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    TaKaRa quantitative real time pcr analysis total rna
    Expression analysis of PtrANR1 and PtrLAR1 in P. trichocarpa tissues. (A) Semi-quantitative <t>RT-PCR</t> analysis of PtrANR1 and PtrLAR1 expression in various tissues of P. trichocarpa . (B) Quantitative real-time PCR analysis of PtrANR1 transcript levels in various tissues of P. trichocarpa. (C) Quantitative real-time PCR analysis of PtrLAR1 transcript levels in various tissues of P. trichocarpa . Poplar 18S expression was used as a control. Total <t>RNA</t> was isolated from roots (R), stems (S), petioles (P), mature leaves (ML), and young leaves (YL).
    Quantitative Real Time Pcr Analysis Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa"

    Article Title: Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064664

    Expression analysis of PtrANR1 and PtrLAR1 in P. trichocarpa tissues. (A) Semi-quantitative RT-PCR analysis of PtrANR1 and PtrLAR1 expression in various tissues of P. trichocarpa . (B) Quantitative real-time PCR analysis of PtrANR1 transcript levels in various tissues of P. trichocarpa. (C) Quantitative real-time PCR analysis of PtrLAR1 transcript levels in various tissues of P. trichocarpa . Poplar 18S expression was used as a control. Total RNA was isolated from roots (R), stems (S), petioles (P), mature leaves (ML), and young leaves (YL).
    Figure Legend Snippet: Expression analysis of PtrANR1 and PtrLAR1 in P. trichocarpa tissues. (A) Semi-quantitative RT-PCR analysis of PtrANR1 and PtrLAR1 expression in various tissues of P. trichocarpa . (B) Quantitative real-time PCR analysis of PtrANR1 transcript levels in various tissues of P. trichocarpa. (C) Quantitative real-time PCR analysis of PtrLAR1 transcript levels in various tissues of P. trichocarpa . Poplar 18S expression was used as a control. Total RNA was isolated from roots (R), stems (S), petioles (P), mature leaves (ML), and young leaves (YL).

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

    2) Product Images from "Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants"

    Article Title: Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0153494

    Seed size comparison and expression profile of TaEXPA2 during wheat-grain-filling in WT and transgenic plants. (A) The level of TaEXPA2 expression during wheat-grain-filling by RT-PCR. The wheat grains were collected after pollination at intervals of 7 days to isolate total RNA. TaTubulin was used as an internal control. (B) Comparison of seed size in WT and TaEXPA2 transgenic tobacco plants. These images were obtained under the same magnification using a dissecting microscope (OLYMPUS SZX12). (C) Total weight of one thousand seeds. Data represent the mean values for the six independent biological replicates.
    Figure Legend Snippet: Seed size comparison and expression profile of TaEXPA2 during wheat-grain-filling in WT and transgenic plants. (A) The level of TaEXPA2 expression during wheat-grain-filling by RT-PCR. The wheat grains were collected after pollination at intervals of 7 days to isolate total RNA. TaTubulin was used as an internal control. (B) Comparison of seed size in WT and TaEXPA2 transgenic tobacco plants. These images were obtained under the same magnification using a dissecting microscope (OLYMPUS SZX12). (C) Total weight of one thousand seeds. Data represent the mean values for the six independent biological replicates.

    Techniques Used: Expressing, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Microscopy

    Expression profiles of TaEXPA2 in different tissues of wheat plants in response to abiotic stresses and signaling molecules, as detected by qRT-PCR. (A) Expression of TaEXPA2 in different organs/tissues of wheat. (B-D) Total RNA was isolated from 2-week-old wheat seedling leaves that were collected after exposure to 20% PEG and 150 mM NaCl (B), 2 mM ABA, 50 mM GA (C), 10 mM MeJA, and 10 mM SA (D). Wheat seedlings incubated in sterile water (CK) were used as controls. The α -tubulin gene was used as an internal reference. The experiments were repeated at least three times.
    Figure Legend Snippet: Expression profiles of TaEXPA2 in different tissues of wheat plants in response to abiotic stresses and signaling molecules, as detected by qRT-PCR. (A) Expression of TaEXPA2 in different organs/tissues of wheat. (B-D) Total RNA was isolated from 2-week-old wheat seedling leaves that were collected after exposure to 20% PEG and 150 mM NaCl (B), 2 mM ABA, 50 mM GA (C), 10 mM MeJA, and 10 mM SA (D). Wheat seedlings incubated in sterile water (CK) were used as controls. The α -tubulin gene was used as an internal reference. The experiments were repeated at least three times.

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

    3) Product Images from "RNA Interference against Discoidin Domain Receptor 2 Ameliorates Alcoholic Liver Disease in Rats"

    Article Title: RNA Interference against Discoidin Domain Receptor 2 Ameliorates Alcoholic Liver Disease in Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0055860

    Screening of discoidin domain receptor 2 (DDR2) siRNA and transfection efficiency of short hairpin RNA plasmid (p.DDR2.shRNA). (a) RT-PCR analysis of mRNA level of DDR2 in siRNA-transfected immortalized rat hepatic stellate cell line HSC-T6. Arrows indicate the position of DDR2 and actin. (b) Quantification of mRNA level of DDR2 relative to that of actin. Data are mean ± SD (n = 3). **P
    Figure Legend Snippet: Screening of discoidin domain receptor 2 (DDR2) siRNA and transfection efficiency of short hairpin RNA plasmid (p.DDR2.shRNA). (a) RT-PCR analysis of mRNA level of DDR2 in siRNA-transfected immortalized rat hepatic stellate cell line HSC-T6. Arrows indicate the position of DDR2 and actin. (b) Quantification of mRNA level of DDR2 relative to that of actin. Data are mean ± SD (n = 3). **P

