipogenic gene expression. Quantitative real-time PCR data comparing adipogenic gene transcript levels in RNA from human adipocytes differentiated in ECM for 14 days to undifferentiated preadipocytes (PA) in ECM cultured for 72 hours in nonadipogenic media. Ordinate: Fold difference in transcript level in mature adipocytes relative to undifferentiated preadipocyte referent = 1; all fold differences were significant ( P

    Techniques Used: Immunohistochemistry, Staining, Formalin-fixed Paraffin-Embedded, Expressing, Real-time Polymerase Chain Reaction, Cell Culture

    3) Product Images from "MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells"

    Article Title: MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgr223

    Inverse correlation of expression of miR-150 and MUC4 in PC. ( A ) Expression of mature miR-150 was examined in normal and pancreatic cancerous tissues by quantitative reverse transcription–PCR. U6 small nuclear RNA was used as an internal control for relative quantitation. A reduced expression of miR-150 ( > 2.0-fold) was observed in 14 of 20 cancer tissues, whereas one normal tissue (#3) also exhibited decreased expression. ( B and C ) Expression analyses of MUC4 in normal and cancerous pancreatic tissue at mRNA (B) and protein (C) levels. An aberrant expression of MUC4 was detected in malignant pancreatic tissues, whereas it was not expressed in any of the NP sample even at the transcript level. Furthermore, we observed discordance in the expression of MUC4 at transcript and protein levels that correlated with dysregulated expression of miR-150.
    Figure Legend Snippet: Inverse correlation of expression of miR-150 and MUC4 in PC. ( A ) Expression of mature miR-150 was examined in normal and pancreatic cancerous tissues by quantitative reverse transcription–PCR. U6 small nuclear RNA was used as an internal control for relative quantitation. A reduced expression of miR-150 ( > 2.0-fold) was observed in 14 of 20 cancer tissues, whereas one normal tissue (#3) also exhibited decreased expression. ( B and C ) Expression analyses of MUC4 in normal and cancerous pancreatic tissue at mRNA (B) and protein (C) levels. An aberrant expression of MUC4 was detected in malignant pancreatic tissues, whereas it was not expressed in any of the NP sample even at the transcript level. Furthermore, we observed discordance in the expression of MUC4 at transcript and protein levels that correlated with dysregulated expression of miR-150.

    Techniques Used: Expressing, Polymerase Chain Reaction, Quantitation Assay

    miR-150 negatively regulates the expression of MUC4. ( A ) Identification of a putative miR-150-binding site in the MUC4 3′ UTR at position. Eight bases (71 through 78) of the MUC4 3′ UTR are perfect matches (seed sequence) for the miR-150. ( B ) Comparison of the MUC4 -binding element among mammals demonstrates a high degree of conservation. ( C and D ) Posttranscriptional regulation of MUC4 by miR-150. HPAF, Panc10.05 and Colo357 PC cells treated with different concentration of miR-150 or non-targeting control (miR-NC) mimic for 48 h. Mock-transfected cells represent cells treated with Lipofectamine 2000 alone. Expression of MUC4 was examined at mRNA (C) and protein (D) levels by quantitative reverse transcription–PCR and western blot analyses, respectively. GAPDH (for RNA) and β-actin (for protein) were used as internal controls. Amplified products from one of replicate wells of MUC4 and GAPDH quantitative PCR were also run on 1% agarose gel (C). Intensities of the immunoreactive bands in western blots were quantified by densitometry (D). Bars represent relative MUC4 expression after normalization with the relative internal control ± SD, * P
    Figure Legend Snippet: miR-150 negatively regulates the expression of MUC4. ( A ) Identification of a putative miR-150-binding site in the MUC4 3′ UTR at position. Eight bases (71 through 78) of the MUC4 3′ UTR are perfect matches (seed sequence) for the miR-150. ( B ) Comparison of the MUC4 -binding element among mammals demonstrates a high degree of conservation. ( C and D ) Posttranscriptional regulation of MUC4 by miR-150. HPAF, Panc10.05 and Colo357 PC cells treated with different concentration of miR-150 or non-targeting control (miR-NC) mimic for 48 h. Mock-transfected cells represent cells treated with Lipofectamine 2000 alone. Expression of MUC4 was examined at mRNA (C) and protein (D) levels by quantitative reverse transcription–PCR and western blot analyses, respectively. GAPDH (for RNA) and β-actin (for protein) were used as internal controls. Amplified products from one of replicate wells of MUC4 and GAPDH quantitative PCR were also run on 1% agarose gel (C). Intensities of the immunoreactive bands in western blots were quantified by densitometry (D). Bars represent relative MUC4 expression after normalization with the relative internal control ± SD, * P

    Techniques Used: Expressing, Binding Assay, Sequencing, Concentration Assay, Transfection, Polymerase Chain Reaction, Western Blot, Amplification, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis

    4) Product Images from "Survivin Is a Therapeutic Target in Merkel Cell Carcinoma"