    Techniques Used: Transfection, shRNA, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    4) Product Images from "Berberine increases adipose triglyceride lipase in 3T3-L1 adipocytes through the AMPK pathway"

    Article Title: Berberine increases adipose triglyceride lipase in 3T3-L1 adipocytes through the AMPK pathway

    Journal: Lipids in Health and Disease

    doi: 10.1186/s12944-016-0383-4

    On D12, cells were treated with 10 μM and 100 μM BBR for 24 h or 48 h in serum-starved DMEM. a Proteins were separated by SDS-PAGE and immunoblotted for p-HSL (Ser 565), HSL, ATGL, GPAT3 and β-actin. The values of p-HSL were quantified using densitometry and normalized with HSL, while those of ATGL and GPAT3 were quantified by densitometry and normalized with β-actin. Representative Western blot results are shown. b Total RNA was extracted from differentiated cells treated with 10 μM and 100 μM BBR for 24 h or 48 h in serum-free DMEM. mRNA levels of ATGL and HSL were determined by real-time PCR and normalized with β-actin. Values are reported as the fold-change relative to the control group. The data are from 3 independent experiments and are presented as the mean ± SD. * p
    Figure Legend Snippet: On D12, cells were treated with 10 μM and 100 μM BBR for 24 h or 48 h in serum-starved DMEM. a Proteins were separated by SDS-PAGE and immunoblotted for p-HSL (Ser 565), HSL, ATGL, GPAT3 and β-actin. The values of p-HSL were quantified using densitometry and normalized with HSL, while those of ATGL and GPAT3 were quantified by densitometry and normalized with β-actin. Representative Western blot results are shown. b Total RNA was extracted from differentiated cells treated with 10 μM and 100 μM BBR for 24 h or 48 h in serum-free DMEM. mRNA levels of ATGL and HSL were determined by real-time PCR and normalized with β-actin. Values are reported as the fold-change relative to the control group. The data are from 3 independent experiments and are presented as the mean ± SD. * p

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

    5) Product Images from "Non-Coding RNAs Participate in the Regulation of CRY-DASH in the Growth and Early Development of Saccharina japonica (Laminariales, Phaeophyceae)"

    Article Title: Non-Coding RNAs Participate in the Regulation of CRY-DASH in the Growth and Early Development of Saccharina japonica (Laminariales, Phaeophyceae)

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21010309

    Transcription changes of sjCRY-DASH induced by white light, blue light, and red light. Transcription accumulation was quantified by qRT-PCR. The changes in transcript levels after exposure to different light conditions are presented as fold changes relative to the RNA from dark-grown sporophytes. Each test was performed in six biological samples. The data in the figures represent the averages ± standard deviation. Data were analyzed by two-way ANOVA followed by Turkey’s multiple comparison test. * p
    Figure Legend Snippet: Transcription changes of sjCRY-DASH induced by white light, blue light, and red light. Transcription accumulation was quantified by qRT-PCR. The changes in transcript levels after exposure to different light conditions are presented as fold changes relative to the RNA from dark-grown sporophytes. Each test was performed in six biological samples. The data in the figures represent the averages ± standard deviation. Data were analyzed by two-way ANOVA followed by Turkey’s multiple comparison test. * p

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    Identification of sjCRY-DASH as a target gene of novel-m3234 in S. japonica . ( A ) Base-pairing interaction between novel-m3234-5p and sjCRY-DASH . ( B ) Secondary structure of the predator sequence of novel-m3234-5p . ( C ) qRT-PCR analysis of miRNA and lncRNA expression that targeted sjCRY-DASH genes. Non-coding RNA expression levels in the dark were used for data normalization. Each value was performed in six biological samples. The data in the figures represent the averages ± standard deviation. One-way ANOVA was used for statistical comparisons between groups. * p
    Figure Legend Snippet: Identification of sjCRY-DASH as a target gene of novel-m3234 in S. japonica . ( A ) Base-pairing interaction between novel-m3234-5p and sjCRY-DASH . ( B ) Secondary structure of the predator sequence of novel-m3234-5p . ( C ) qRT-PCR analysis of miRNA and lncRNA expression that targeted sjCRY-DASH genes. Non-coding RNA expression levels in the dark were used for data normalization. Each value was performed in six biological samples. The data in the figures represent the averages ± standard deviation. One-way ANOVA was used for statistical comparisons between groups. * p

    Techniques Used: Sequencing, Quantitative RT-PCR, Expressing, RNA Expression, Standard Deviation

    6) Product Images from "Thyroid-Stimulating Hormone Inhibits Adipose Triglyceride Lipase in 3T3-L1 Adipocytes through the PKA Pathway"

    Article Title: Thyroid-Stimulating Hormone Inhibits Adipose Triglyceride Lipase in 3T3-L1 Adipocytes through the PKA Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116439

    TSH decreased ATGL expression in mature differentiated cells. (A) On D12, the cells were treated with 0.1 μM bTSH, 1 μM bTSH or 2 μM bTSH for 24 h or 48 h in serum-starved DMEM. Proteins were separated by SDS-PAGE and immunoblotted for ATGL and GAPDH. Values are quantified by densitometry and normalized with GAPDH. Representative Western blot results are shown. (B) Total RNA was extracted from differentiated cells treated with 2 μM bTSH for 48 h in serum-free DMEM. ATGL mRNA levels were determined by real-time PCR and normalized with β-actin. Values are reported as the fold change relative to the control group. The data are from 3 independent experiments and are presented as the mean ± SD. ** p
    Figure Legend Snippet: TSH decreased ATGL expression in mature differentiated cells. (A) On D12, the cells were treated with 0.1 μM bTSH, 1 μM bTSH or 2 μM bTSH for 24 h or 48 h in serum-starved DMEM. Proteins were separated by SDS-PAGE and immunoblotted for ATGL and GAPDH. Values are quantified by densitometry and normalized with GAPDH. Representative Western blot results are shown. (B) Total RNA was extracted from differentiated cells treated with 2 μM bTSH for 48 h in serum-free DMEM. ATGL mRNA levels were determined by real-time PCR and normalized with β-actin. Values are reported as the fold change relative to the control group. The data are from 3 independent experiments and are presented as the mean ± SD. ** p

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

    7) Product Images from "Rice Premature Leaf Senescence 2, Encoding a Glycosyltransferase (GT), Is Involved in Leaf Senescence"

    Article Title: Rice Premature Leaf Senescence 2, Encoding a Glycosyltransferase (GT), Is Involved in Leaf Senescence

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00560

    Expression and localization of PLS2. (A) Expression levels in various tissues revealed by qRT-PCR using the UBQ as the reference gene. Data is presented as the mean ± standard deviation ( n = 9). (B–E) GUS expression patten of P PLS2 :GUS transgenic rice plants on 5-day-old seedlings (B) , root cross-scection of the box area in B (C) , leaf sheath (D) , leaf ( E ; left, P PLS2 :GUS transgenic leaf; right, control). (G,H) RNA in situ hybridization analysis of PLS2 . Flag leaves of wild type plants at heading were cross-sectioned and hybridized with PLS2 -specific antisense (G) or sense (H) probes. x, xylem; p, phloem; ep, epidermis; m, mesophyll. (I) Co-expression of PLS2-GFP fusion protein with HDEL-mRFP (ER marker). ER, endoplasmic reticulum. Scar bars: 12 mm in (B) , 100 μm in (C) , 1 cm in (D,E) , 3 mm in (F) , 200 μm in (G,H) , 10 μm in (I) .
    Figure Legend Snippet: Expression and localization of PLS2. (A) Expression levels in various tissues revealed by qRT-PCR using the UBQ as the reference gene. Data is presented as the mean ± standard deviation ( n = 9). (B–E) GUS expression patten of P PLS2 :GUS transgenic rice plants on 5-day-old seedlings (B) , root cross-scection of the box area in B (C) , leaf sheath (D) , leaf ( E ; left, P PLS2 :GUS transgenic leaf; right, control). (G,H) RNA in situ hybridization analysis of PLS2 . Flag leaves of wild type plants at heading were cross-sectioned and hybridized with PLS2 -specific antisense (G) or sense (H) probes. x, xylem; p, phloem; ep, epidermis; m, mesophyll. (I) Co-expression of PLS2-GFP fusion protein with HDEL-mRFP (ER marker). ER, endoplasmic reticulum. Scar bars: 12 mm in (B) , 100 μm in (C) , 1 cm in (D,E) , 3 mm in (F) , 200 μm in (G,H) , 10 μm in (I) .

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation, Transgenic Assay, RNA In Situ Hybridization, Marker

    8) Product Images from "Lysophosphatidic Acid Stimulates MCP-1 Secretion from C2C12 Myoblast"

    Article Title: Lysophosphatidic Acid Stimulates MCP-1 Secretion from C2C12 Myoblast

    Journal: ISRN inflammation

    doi: 10.5402/2012/983420

    LPA induction of the secretion of MCP-1 was confirmed by RT-PCR and western blot analysis. (a) C2C12 cells were exposed to vehicle, unsaturated forms of LPA (18 : 1) (3 μ M), and cPA (18 : 1) (3 μ M) for 24 h, and RNA was isolated. The mRNA levels of MCP-1 were determined by real-time PCR. MCP-1 mRNA levels were normalized to 18S rRNA (mean ± SEM, n = 3; ** P
    Figure Legend Snippet: LPA induction of the secretion of MCP-1 was confirmed by RT-PCR and western blot analysis. (a) C2C12 cells were exposed to vehicle, unsaturated forms of LPA (18 : 1) (3 μ M), and cPA (18 : 1) (3 μ M) for 24 h, and RNA was isolated. The mRNA levels of MCP-1 were determined by real-time PCR. MCP-1 mRNA levels were normalized to 18S rRNA (mean ± SEM, n = 3; ** P

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

    9) Product Images from "Estrogen stimulates SREBP2 expression in hepatic cell lines via an estrogen response element in the SREBP2 promoter"

    Article Title: Estrogen stimulates SREBP2 expression in hepatic cell lines via an estrogen response element in the SREBP2 promoter