    Article Title: Survivin Is a Therapeutic Target in Merkel Cell Carcinoma

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.3003713

    Survivin oncoprotein mRNA expression is increased in MCV-positive MCC. ( A ) Digital transcriptome subtraction comparison of 64 genes involved in programmed cell death and cell cycle regulation showed that survivin ( BIRC5 ). ( B ) MCV T antigen is required for survivin expression. Lentiviral MCV T antigen exon 1 knockdown (panT1) decreased survivin protein expression (left) but did not alter XIAP, BCL-2, BAX, or p53 protein levels (right) in four MCV-positive MCC cell lines: MKL-1, MKL-2, MS-1, and WaGa. UISO, MCV-negative cell line; shCntrl, scrambled shRNA control lentivirus; LT, large T antigen. ( C ) MCV T antigen is required for survivin transcription. Survivin mRNA levels were reduced in the MKL-1 but not the UISO cell line after T antigen knockdown, indicating that T antigen activates survivin transcription. Survivin mRNA was measured by qRT-PCR and normalized to β-actin mRNA. The experiments were performed in triplicate and repeated twice (mean ± SEM, two-tailed t test). ( D ) Survivin expression is required for MCV-positive MCC tumor cell survival. Survivin was targeted for knockdown using two shRNA lentiviral vectors, shsur1 and shsur2, in MKL-1 and UISO cells. MKL-1 cells initiate apoptosis after survivin knockdown, with increased expression of cleaved PARP (cPARP) and caspase 3 (cCasp3), whereas UISO cells are resistant to survivin knockdown-induced apoptosis. α-Tubulin is used as a loading control.
    Figure Legend Snippet: Survivin oncoprotein mRNA expression is increased in MCV-positive MCC. ( A ) Digital transcriptome subtraction comparison of 64 genes involved in programmed cell death and cell cycle regulation showed that survivin ( BIRC5 ). ( B ) MCV T antigen is required for survivin expression. Lentiviral MCV T antigen exon 1 knockdown (panT1) decreased survivin protein expression (left) but did not alter XIAP, BCL-2, BAX, or p53 protein levels (right) in four MCV-positive MCC cell lines: MKL-1, MKL-2, MS-1, and WaGa. UISO, MCV-negative cell line; shCntrl, scrambled shRNA control lentivirus; LT, large T antigen. ( C ) MCV T antigen is required for survivin transcription. Survivin mRNA levels were reduced in the MKL-1 but not the UISO cell line after T antigen knockdown, indicating that T antigen activates survivin transcription. Survivin mRNA was measured by qRT-PCR and normalized to β-actin mRNA. The experiments were performed in triplicate and repeated twice (mean ± SEM, two-tailed t test). ( D ) Survivin expression is required for MCV-positive MCC tumor cell survival. Survivin was targeted for knockdown using two shRNA lentiviral vectors, shsur1 and shsur2, in MKL-1 and UISO cells. MKL-1 cells initiate apoptosis after survivin knockdown, with increased expression of cleaved PARP (cPARP) and caspase 3 (cCasp3), whereas UISO cells are resistant to survivin knockdown-induced apoptosis. α-Tubulin is used as a loading control.

    Techniques Used: Expressing, Mass Spectrometry, shRNA, Quantitative RT-PCR, Two Tailed Test

    MCV large T antigen isoform induces survivin oncoprotein expression in human BJ cells by targeting RB. ( A ) BJ cells were transduced with either empty vector, a tumor-derived large T antigen cDNA (LT.339), or a large T antigen cDNA with an inactive RB binding domain (LT.339LFCDK). Immunoblotting reveals that MCV LT.339 induces survivin expression but LT.339LFCDK does not. A similar pattern is seen for other S-phase cell cycle proteins such as E2F1 and cyclin E that are also transcriptionally repressed by RB. ( B ) LI-COR quantitative immunoblotting for survivin in (A), normalized to α-tubulin (arbitrary units). ( C ) Survivin mRNA levels increased in BJ cells expressing LT.339 protein but not in cells expressing the RB1 binding mutant LT.339LFCDK. BJ cells expressing either empty vector, LT.339, or LT.339LFCDK were serum-starved for 48 hours and then harvested for RNA. Survivin mRNA was measured by qRT-PCR and normalized to β-actin mRNA. The experiments were performed three times in duplicate (mean ± SEM, two-tailed t test).
    Figure Legend Snippet: MCV large T antigen isoform induces survivin oncoprotein expression in human BJ cells by targeting RB. ( A ) BJ cells were transduced with either empty vector, a tumor-derived large T antigen cDNA (LT.339), or a large T antigen cDNA with an inactive RB binding domain (LT.339LFCDK). Immunoblotting reveals that MCV LT.339 induces survivin expression but LT.339LFCDK does not. A similar pattern is seen for other S-phase cell cycle proteins such as E2F1 and cyclin E that are also transcriptionally repressed by RB. ( B ) LI-COR quantitative immunoblotting for survivin in (A), normalized to α-tubulin (arbitrary units). ( C ) Survivin mRNA levels increased in BJ cells expressing LT.339 protein but not in cells expressing the RB1 binding mutant LT.339LFCDK. BJ cells expressing either empty vector, LT.339, or LT.339LFCDK were serum-starved for 48 hours and then harvested for RNA. Survivin mRNA was measured by qRT-PCR and normalized to β-actin mRNA. The experiments were performed three times in duplicate (mean ± SEM, two-tailed t test).

    Techniques Used: Expressing, Transduction, Plasmid Preparation, Derivative Assay, Binding Assay, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

    5) Product Images from "The Immunoregulatory Role of Corneal Epithelium-Derived Thrombospondin-1 in Dry Eye Disease"

    Article Title: The Immunoregulatory Role of Corneal Epithelium-Derived Thrombospondin-1 in Dry Eye Disease

    Journal: The ocular surface

    doi: 10.1016/j.jtos.2018.07.005

    Expression of corneal epithelial TSP-1 is increased in DED mice. Corneas were harvested on days 3, 7 and 14 after DED induction. The epithelial layer was peeled off and single cell suspension was prepared. Healthy mice served as the control. (A) TSP-1 mRNA expression was quantified using real-time PCR. (B) Representative flow cytometry plots showing the frequencies of TSP-1 + CD45 − epithelial cells in healthy and DED mice. (C) Protein expression (MFI) of TSP-1 by corneal epithelial cells was assessed in DED mice on days 7 and 14 after desiccating stress using flow cytometry. Data are presented as mean ± SEM of three independent experiments, each consisting of six corneas per group. MFI: mean fluorescence intensity.
    Figure Legend Snippet: Expression of corneal epithelial TSP-1 is increased in DED mice. Corneas were harvested on days 3, 7 and 14 after DED induction. The epithelial layer was peeled off and single cell suspension was prepared. Healthy mice served as the control. (A) TSP-1 mRNA expression was quantified using real-time PCR. (B) Representative flow cytometry plots showing the frequencies of TSP-1 + CD45 − epithelial cells in healthy and DED mice. (C) Protein expression (MFI) of TSP-1 by corneal epithelial cells was assessed in DED mice on days 7 and 14 after desiccating stress using flow cytometry. Data are presented as mean ± SEM of three independent experiments, each consisting of six corneas per group. MFI: mean fluorescence intensity.