    Journal: Cellular & Molecular Biology Letters

    doi: 10.1186/s11658-019-0194-5

    Existence of an ERE within the SREBP2 promoter. a ChIP analysis was performed using anti-ERα or anti-RNA polymerase II antibody to ascertain the existence of the ERE in the promoter of the SREBP2 gene. The PCR results show that one fragment containing the putative ERE could be precipitated after treatment of HepG2 and HuH-7 with E 2 (10 − 7 mol/l) for 24 h. b The pulled-down band was excised from the gel and sequenced. SREBP2: sterol regulatory element-binding protein; E 2 : estradiol; ERE: estrogen response element; ChIP: chromatin immunoprecipitation
    Figure Legend Snippet: Existence of an ERE within the SREBP2 promoter. a ChIP analysis was performed using anti-ERα or anti-RNA polymerase II antibody to ascertain the existence of the ERE in the promoter of the SREBP2 gene. The PCR results show that one fragment containing the putative ERE could be precipitated after treatment of HepG2 and HuH-7 with E 2 (10 − 7 mol/l) for 24 h. b The pulled-down band was excised from the gel and sequenced. SREBP2: sterol regulatory element-binding protein; E 2 : estradiol; ERE: estrogen response element; ChIP: chromatin immunoprecipitation

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay

    10) Product Images from "Transcriptome Analyses Provide Novel Insights into Heat Stress Responses in Chieh-Qua (Benincasa hispida Cogn. var. Chieh-Qua How)"

    Article Title: Transcriptome Analyses Provide Novel Insights into Heat Stress Responses in Chieh-Qua (Benincasa hispida Cogn. var. Chieh-Qua How)

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20040883

    Validation of DEGs under heat stress. Correlation between the fold change analyzed by RNA-seq ( y -axis) and data obtained using qRT-PCR ( x -axis).
    Figure Legend Snippet: Validation of DEGs under heat stress. Correlation between the fold change analyzed by RNA-seq ( y -axis) and data obtained using qRT-PCR ( x -axis).

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR

    11) Product Images from "Effects of Long-Chain Fatty Acyl-CoA Synthetase 1 on Diglyceride Synthesis and Arachidonic Acid Metabolism in Sheep Adipocytes"

    Article Title: Effects of Long-Chain Fatty Acyl-CoA Synthetase 1 on Diglyceride Synthesis and Arachidonic Acid Metabolism in Sheep Adipocytes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21062044

    Transcriptome sequencing: ( a ) heat map; ( b ) volcano plots; ( c ) KEGG analysis; ( d ) RNA-seq data and qRT-PCR of sheep adipocytes (* p
    Figure Legend Snippet: Transcriptome sequencing: ( a ) heat map; ( b ) volcano plots; ( c ) KEGG analysis; ( d ) RNA-seq data and qRT-PCR of sheep adipocytes (* p

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

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Metformin Inhibits Advanced Glycation End Products-Induced Inflammatory Response in Murine Macrophages Partly through AMPK Activation and RAGE/NFκB Pathway Suppression
    Article Snippet: .. Real-Time PCR Analysis Total RNA was extracted from BMDMs using TRIzol reagent (Takara, Liaoning, China, Cat# 9109), and 1 μ g of total RNA was reverse transcribed to cDNA using the PrimeScript RT Master Mix kit (Takara, Liaoning, China, Cat# RR036A). .. Real-time PCR array analysis was performed by using the SYBR Premix Ex Taq™ kit (Takara, Liaoning, China, Cat# RR420A) in a total volume of 20 μ L, with 2 μ L of cDNA primers (0.2 mM each), 10 μ L of SYBR Green, and 0.4 μ L of Rox Dye II.

    Article Title: RNA Interference against Discoidin Domain Receptor 2 Ameliorates Alcoholic Liver Disease in Rats
    Article Snippet: .. Quantitative Real-time PCR Analysis Total RNA was homogenized and extracted from cells and frozen specimens by use of Trizol (Takara, Shiga, Japan). .. The RNA samples underwent reverse transcription with M-MLV reverse transcriptase (Invitrogen, Shanghai) and random primers.

    Article Title: Berberine increases adipose triglyceride lipase in 3T3-L1 adipocytes through the AMPK pathway
    Article Snippet: .. RNA extraction and quantitative real-time PCR analysis Total RNA was isolated from cells using TRIzol reagent (Takara, Tokyo, Japan) according to the manufacturer’s instructions. ..

    Article Title: Production of viable seeds from the seedling lethal mutant ppi2-2 lacking the atToc159 chloroplast protein import receptor using plastic containers, and characterization of the homozygous mutant progeny
    Article Snippet: .. RNA ISOLATION AND REAL-TIME PCR ANALYSIS Total RNA was extracted from aerial tissues of wild-type and mutant plants using an RNAiso plus reagent (Takara) as described elsewhere ( ). ..

    Article Title: Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants
    Article Snippet: .. Generation of TaEXPA2 -overexpressing tobacco lines and quantitative real-time PCR analysis Total RNA was extracted from wheat leaves with Trizol reagent (TaKaRa, Japan) according to the manufacturer’s instructions. .. Total RNA (2 μg) was reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA).

    Article Title: Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa
    Article Snippet: .. Semi-quantitative RT-PCR and Quantitative Real-time PCR Analysis Total RNA was extracted from leaves, roots, stems, and petioles of poplar plants and treated with DNase I (TaKaRa) according to the manufacturer’s instructions. .. All RNA was purified and first-strand cDNA was synthesized as described above.

    Article Title: Thyroid-Stimulating Hormone Inhibits Adipose Triglyceride Lipase in 3T3-L1 Adipocytes through the PKA Pathway
    Article Snippet: .. RNA extraction and quantitative real-time PCR analysis Total RNA from the cells and fresh mouse adipose tissues was isolated using TRIzol reagent (Takara, Tokyo, Japan) following the manufacturer’s instructions. ..