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    6) Product Images from "Regulation of Membrane-Type 4 Matrix Metalloproteinase by SLUG Contributes to Hypoxia-Mediated Metastasis 1 by SLUG Contributes to Hypoxia-Mediated Metastasis 1 2"

    Article Title: Regulation of Membrane-Type 4 Matrix Metalloproteinase by SLUG Contributes to Hypoxia-Mediated Metastasis 1 by SLUG Contributes to Hypoxia-Mediated Metastasis 1 2

    Journal:

    doi:

    Hypoxia or constitutive expression of HIF-1α(ΔODD) upregulates MT4-MMP expression. (A) Upper: Fold change of mRNA levels of HIF-1α and MT4-MMP by real-time RT-PCR analysis in FADU, SAS, and OECM-1 cells under normoxia versus hypoxia.
    Figure Legend Snippet: Hypoxia or constitutive expression of HIF-1α(ΔODD) upregulates MT4-MMP expression. (A) Upper: Fold change of mRNA levels of HIF-1α and MT4-MMP by real-time RT-PCR analysis in FADU, SAS, and OECM-1 cells under normoxia versus hypoxia.

    Techniques Used: Expressing, Quantitative RT-PCR

    7) Product Images from "miR-125a-3p and miR-483-5p promote adipogenesis via suppressing the RhoA/ROCK1/ERK1/2 pathway in multiple symmetric lipomatosis"

    Article Title: miR-125a-3p and miR-483-5p promote adipogenesis via suppressing the RhoA/ROCK1/ERK1/2 pathway in multiple symmetric lipomatosis

    Journal: Scientific Reports

    doi: 10.1038/srep11909

    The expression of miR-125a-3p and miR-483-5p are increased with the induction of hADSCs. ( A ) The hADSCs were induced to mature adipocytes for 12 days. The miR-125a-3p and miR-483-5p expression levels were detected by quantitative real-time PCR at days 0, 6, and 12. ( B ) The hADSCs were induced to mature adipocytes for 12 days. Total protein was extracted for immunoblotting of RhoA, ROCK1, total ERK1/2 (T-EKR1/2), and phosphorylated ERK1/2 (T-p-EKR1/2) by western blot. ( C ) The expression of RhoA, ROCK1, T-EKR1/2, and T-p-EKR1/2 in the adipose tissues of MSL patients (n = 3) and controls (n = 3) were determined by western blot. *p
    Figure Legend Snippet: The expression of miR-125a-3p and miR-483-5p are increased with the induction of hADSCs. ( A ) The hADSCs were induced to mature adipocytes for 12 days. The miR-125a-3p and miR-483-5p expression levels were detected by quantitative real-time PCR at days 0, 6, and 12. ( B ) The hADSCs were induced to mature adipocytes for 12 days. Total protein was extracted for immunoblotting of RhoA, ROCK1, total ERK1/2 (T-EKR1/2), and phosphorylated ERK1/2 (T-p-EKR1/2) by western blot. ( C ) The expression of RhoA, ROCK1, T-EKR1/2, and T-p-EKR1/2 in the adipose tissues of MSL patients (n = 3) and controls (n = 3) were determined by western blot. *p

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

    miR-125a-3p and miR-483-5p regulate RhoA/ROCK1/ERK1/2 signaling and promote mouse de novo adipogenesis. hADSCs were transfected with a lentiviral vector containing pre-miRs of miR-125a (LV-miR-125a), miR-483 (LV-miR-483), or negative control miR (LV-NC) and transplanted to the back subcutaneous tissues of nude mice with the transfected hADSCs or non-tranfected hADSC as control group (Con) or injection of PBS. After 5 weeks of self-differentiation, ( A ) De novo adipose tissue formation was observed (n = 5); ( B ) The weights of the de novo adipose tissues were measured (n = 5); ( C ) The expression levels of miR-125a-3p/5p and miR-483-3p/5p in de novo adipose tissue were detected by real-time PCR (n = 5); ( D ) The de novo adipose tissue was stained to observe the histology (n = 5); ( E ) Adipoctye size were analyzed by ImageJ software ( F ) The protein expression levels of RhoA, ROCK1, total ERK1/2 (T-EKR1/2), and phosphorylated ERK1/2 (T-p-EKR1/2) were analyzed by western blot; and ( G ) Total nuclear ERK1/2 (n-T-ERK1/2) and p-ERK1/2 (n-p-ERK1/2) were analyzed by western blot. All measurements were preformed by three independent experiments. *p
    Figure Legend Snippet: miR-125a-3p and miR-483-5p regulate RhoA/ROCK1/ERK1/2 signaling and promote mouse de novo adipogenesis. hADSCs were transfected with a lentiviral vector containing pre-miRs of miR-125a (LV-miR-125a), miR-483 (LV-miR-483), or negative control miR (LV-NC) and transplanted to the back subcutaneous tissues of nude mice with the transfected hADSCs or non-tranfected hADSC as control group (Con) or injection of PBS. After 5 weeks of self-differentiation, ( A ) De novo adipose tissue formation was observed (n = 5); ( B ) The weights of the de novo adipose tissues were measured (n = 5); ( C ) The expression levels of miR-125a-3p/5p and miR-483-3p/5p in de novo adipose tissue were detected by real-time PCR (n = 5); ( D ) The de novo adipose tissue was stained to observe the histology (n = 5); ( E ) Adipoctye size were analyzed by ImageJ software ( F ) The protein expression levels of RhoA, ROCK1, total ERK1/2 (T-EKR1/2), and phosphorylated ERK1/2 (T-p-EKR1/2) were analyzed by western blot; and ( G ) Total nuclear ERK1/2 (n-T-ERK1/2) and p-ERK1/2 (n-p-ERK1/2) were analyzed by western blot. All measurements were preformed by three independent experiments. *p

    Techniques Used: Transfection, Plasmid Preparation, Negative Control, Mouse Assay, Injection, Expressing, Real-time Polymerase Chain Reaction, Staining, Software, Western Blot

    Photographs of two MSL patients and TaqMan miR array analysis of SAT. ( A ) There was symmetrical SAT accumulation in the neck, upper arms, bilateral shoulders, upper thorax, back, and abdomen. ( B ) Relative expression of ten miRs were verified by quantitative real-time PCR (n = 3). ( C ) KEGG pathway analysis of the upregulated miRs in the SAT of MSL patients.
    Figure Legend Snippet: Photographs of two MSL patients and TaqMan miR array analysis of SAT. ( A ) There was symmetrical SAT accumulation in the neck, upper arms, bilateral shoulders, upper thorax, back, and abdomen. ( B ) Relative expression of ten miRs were verified by quantitative real-time PCR (n = 3). ( C ) KEGG pathway analysis of the upregulated miRs in the SAT of MSL patients.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    8) Product Images from "Decrease in Proportion of CD19+CD24hiCD27+ B Cells and Impairment of Their Suppressive Function in Graves' Disease"

    Article Title: Decrease in Proportion of CD19+CD24hiCD27+ B Cells and Impairment of Their Suppressive Function in Graves' Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049835

    Frequencies of B10 cells from the blood of healthy individuals and GD patients. (A) Representative intracellular IL-10 expression in CD19 + B cells of healthy individuals, new-onset GD patients, and recovered GD patients after in vitro CpG+PIB stimulation for 5 h. (B). Dots represent B10 cell frequencies in CD19 + B cells and total PBMCs after in vitro CpG+PIB stimulation from 24 healthy individuals, 15 new-onset GD patients, and 10 recovered GD patients. (C) IL-10 mRNA transcript expression in CD19 + B cells. The RNA of freshly purified CD19 + B cells was isolated (left) and IL-10 transcripts quantified by quantitative real-time PCR. Dots represent results from 10 individuals of each group (right). *P
    Figure Legend Snippet: Frequencies of B10 cells from the blood of healthy individuals and GD patients. (A) Representative intracellular IL-10 expression in CD19 + B cells of healthy individuals, new-onset GD patients, and recovered GD patients after in vitro CpG+PIB stimulation for 5 h. (B). Dots represent B10 cell frequencies in CD19 + B cells and total PBMCs after in vitro CpG+PIB stimulation from 24 healthy individuals, 15 new-onset GD patients, and 10 recovered GD patients. (C) IL-10 mRNA transcript expression in CD19 + B cells. The RNA of freshly purified CD19 + B cells was isolated (left) and IL-10 transcripts quantified by quantitative real-time PCR. Dots represent results from 10 individuals of each group (right). *P

    Techniques Used: Expressing, In Vitro, Purification, Isolation, Real-time Polymerase Chain Reaction

    9) Product Images from "Thrombin-Induced Endothelin-1 Synthesis and Secretion in Retinal Pigment Epithelial Cells Is Rho Kinase Dependent"

    Article Title: Thrombin-Induced Endothelin-1 Synthesis and Secretion in Retinal Pigment Epithelial Cells Is Rho Kinase Dependent

    Journal: Journal of Ocular Pharmacology and Therapeutics

    doi: 10.1089/jop.2010.0072

    Measurement of preproET-1 (ppET-1) mRNA by quantitative polymerase chain reaction (PCR) in mature ARPE-19 and primary hRPE cells. Quantitative RT-PCR was performed using the SYBR-green PCR core reagents. Quantitation of ppET-1 transcripts was done by
    Figure Legend Snippet: Measurement of preproET-1 (ppET-1) mRNA by quantitative polymerase chain reaction (PCR) in mature ARPE-19 and primary hRPE cells. Quantitative RT-PCR was performed using the SYBR-green PCR core reagents. Quantitation of ppET-1 transcripts was done by

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, Quantitation Assay

    10) Product Images from "Co-clinical Analysis of a Genetically Engineered Mouse Model and Human Prostate Cancer Reveals Significance of NKX3.1 Expression for Response to 5α-reductase Inhibition"

    Article Title: Co-clinical Analysis of a Genetically Engineered Mouse Model and Human Prostate Cancer Reveals Significance of NKX3.1 Expression for Response to 5α-reductase Inhibition

    Journal: European urology

    doi: 10.1016/j.eururo.2017.03.031

    Finasteride abrogates prostatic intraepithelial neoplasia (PIN) in a genetically engineered mouse model of prostate cancer. (A) Preclinical trial design. Cohorts of Nkx3.1 +/+ and Nkx3.1 −/− mice aged 4 mo were randomly assigned to treatment with finasteride (1 mg/ml in phosphate-buffered saline) or vehicle once a day on a schedule of 5 d/wk for 8 mo, until the mice were aged 12 mo. At the conclusion of the study, mice were sacrificed and analysis of the endpoints indicated was performed. PCR = polymerase chain reaction. (B) Levels of dihydrotestosterone (DHT) in serum as indicated ( n = 5 per group); p values represent comparisons between bracketed groups and were estimated using a two-tailed two-sample t test. Veh = vehicle; Fin = finasteride. (C) Histological analysis. Shown are representative images of hematoxylin and eosin (H E) staining and immunohistochemical (IHC) staining of anterior prostate from Nkx3.1 +/+ and Nkx3.1 −/− mice treated with finasteride or vehicle, as indicated ( n ]. Scale bars represent 100 μm (H E low power) or 50 μm (H E high power and IHC staining). (D) Summary of PIN phenotype following treatment. Shown is the percentage area of prostatic tissue that is normal/PIN1, PIN2, PIN3, and PIN4 following treatment with finasteride in Nkx3.1 +/+ ( n = 5 per group) and Nkx3.1 −/− ( n = 15 per group) mice; p values were estimated using a two-tailed two-sample t test. ns = not significant. (E) Quantitative real-time PCR was carried out using total RNA from Nkx3.1 +/+ and Nkx3.1 −/− prostate treated with vehicle or finasteride, as indicated. Analyses were performed in triplicate and normalized to GAPDH; p values were estimated using a two-tailed two-sample t test.
    Figure Legend Snippet: Finasteride abrogates prostatic intraepithelial neoplasia (PIN) in a genetically engineered mouse model of prostate cancer. (A) Preclinical trial design. Cohorts of Nkx3.1 +/+ and Nkx3.1 −/− mice aged 4 mo were randomly assigned to treatment with finasteride (1 mg/ml in phosphate-buffered saline) or vehicle once a day on a schedule of 5 d/wk for 8 mo, until the mice were aged 12 mo. At the conclusion of the study, mice were sacrificed and analysis of the endpoints indicated was performed. PCR = polymerase chain reaction. (B) Levels of dihydrotestosterone (DHT) in serum as indicated ( n = 5 per group); p values represent comparisons between bracketed groups and were estimated using a two-tailed two-sample t test. Veh = vehicle; Fin = finasteride. (C) Histological analysis. Shown are representative images of hematoxylin and eosin (H E) staining and immunohistochemical (IHC) staining of anterior prostate from Nkx3.1 +/+ and Nkx3.1 −/− mice treated with finasteride or vehicle, as indicated ( n ]. Scale bars represent 100 μm (H E low power) or 50 μm (H E high power and IHC staining). (D) Summary of PIN phenotype following treatment. Shown is the percentage area of prostatic tissue that is normal/PIN1, PIN2, PIN3, and PIN4 following treatment with finasteride in Nkx3.1 +/+ ( n = 5 per group) and Nkx3.1 −/− ( n = 15 per group) mice; p values were estimated using a two-tailed two-sample t test. ns = not significant. (E) Quantitative real-time PCR was carried out using total RNA from Nkx3.1 +/+ and Nkx3.1 −/− prostate treated with vehicle or finasteride, as indicated. Analyses were performed in triplicate and normalized to GAPDH; p values were estimated using a two-tailed two-sample t test.