    Article Title: Non-Coding RNAs Participate in the Regulation of CRY-DASH in the Growth and Early Development of Saccharina japonica (Laminariales, Phaeophyceae)
    Article Snippet: .. Quantitative Real-Time PCR Analysis Total RNA (1 µg) was used for reverse transcription using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Tokyo, Japan). .. Real-time PCR was performed with SYBR Premix Ex Taq II (TaKaRa, Tokyo, Japan) on a TP800 Thermal Cycler Dice Real-Time System (TaKaRa, Japan).

    Isolation:

    Article Title: Berberine increases adipose triglyceride lipase in 3T3-L1 adipocytes through the AMPK pathway
    Article Snippet: .. RNA extraction and quantitative real-time PCR analysis Total RNA was isolated from cells using TRIzol reagent (Takara, Tokyo, Japan) according to the manufacturer’s instructions. ..

    Article Title: Production of viable seeds from the seedling lethal mutant ppi2-2 lacking the atToc159 chloroplast protein import receptor using plastic containers, and characterization of the homozygous mutant progeny
    Article Snippet: .. RNA ISOLATION AND REAL-TIME PCR ANALYSIS Total RNA was extracted from aerial tissues of wild-type and mutant plants using an RNAiso plus reagent (Takara) as described elsewhere ( ). ..

    Article Title: Thyroid-Stimulating Hormone Inhibits Adipose Triglyceride Lipase in 3T3-L1 Adipocytes through the PKA Pathway
    Article Snippet: .. RNA extraction and quantitative real-time PCR analysis Total RNA from the cells and fresh mouse adipose tissues was isolated using TRIzol reagent (Takara, Tokyo, Japan) following the manufacturer’s instructions. ..

    RNA Extraction:

    Article Title: Berberine increases adipose triglyceride lipase in 3T3-L1 adipocytes through the AMPK pathway
    Article Snippet: .. RNA extraction and quantitative real-time PCR analysis Total RNA was isolated from cells using TRIzol reagent (Takara, Tokyo, Japan) according to the manufacturer’s instructions. ..

    Article Title: Thyroid-Stimulating Hormone Inhibits Adipose Triglyceride Lipase in 3T3-L1 Adipocytes through the PKA Pathway
    Article Snippet: .. RNA extraction and quantitative real-time PCR analysis Total RNA from the cells and fresh mouse adipose tissues was isolated using TRIzol reagent (Takara, Tokyo, Japan) following the manufacturer’s instructions. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa
    Article Snippet: .. Semi-quantitative RT-PCR and Quantitative Real-time PCR Analysis Total RNA was extracted from leaves, roots, stems, and petioles of poplar plants and treated with DNase I (TaKaRa) according to the manufacturer’s instructions. .. All RNA was purified and first-strand cDNA was synthesized as described above.

    Mutagenesis:

    Article Title: Production of viable seeds from the seedling lethal mutant ppi2-2 lacking the atToc159 chloroplast protein import receptor using plastic containers, and characterization of the homozygous mutant progeny
    Article Snippet: .. RNA ISOLATION AND REAL-TIME PCR ANALYSIS Total RNA was extracted from aerial tissues of wild-type and mutant plants using an RNAiso plus reagent (Takara) as described elsewhere ( ). ..

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    TaKaRa quantitative real time pcr qrt pcr analysis total rna
    Comparisons of the protein and mRNA expression patterns of three representative DEPs at four artificial ageing stages (WH98, WH50, WH20, and WH01) by iTRAQ and <t>qRT-PCR.</t> Solid lines represent mRNA expression patterns, and dotted lines represent protein expression patterns.
    Quantitative Real Time Pcr Qrt Pcr Analysis Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative real time polymerase chain reaction pcr analysis total rna
    (A) The effect of small interfering <t>RNA</t> on heart-type fatty-acid-binding protein (FABP3) mRNA expression in human coronary artery endothelial cells (HCAECs). The efficiency of FABP3 knockdown was calculated to be 75% by real-time quantitative <t>RT-PCR.</t> Data are presented as the mean ± SEM ( n = 3). Protein levels were analyzed by SDS–PAGE and visualized with the enhanced chemiluminescence reagent. Each lane was loaded with 20 μg of whole-cell lysate. β-Actin was used as the loading control. (B) Effect of lysophosphatidic acid (LPA) on reporter activation in FABP3-knocked-down HCAECs. FABP3-knocked-down cells were transiently transfected with a pGL3-PPRE-acyl-CoA oxidase luciferase reporter vector. The cells were treated with 1–30 μM LPA for 20 h. Luciferase activity was normalized to Renilla luciferase activity. The synthetic peroxisome proliferator-activated receptor gamma antagonist T0070907 (10 μM) was used as the positive control. Data are expressed as the mean ± SEM ( n = 4), ∗∗ P
    Quantitative Real Time Polymerase Chain Reaction Pcr Analysis Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa rt qpcr analysis total rna
    SCOTIN restricts HCV replication through autophagy-mediated protein degradation. ( a – d ) Huh-7 cells were transfected with the indicated plasmids or with siRNA, followed by HCVcc infection (10 MOI) for 3 days. The cells were treated with 3-MA (10 mM; a ), BFA (100 μM; c ) or CQ (50 μM; d ) for 12 h before harvesting. The intracellular HCV <t>RNA</t> levels were measured with <t>RT–qPCR,</t> and total cell lysates were subjected to immunoblotting using the indicated antibodies. DW was used as a control for 3-MA and CQ, and DMSO was used as a control for BFA. The bars indicate the average value±s.d. obtained from three experiments. The asterisks denote the P values calculated using the t -test (*** P value
    Rt Qpcr Analysis Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Comparisons of the protein and mRNA expression patterns of three representative DEPs at four artificial ageing stages (WH98, WH50, WH20, and WH01) by iTRAQ and qRT-PCR. Solid lines represent mRNA expression patterns, and dotted lines represent protein expression patterns.