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Two Tailed Test, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

    11) Product Images from "ATF4 Targets RET for Degradation and Is a Candidate Tumor Suppressor Gene in Medullary Thyroid Cancer"

    Article Title: ATF4 Targets RET for Degradation and Is a Candidate Tumor Suppressor Gene in Medullary Thyroid Cancer

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi: 10.1210/jc.2016-2878

    ATF4 expression promotes RET ubiquitination. (A and B) HEK 293T cells were transfected with the indicated plasmids, and western blot (WB) analysis was performed with the indicated antibodies. (C) Western blot analysis of TT cells infected with the lentivirus harboring ATF4 with the indicated antibodies. (D) RNA was isolated from cells treated as in (C) and subjected to real-time PCR using ATF4 or RET Taqman primers probes. (E) MTC cells expressing lentiviral-ATF4 were treated with MG132 (4 h), and denatured extracts were immunoprecipitated with an anti-RET antibody followed by western blot analysis with antiubiquitin antibody. (F) ATF4-shRNA (clones of 74 and 76) and nontargeting shRNA (Con) are treated with tunicamycin (2 μg/mL) for 24 h, and western blot analysis was performed with the indicated antibodies. (G) ATF4 expression inhibits RET signaling. MTC cells expressing lentiviral-ATF4 were immunoblotted with the indicated antibodies. IP, immunoprecipitation.
    Figure Legend Snippet: ATF4 expression promotes RET ubiquitination. (A and B) HEK 293T cells were transfected with the indicated plasmids, and western blot (WB) analysis was performed with the indicated antibodies. (C) Western blot analysis of TT cells infected with the lentivirus harboring ATF4 with the indicated antibodies. (D) RNA was isolated from cells treated as in (C) and subjected to real-time PCR using ATF4 or RET Taqman primers probes. (E) MTC cells expressing lentiviral-ATF4 were treated with MG132 (4 h), and denatured extracts were immunoprecipitated with an anti-RET antibody followed by western blot analysis with antiubiquitin antibody. (F) ATF4-shRNA (clones of 74 and 76) and nontargeting shRNA (Con) are treated with tunicamycin (2 μg/mL) for 24 h, and western blot analysis was performed with the indicated antibodies. (G) ATF4 expression inhibits RET signaling. MTC cells expressing lentiviral-ATF4 were immunoblotted with the indicated antibodies. IP, immunoprecipitation.

    Techniques Used: Expressing, Transfection, Western Blot, Infection, Isolation, Real-time Polymerase Chain Reaction, Immunoprecipitation, shRNA

    Related Articles

    Real-time Polymerase Chain Reaction:

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    Article Snippet: .. Quantitative real-time polymerase chain reaction (PCR) was performed using 5 μl (for MUC4) and 2.5 μl (for GAPDH) of 1:10 dilution first-strand complementary DNA in 96-well plates using SYBR Green Master Mix (Applied Biosystems) on an iCycler system (Bio-Rad Laboratories, Hercules, CA). .. To examine the expression level of mature miR-150, we followed the strategy developed by Chen et al. ( ).

    Article Title: Survivin Is a Therapeutic Target in Merkel Cell Carcinoma
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    Article Title: The Immunoregulatory Role of Corneal Epithelium-Derived Thrombospondin-1 in Dry Eye Disease
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    Article Title: Regulation of Membrane-Type 4 Matrix Metalloproteinase by SLUG Contributes to Hypoxia-Mediated Metastasis 1 by SLUG Contributes to Hypoxia-Mediated Metastasis 1 2
    Article Snippet: .. To evaluate HIF-1α and MT4-MMP mRNA expression, quantitative real-time polymerase chain reaction (PCR) was performed using a PRISM7700 Sequence Detection System (Applied Biosystems, Foster City, CA) with the preset PCR program. ..

    Article Title: Decrease in Proportion of CD19+CD24hiCD27+ B Cells and Impairment of Their Suppressive Function in Graves' Disease
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    Article Title: Diabetes-Specific Regulation of Adipocyte Metabolism by the Adipose Tissue Extracellular Matrix
    Article Snippet: .. Equal amounts of input RNA were reverse-transcribed, and quantitative real-time polymerase chain reaction (PCR) was performed using Taqman primer-probes (Life Technologies, Inc., Carlsbad, CA) with actin as an endogenous control on a StepOnePlus thermocycler (Applied Biosystems, Inc., Foster City, CA). ..

    Synthesized:

    Article Title: The Immunoregulatory Role of Corneal Epithelium-Derived Thrombospondin-1 in Dry Eye Disease
    Article Snippet: .. Total RNA was extracted from corneal or conjunctival tissue using a commercial reagent (TRIzol; Invitrogen, Carlsbad, CA) and an RNA purification kit (RNeasy Micro Kit; Qiagen, Germantown, MD).First-strand cDNA was synthesized with random hexamers using reverse transcriptase (SuperScript III; Invitrogen), and quantitative real-time polymerase chain reaction (PCR) was performed using predesigned primers (Taqman PCR Mastermix and FAM dye-labeled primers; Applied Biosystems, Foster City, CA) for TSP-1 (Mm00449032_g1), IL-1β (Mm00434228_m1), IL-6 (Mm00446190_m1), IL-23 ((Mm00518984_m1), IL-17A (Mm00439619_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1). ..

    Purification:

    Article Title: The Immunoregulatory Role of Corneal Epithelium-Derived Thrombospondin-1 in Dry Eye Disease
    Article Snippet: .. Total RNA was extracted from corneal or conjunctival tissue using a commercial reagent (TRIzol; Invitrogen, Carlsbad, CA) and an RNA purification kit (RNeasy Micro Kit; Qiagen, Germantown, MD).First-strand cDNA was synthesized with random hexamers using reverse transcriptase (SuperScript III; Invitrogen), and quantitative real-time polymerase chain reaction (PCR) was performed using predesigned primers (Taqman PCR Mastermix and FAM dye-labeled primers; Applied Biosystems, Foster City, CA) for TSP-1 (Mm00449032_g1), IL-1β (Mm00434228_m1), IL-6 (Mm00446190_m1), IL-23 ((Mm00518984_m1), IL-17A (Mm00439619_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1). ..

    SYBR Green Assay:

    Article Title: MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells
    Article Snippet: .. Quantitative real-time polymerase chain reaction (PCR) was performed using 5 μl (for MUC4) and 2.5 μl (for GAPDH) of 1:10 dilution first-strand complementary DNA in 96-well plates using SYBR Green Master Mix (Applied Biosystems) on an iCycler system (Bio-Rad Laboratories, Hercules, CA). .. To examine the expression level of mature miR-150, we followed the strategy developed by Chen et al. ( ).

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    Article Title: Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity
    Article Snippet: .. Quantitative real-time polymerase chain reaction (PCR) was performed using Superscript III kit for reverse-transcription PCR (RT-PCR; Invitrogen/Life Technologies) and Power SYBR Green reagent (Life Technologies/Thermo Fisher Scientific). ..

    Sequencing:

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    Article Snippet: .. To evaluate HIF-1α and MT4-MMP mRNA expression, quantitative real-time polymerase chain reaction (PCR) was performed using a PRISM7700 Sequence Detection System (Applied Biosystems, Foster City, CA) with the preset PCR program. ..

    Polymerase Chain Reaction:

    Article Title: MicroRNA-150 directly targets MUC4 and suppresses growth and malignant behavior of pancreatic cancer cells
    Article Snippet: .. Quantitative real-time polymerase chain reaction (PCR) was performed using 5 μl (for MUC4) and 2.5 μl (for GAPDH) of 1:10 dilution first-strand complementary DNA in 96-well plates using SYBR Green Master Mix (Applied Biosystems) on an iCycler system (Bio-Rad Laboratories, Hercules, CA). .. To examine the expression level of mature miR-150, we followed the strategy developed by Chen et al. ( ).

    Article Title: Survivin Is a Therapeutic Target in Merkel Cell Carcinoma
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    Article Title: The Immunoregulatory Role of Corneal Epithelium-Derived Thrombospondin-1 in Dry Eye Disease
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    Article Title: Regulation of Membrane-Type 4 Matrix Metalloproteinase by SLUG Contributes to Hypoxia-Mediated Metastasis 1 by SLUG Contributes to Hypoxia-Mediated Metastasis 1 2
    Article Snippet: .. To evaluate HIF-1α and MT4-MMP mRNA expression, quantitative real-time polymerase chain reaction (PCR) was performed using a PRISM7700 Sequence Detection System (Applied Biosystems, Foster City, CA) with the preset PCR program. ..

    Article Title: Decrease in Proportion of CD19+CD24hiCD27+ B Cells and Impairment of Their Suppressive Function in Graves' Disease
    Article Snippet: .. The expression of IL-10 mRNA was analyzed using quantitative real-time polymerase chain reaction (PCR) methods according to the manufacturer’s instructions (Applied Biosystems). .. The sense GAPDH primer was 5′-GCC ACC CAG AAG ACT GTG GAT GGC-3′ and the antisense primer was 5′-CAT GTA GGC CAT GAG GTC CAC CAC-3′ .

    Article Title: miR-125a-3p and miR-483-5p promote adipogenesis via suppressing the RhoA/ROCK1/ERK1/2 pathway in multiple symmetric lipomatosis
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    Article Title: Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity
    Article Snippet: .. Quantitative real-time polymerase chain reaction (PCR) was performed using Superscript III kit for reverse-transcription PCR (RT-PCR; Invitrogen/Life Technologies) and Power SYBR Green reagent (Life Technologies/Thermo Fisher Scientific). ..

    Article Title: Diabetes-Specific Regulation of Adipocyte Metabolism by the Adipose Tissue Extracellular Matrix
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    Expressing:

    Article Title: Regulation of Membrane-Type 4 Matrix Metalloproteinase by SLUG Contributes to Hypoxia-Mediated Metastasis 1 by SLUG Contributes to Hypoxia-Mediated Metastasis 1 2
    Article Snippet: .. To evaluate HIF-1α and MT4-MMP mRNA expression, quantitative real-time polymerase chain reaction (PCR) was performed using a PRISM7700 Sequence Detection System (Applied Biosystems, Foster City, CA) with the preset PCR program. ..

    Article Title: Decrease in Proportion of CD19+CD24hiCD27+ B Cells and Impairment of Their Suppressive Function in Graves' Disease
    Article Snippet: .. The expression of IL-10 mRNA was analyzed using quantitative real-time polymerase chain reaction (PCR) methods according to the manufacturer’s instructions (Applied Biosystems). .. The sense GAPDH primer was 5′-GCC ACC CAG AAG ACT GTG GAT GGC-3′ and the antisense primer was 5′-CAT GTA GGC CAT GAG GTC CAC CAC-3′ .

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity
    Article Snippet: .. Quantitative real-time polymerase chain reaction (PCR) was performed using Superscript III kit for reverse-transcription PCR (RT-PCR; Invitrogen/Life Technologies) and Power SYBR Green reagent (Life Technologies/Thermo Fisher Scientific). ..