    Journal: PLoS ONE

    Article Title: Quantitative Proteomic Analysis of Wheat Seeds during Artificial Ageing and Priming Using the Isobaric Tandem Mass Tag Labeling

    doi: 10.1371/journal.pone.0162851

    Figure Lengend Snippet: Comparisons of the protein and mRNA expression patterns of three representative DEPs at four artificial ageing stages (WH98, WH50, WH20, and WH01) by iTRAQ and qRT-PCR. Solid lines represent mRNA expression patterns, and dotted lines represent protein expression patterns.

    Article Snippet: RNA isolation and quantitative real-time PCR (qRT-PCR) analysis Total RNA from wheat embryos of WH98, WH50, WH20, and WH01 were extracted by using RNAiso Plus reagent (Takara, Tokyo, Japan), and genomic DNA was removed by treating with DNase I (Takara) following the manufacturer’s protocol.

    Techniques: Expressing, Quantitative RT-PCR

    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

    (A) The effect of small interfering RNA on heart-type fatty-acid-binding protein (FABP3) mRNA expression in human coronary artery endothelial cells (HCAECs). The efficiency of FABP3 knockdown was calculated to be 75% by real-time quantitative RT-PCR. Data are presented as the mean ± SEM ( n = 3). Protein levels were analyzed by SDS–PAGE and visualized with the enhanced chemiluminescence reagent. Each lane was loaded with 20 μg of whole-cell lysate. β-Actin was used as the loading control. (B) Effect of lysophosphatidic acid (LPA) on reporter activation in FABP3-knocked-down HCAECs. FABP3-knocked-down cells were transiently transfected with a pGL3-PPRE-acyl-CoA oxidase luciferase reporter vector. The cells were treated with 1–30 μM LPA for 20 h. Luciferase activity was normalized to Renilla luciferase activity. The synthetic peroxisome proliferator-activated receptor gamma antagonist T0070907 (10 μM) was used as the positive control. Data are expressed as the mean ± SEM ( n = 4), ∗∗ P

    Journal: FEBS Open Bio

    Article Title: Heart-type fatty-acid-binding protein (FABP3) is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

    doi: 10.1016/j.fob.2014.10.014

    Figure Lengend Snippet: (A) The effect of small interfering RNA on heart-type fatty-acid-binding protein (FABP3) mRNA expression in human coronary artery endothelial cells (HCAECs). The efficiency of FABP3 knockdown was calculated to be 75% by real-time quantitative RT-PCR. Data are presented as the mean ± SEM ( n = 3). Protein levels were analyzed by SDS–PAGE and visualized with the enhanced chemiluminescence reagent. Each lane was loaded with 20 μg of whole-cell lysate. β-Actin was used as the loading control. (B) Effect of lysophosphatidic acid (LPA) on reporter activation in FABP3-knocked-down HCAECs. FABP3-knocked-down cells were transiently transfected with a pGL3-PPRE-acyl-CoA oxidase luciferase reporter vector. The cells were treated with 1–30 μM LPA for 20 h. Luciferase activity was normalized to Renilla luciferase activity. The synthetic peroxisome proliferator-activated receptor gamma antagonist T0070907 (10 μM) was used as the positive control. Data are expressed as the mean ± SEM ( n = 4), ∗∗ P

    Article Snippet: 2.7 Quantitative real-time polymerase chain reaction (PCR) analysis Total RNA was prepared from HCAECs cells using NucleoSpin® RNA II (Takara, Shiga, Japan).

    Techniques: Small Interfering RNA, Binding Assay, Expressing, Quantitative RT-PCR, SDS Page, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Positive Control

    SCOTIN restricts HCV replication through autophagy-mediated protein degradation. ( a – d ) Huh-7 cells were transfected with the indicated plasmids or with siRNA, followed by HCVcc infection (10 MOI) for 3 days. The cells were treated with 3-MA (10 mM; a ), BFA (100 μM; c ) or CQ (50 μM; d ) for 12 h before harvesting. The intracellular HCV RNA levels were measured with RT–qPCR, and total cell lysates were subjected to immunoblotting using the indicated antibodies. DW was used as a control for 3-MA and CQ, and DMSO was used as a control for BFA. The bars indicate the average value±s.d. obtained from three experiments. The asterisks denote the P values calculated using the t -test (*** P value

    Journal: Nature Communications

    Article Title: Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A

    doi: 10.1038/ncomms10631

    Figure Lengend Snippet: SCOTIN restricts HCV replication through autophagy-mediated protein degradation. ( a – d ) Huh-7 cells were transfected with the indicated plasmids or with siRNA, followed by HCVcc infection (10 MOI) for 3 days. The cells were treated with 3-MA (10 mM; a ), BFA (100 μM; c ) or CQ (50 μM; d ) for 12 h before harvesting. The intracellular HCV RNA levels were measured with RT–qPCR, and total cell lysates were subjected to immunoblotting using the indicated antibodies. DW was used as a control for 3-MA and CQ, and DMSO was used as a control for BFA. The bars indicate the average value±s.d. obtained from three experiments. The asterisks denote the P values calculated using the t -test (*** P value

    Article Snippet: RNA extraction and RT–qPCR analysis Total RNA was extracted using RNAiso Plus (Takara), and 1 μg of RNA was reverse-transcribed into cDNA using random primers and an ImProm-II Reverse Transcription Kit (Promega).