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  • 91
    Thermo Fisher quantitative real time reverse transcription polymerase chain reaction qrt pcr
    Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) <t>qRT-PCR</t> indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p
    Quantitative Real Time Reverse Transcription Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time reverse transcription polymerase chain reaction qrt pcr/product/Thermo Fisher
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    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr total rna
    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by <t>qRT-PCR;</t> b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 433 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher real time quantitative reverse transcription polymerase chain reaction qrt pcr total rna
    MiR-194 reveals no significant effect on CRC cell proliferation. Notes: ( A ) The changes in morphology before and after transfection were observed by inverted microscopy (×100). ( B ) SW480 cells were infected with miR-194 or negative control (miR-NC) or mock transfection control (miR-MT) lentivirus, and the expression of miR-194 was analyzed by <t>qRT-PCR.</t> ( C ) MTT assays were performed to investigate the proliferation ability of CRC cells. * P
    Real Time Quantitative Reverse Transcription Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 34 article reviews
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    Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

    Journal: OncoTargets and therapy

    Article Title: Nuclear factor-κB p65 regulates glutaminase 1 expression in human hepatocellular carcinoma

    doi: 10.2147/OTT.S167408

    Figure Lengend Snippet: Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

    Article Snippet: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Upon relative treatment, cells were lysed, and total RNA was isolated using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

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

    PART1 was highly expressed in HCC tissues and cell lines. ( A – B ) The expression of PART1 was measured in 46 pairs of HCC tissues and adjacent normal tissues, as well as human normal liver cell line (THLE-2) and HCC cell lines (SK-HEP-1, Huh-1, Hep3B and Huh-7) by qRT-PCR assay. **P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA PART1 Promotes Proliferation, Migration and Invasion of Hepatocellular Carcinoma Cells via miR-149-5p/MAP2K1 Axis

    doi: 10.2147/CMAR.S246311

    Figure Lengend Snippet: PART1 was highly expressed in HCC tissues and cell lines. ( A – B ) The expression of PART1 was measured in 46 pairs of HCC tissues and adjacent normal tissues, as well as human normal liver cell line (THLE-2) and HCC cell lines (SK-HEP-1, Huh-1, Hep3B and Huh-7) by qRT-PCR assay. **P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay Total RNA was isolated from HCC tumor tissues and cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Expressing, Quantitative RT-PCR

    PART1 regulated proliferation, migration and invasion of HCC cells via miR-149-5p. SK-HEP-1 and Hep3B cells were transfected with si-NC, si-PART1#1, si-PART1#1+anti-miR-NC, or si-PART1#1+miR-149-5p inhibitor. ( A ) QRT-PCR was carried out to detect the expression of miR-149-5p in transfected HCC cells. ( B, C ) CCK-8 was used to assess the proliferation. ( D, E ) The migration and invasion were detected by transwell assay. **P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA PART1 Promotes Proliferation, Migration and Invasion of Hepatocellular Carcinoma Cells via miR-149-5p/MAP2K1 Axis

    doi: 10.2147/CMAR.S246311

    Figure Lengend Snippet: PART1 regulated proliferation, migration and invasion of HCC cells via miR-149-5p. SK-HEP-1 and Hep3B cells were transfected with si-NC, si-PART1#1, si-PART1#1+anti-miR-NC, or si-PART1#1+miR-149-5p inhibitor. ( A ) QRT-PCR was carried out to detect the expression of miR-149-5p in transfected HCC cells. ( B, C ) CCK-8 was used to assess the proliferation. ( D, E ) The migration and invasion were detected by transwell assay. **P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay Total RNA was isolated from HCC tumor tissues and cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Migration, Transfection, Quantitative RT-PCR, Expressing, CCK-8 Assay, Transwell Assay

    PART1 could bind miR-149-5p specifically and suppressed the miR-149-5p expression. ( A ) The putative binding sites between PART1 and miR-149-5p and the mutant sequences of PART1 were shown. ( B, C ) Luciferase activity was detected in SK-HEP-1 and Hep3B cells co-transfected with PART1-WT or PART1-MUT and miR-149-5p mimics or miR-NC. ( D, E ) The expression of miR-149-5p was examined by qRT-PCR in SK-HEP-1 and Hep3B cells transfected with si-NC, si-PART1#1, Vector or PART1. ( F, G ) The expression level of PART1 was detected in SK-HEP-1 and Hep3B cells transfected with miR-NC, miR-149-5p mimics, anti-miR-NC or miR-149-5p inhibitor. ( H ) The miR-149-5p expression was measured in HCC tissues and adjacent normal tissues. ( I ) Correlation analysis between PART1 and miR-149-5p expression was conducted by Pearson analysis. **P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA PART1 Promotes Proliferation, Migration and Invasion of Hepatocellular Carcinoma Cells via miR-149-5p/MAP2K1 Axis

    doi: 10.2147/CMAR.S246311

    Figure Lengend Snippet: PART1 could bind miR-149-5p specifically and suppressed the miR-149-5p expression. ( A ) The putative binding sites between PART1 and miR-149-5p and the mutant sequences of PART1 were shown. ( B, C ) Luciferase activity was detected in SK-HEP-1 and Hep3B cells co-transfected with PART1-WT or PART1-MUT and miR-149-5p mimics or miR-NC. ( D, E ) The expression of miR-149-5p was examined by qRT-PCR in SK-HEP-1 and Hep3B cells transfected with si-NC, si-PART1#1, Vector or PART1. ( F, G ) The expression level of PART1 was detected in SK-HEP-1 and Hep3B cells transfected with miR-NC, miR-149-5p mimics, anti-miR-NC or miR-149-5p inhibitor. ( H ) The miR-149-5p expression was measured in HCC tissues and adjacent normal tissues. ( I ) Correlation analysis between PART1 and miR-149-5p expression was conducted by Pearson analysis. **P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay Total RNA was isolated from HCC tumor tissues and cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation

    PART1 increased MAP2K1 level by weakening miR-149-5p-mediated inhibitory effect on MAP2K1 in SK-HEP-1 and Hep3B cells. ( A ) Correlation analysis between MAP2K1 and PART1 expression in HCC was performed by Pearson analysis. ( B – D ) SK-HEP-1 and Hep3B cells were transfected with si-NC, si-PART1#1, si-PART1#1+anti-miR-NC, or si-PART1#1+miR-149-5p inhibitor. ( B ) The MAP2K1 mRNA expression was assessed by qRT-PCR. ( C, D ) MAP2K1 protein level was determined by Western blot in transfected SK-HEP-1 and Hep3B cells. **P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA PART1 Promotes Proliferation, Migration and Invasion of Hepatocellular Carcinoma Cells via miR-149-5p/MAP2K1 Axis

    doi: 10.2147/CMAR.S246311

    Figure Lengend Snippet: PART1 increased MAP2K1 level by weakening miR-149-5p-mediated inhibitory effect on MAP2K1 in SK-HEP-1 and Hep3B cells. ( A ) Correlation analysis between MAP2K1 and PART1 expression in HCC was performed by Pearson analysis. ( B – D ) SK-HEP-1 and Hep3B cells were transfected with si-NC, si-PART1#1, si-PART1#1+anti-miR-NC, or si-PART1#1+miR-149-5p inhibitor. ( B ) The MAP2K1 mRNA expression was assessed by qRT-PCR. ( C, D ) MAP2K1 protein level was determined by Western blot in transfected SK-HEP-1 and Hep3B cells. **P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay Total RNA was isolated from HCC tumor tissues and cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.

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

    PART1 knockdown suppressed proliferation, migration and invasion in HCC cells. ( A ) The transfection efficacy of si-PART1#1/#2/#3 was measured by qRT-PCR assay. ( B – E ) SK-HEP-1 and Hep3B cells were transfected with si-PART1#1 or si-NC. ( B – C ) Cell viability was evaluated by CCK-8 assay. ( D, E ) The migration and invasion of transfected HCC cells were detected by transwell assay. **P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA PART1 Promotes Proliferation, Migration and Invasion of Hepatocellular Carcinoma Cells via miR-149-5p/MAP2K1 Axis

    doi: 10.2147/CMAR.S246311

    Figure Lengend Snippet: PART1 knockdown suppressed proliferation, migration and invasion in HCC cells. ( A ) The transfection efficacy of si-PART1#1/#2/#3 was measured by qRT-PCR assay. ( B – E ) SK-HEP-1 and Hep3B cells were transfected with si-PART1#1 or si-NC. ( B – C ) Cell viability was evaluated by CCK-8 assay. ( D, E ) The migration and invasion of transfected HCC cells were detected by transwell assay. **P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay Total RNA was isolated from HCC tumor tissues and cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Migration, Transfection, Quantitative RT-PCR, CCK-8 Assay, Transwell Assay

    miR-149-5p inhibited MAP2K1 expression and regulated proliferation, migration and invasion of HCC cells via MAP2K1. SK-HEP-1 and Hep3B cells were transfected with miR-NC, miR-149-5p mimics, miR-149-5p mimics + Vector, or miR-149-5p mimics+MAP2K1. ( A ) The expression level of MAP2K1 mRNA was measured by qRT-PCR assay. ( B, C ) The MAP2K1 protein level was evaluated by Western blot in transfected SK-HEP-1 and Hep3B cells. ( D, E ) Cell proliferative ability of transfected SK-HEP-1 and Hep3B cells was assessed by CCK-8 assay. ( F, G ) Cell migration and invasion of transfected SK-HEP-1 and Hep3B cells were detected by transwell assay. *P

    Journal: Cancer Management and Research

    Article Title: Long Non-Coding RNA PART1 Promotes Proliferation, Migration and Invasion of Hepatocellular Carcinoma Cells via miR-149-5p/MAP2K1 Axis

    doi: 10.2147/CMAR.S246311

    Figure Lengend Snippet: miR-149-5p inhibited MAP2K1 expression and regulated proliferation, migration and invasion of HCC cells via MAP2K1. SK-HEP-1 and Hep3B cells were transfected with miR-NC, miR-149-5p mimics, miR-149-5p mimics + Vector, or miR-149-5p mimics+MAP2K1. ( A ) The expression level of MAP2K1 mRNA was measured by qRT-PCR assay. ( B, C ) The MAP2K1 protein level was evaluated by Western blot in transfected SK-HEP-1 and Hep3B cells. ( D, E ) Cell proliferative ability of transfected SK-HEP-1 and Hep3B cells was assessed by CCK-8 assay. ( F, G ) Cell migration and invasion of transfected SK-HEP-1 and Hep3B cells were detected by transwell assay. *P

    Article Snippet: RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay Total RNA was isolated from HCC tumor tissues and cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Expressing, Migration, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Transwell Assay

    MiR-194 reveals no significant effect on CRC cell proliferation. Notes: ( A ) The changes in morphology before and after transfection were observed by inverted microscopy (×100). ( B ) SW480 cells were infected with miR-194 or negative control (miR-NC) or mock transfection control (miR-MT) lentivirus, and the expression of miR-194 was analyzed by qRT-PCR. ( C ) MTT assays were performed to investigate the proliferation ability of CRC cells. * P

    Journal: OncoTargets and therapy

    Article Title: MicroRNA-194 modulates epithelial–mesenchymal transition in human colorectal cancer metastasis

    doi: 10.2147/OTT.S125172

    Figure Lengend Snippet: MiR-194 reveals no significant effect on CRC cell proliferation. Notes: ( A ) The changes in morphology before and after transfection were observed by inverted microscopy (×100). ( B ) SW480 cells were infected with miR-194 or negative control (miR-NC) or mock transfection control (miR-MT) lentivirus, and the expression of miR-194 was analyzed by qRT-PCR. ( C ) MTT assays were performed to investigate the proliferation ability of CRC cells. * P

    Article Snippet: RNA extraction and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instruction, and reverse transcribed using the PrimeScript RT Reagent Kit (Takara, Dalian, People’s Republic of China).

    Techniques: Transfection, Inverted Microscopy, Infection, Negative Control, Expressing, Quantitative RT-PCR, MTT Assay

    The auxo-action of miR-194 on the EMT of CRC cells. Notes: qRT-PCR ( A ) and Western blot ( B ) were performed to examine the effects of miR-194 overexpression on EMT markers (MMP-2, MMP-9, E-cadherin, and vimentin). * P

    Journal: OncoTargets and therapy

    Article Title: MicroRNA-194 modulates epithelial–mesenchymal transition in human colorectal cancer metastasis

    doi: 10.2147/OTT.S125172

    Figure Lengend Snippet: The auxo-action of miR-194 on the EMT of CRC cells. Notes: qRT-PCR ( A ) and Western blot ( B ) were performed to examine the effects of miR-194 overexpression on EMT markers (MMP-2, MMP-9, E-cadherin, and vimentin). * P

    Article Snippet: RNA extraction and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instruction, and reverse transcribed using the PrimeScript RT Reagent Kit (Takara, Dalian, People’s Republic of China).

    Techniques: Quantitative RT-PCR, Western Blot, Over Expression