    Techniques: Transfection, Infection, Quantitative RT-PCR

    Physical interaction between NS5A and SCOTIN is required for control of degradation. ( a ) HEK293 cells were transfected with GST-NS5A along with an empty (pcDNA3.1-MYC) or SCOTIN-MYC plasmid for 48 h. GST-NS5A was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( b ) Schematic representation of GST-tagged NS5A deletion constructs. ( c ) HEK293 cells were transfected with the indicated plasmids and subjected to a GST pulldown assay. ( d ) Huh-7 cells were transfected with the indicated plasmids for 48 h, followed by immunoblotting analysis using the indicated antibodies. EGFP was used to monitor transfection efficiency. ( e ) An illustration of the truncated SCOTIN constructs is shown. ( f ) Huh-7 cells were transfected with the indicated SCOTIN mutant constructs, and immunofluorescence analysis was performed using LC3 and MYC antibodies, followed by Hoechst staining. Cellular localization of LC3 (green), MYC (red) and the nucleus (blue) was determined using fluorescence microscopy. Representative images are shown. Higher-magnification images are shown in the right corner. Scale bar, 10 μm. ( g ) Western blot analysis of Huh-7 cells transfected with the indicated plasmids. EGFP-N1 was co-transfected to monitor transfection efficiency. ( h ) HEK293 cells were transfected with GST-NS5A along with the indicated plasmids for 48 h. Total cell lysates were incubated with glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( i , j ) Huh-7 cells were transfected with the indicated plasmids followed by HCVcc infection (10 MOI) for 3 days before harvesting. The intracellular HCV RNA levels were determined using RT–qPCR ( i ), and total cell lysates were subjected to immunoblotting using the indicated antibodies ( j ). The bars indicate the mean value±s.d. obtained from triplicate experiments. The asterisks indicate the P values calculated using the t -test. ** P value

    Journal: Nature Communications

    Article Title: Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A

    doi: 10.1038/ncomms10631

    Figure Lengend Snippet: Physical interaction between NS5A and SCOTIN is required for control of degradation. ( a ) HEK293 cells were transfected with GST-NS5A along with an empty (pcDNA3.1-MYC) or SCOTIN-MYC plasmid for 48 h. GST-NS5A was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( b ) Schematic representation of GST-tagged NS5A deletion constructs. ( c ) HEK293 cells were transfected with the indicated plasmids and subjected to a GST pulldown assay. ( d ) Huh-7 cells were transfected with the indicated plasmids for 48 h, followed by immunoblotting analysis using the indicated antibodies. EGFP was used to monitor transfection efficiency. ( e ) An illustration of the truncated SCOTIN constructs is shown. ( f ) Huh-7 cells were transfected with the indicated SCOTIN mutant constructs, and immunofluorescence analysis was performed using LC3 and MYC antibodies, followed by Hoechst staining. Cellular localization of LC3 (green), MYC (red) and the nucleus (blue) was determined using fluorescence microscopy. Representative images are shown. Higher-magnification images are shown in the right corner. Scale bar, 10 μm. ( g ) Western blot analysis of Huh-7 cells transfected with the indicated plasmids. EGFP-N1 was co-transfected to monitor transfection efficiency. ( h ) HEK293 cells were transfected with GST-NS5A along with the indicated plasmids for 48 h. Total cell lysates were incubated with glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( i , j ) Huh-7 cells were transfected with the indicated plasmids followed by HCVcc infection (10 MOI) for 3 days before harvesting. The intracellular HCV RNA levels were determined using RT–qPCR ( i ), and total cell lysates were subjected to immunoblotting using the indicated antibodies ( j ). The bars indicate the mean value±s.d. obtained from triplicate experiments. The asterisks indicate the P values calculated using the t -test. ** P value

    Article Snippet: RNA extraction and RT–qPCR analysis Total RNA was extracted using RNAiso Plus (Takara), and 1 μg of RNA was reverse-transcribed into cDNA using random primers and an ImProm-II Reverse Transcription Kit (Promega).

    Techniques: Transfection, Plasmid Preparation, Construct, GST Pulldown Assay, Mutagenesis, Immunofluorescence, Staining, Fluorescence, Microscopy, Western Blot, Incubation, Infection, Quantitative RT-PCR

    SCOTIN is degraded via HCV-induced autophagy. ( a , b ) Huh-7 cells were treated with rapamycin (2 μM) for the indicated durations ( a ) or with 3-MA (10 mM), BFA (100 μM) or CQ (50 μM) for 12 h ( b ). Total cell lysates were subjected to immunoblotting using the indicated antibodies. ( c ) Electron microscopic immunogold staining for the SCOTIN protein, showing its localization in autophagosomes (APs). Scale bar, 0.5 μm. Higher-magnification images are shown on the right. The gold particles (30 nm, indicated by the arrows) show the SCOTIN protein in APs. ( d , e ) Huh-7 cells were infected with HCVcc (10 MOI) for the indicated durations. ( d ) The expression levels of IFN-β, SCOTIN and HCV RNA were determined using RT–qPCR and were compared with those of RPL32 RNA. The relative HCV RNA levels shown were determined by comparison with that of the RNA of cells infected with HCV for 48 h, which was set to 1. The relative levels of IFN-β and SCOTIN RNA were determined by comparison with their levels in control cells, which were set to 1. The bars indicate the mean value±s.d. obtained from triplicate experiments. ( e ) Total cell lysates were subjected to immunoblotting using the indicated antibodies. ( f ) Schematic illustration of the proposed mechanism.

    Journal: Nature Communications

    Article Title: Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A

    doi: 10.1038/ncomms10631

    Figure Lengend Snippet: SCOTIN is degraded via HCV-induced autophagy. ( a , b ) Huh-7 cells were treated with rapamycin (2 μM) for the indicated durations ( a ) or with 3-MA (10 mM), BFA (100 μM) or CQ (50 μM) for 12 h ( b ). Total cell lysates were subjected to immunoblotting using the indicated antibodies. ( c ) Electron microscopic immunogold staining for the SCOTIN protein, showing its localization in autophagosomes (APs). Scale bar, 0.5 μm. Higher-magnification images are shown on the right. The gold particles (30 nm, indicated by the arrows) show the SCOTIN protein in APs. ( d , e ) Huh-7 cells were infected with HCVcc (10 MOI) for the indicated durations. ( d ) The expression levels of IFN-β, SCOTIN and HCV RNA were determined using RT–qPCR and were compared with those of RPL32 RNA. The relative HCV RNA levels shown were determined by comparison with that of the RNA of cells infected with HCV for 48 h, which was set to 1. The relative levels of IFN-β and SCOTIN RNA were determined by comparison with their levels in control cells, which were set to 1. The bars indicate the mean value±s.d. obtained from triplicate experiments. ( e ) Total cell lysates were subjected to immunoblotting using the indicated antibodies. ( f ) Schematic illustration of the proposed mechanism.

    Article Snippet: RNA extraction and RT–qPCR analysis Total RNA was extracted using RNAiso Plus (Takara), and 1 μg of RNA was reverse-transcribed into cDNA using random primers and an ImProm-II Reverse Transcription Kit (Promega).

    Techniques: Staining, Infection, Expressing, Quantitative RT-PCR

    The ER protein SCOTIN inhibits HCV replication. ( a , b ) Huh-7 cells were incubated with IL-1β (10 ng ml −1 ), IL-6 (10 ng ml −1 ), tumour-necrosis factor (TNF)-α (10 ng ml −1 ) or IFN-β (100 U ml −1 ) for 12 h, and the expression of SCOTIN mRNA was measured with RT–qPCR and compared with that of RPL32 mRNA ( a ). Endogenous SCOTIN protein levels were measured by immunoblotting. Lamin B2 was used as a loading control ( b ). The bars indicate the mean value±s.d. obtained from three experiments. ( c – h ) Huh-7 cells were infected with HCVcc (0.5 multiplicity of infection (MOI)) for 2 days, followed by transfection of the indicated plasmids or siRNAs for 72 h. ( c , f ) The intracellular HCV RNA titre was measured using reverse transcriptase–quantitative PCR (RT–qPCR) and normalized to β-actin. ( d , g ) Infectious HCV virions were analysed using a colorimetric focus-forming assay. The results are presented as focus-forming units (FFU) per ml of culture supernatant. ( e , h ) Total cell lysates were subjected to immunoblotting using the indicated antibodies. The bars indicate the mean value±s.e. obtained from triplicate experiments. The asterisks indicate the P values calculated using the t -test. * P value

    Journal: Nature Communications

    Article Title: Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A

    doi: 10.1038/ncomms10631

    Figure Lengend Snippet: The ER protein SCOTIN inhibits HCV replication. ( a , b ) Huh-7 cells were incubated with IL-1β (10 ng ml −1 ), IL-6 (10 ng ml −1 ), tumour-necrosis factor (TNF)-α (10 ng ml −1 ) or IFN-β (100 U ml −1 ) for 12 h, and the expression of SCOTIN mRNA was measured with RT–qPCR and compared with that of RPL32 mRNA ( a ). Endogenous SCOTIN protein levels were measured by immunoblotting. Lamin B2 was used as a loading control ( b ). The bars indicate the mean value±s.d. obtained from three experiments. ( c – h ) Huh-7 cells were infected with HCVcc (0.5 multiplicity of infection (MOI)) for 2 days, followed by transfection of the indicated plasmids or siRNAs for 72 h. ( c , f ) The intracellular HCV RNA titre was measured using reverse transcriptase–quantitative PCR (RT–qPCR) and normalized to β-actin. ( d , g ) Infectious HCV virions were analysed using a colorimetric focus-forming assay. The results are presented as focus-forming units (FFU) per ml of culture supernatant. ( e , h ) Total cell lysates were subjected to immunoblotting using the indicated antibodies. The bars indicate the mean value±s.e. obtained from triplicate experiments. The asterisks indicate the P values calculated using the t -test. * P value

    Article Snippet: RNA extraction and RT–qPCR analysis Total RNA was extracted using RNAiso Plus (Takara), and 1 μg of RNA was reverse-transcribed into cDNA using random primers and an ImProm-II Reverse Transcription Kit (Promega).

    Techniques: Incubation, Expressing, Quantitative RT-PCR, Infection, Transfection, Real-time Polymerase Chain Reaction, Focus Forming Assay