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TaKaRa sybr qrt pcr
IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by <t>qRT-PCR.</t> All data are expressed as the means ± SD (n = 4). *p
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1) Product Images from "Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA"

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2016.12.007

IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4). *p
Figure Legend Snippet: IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4). *p

Techniques Used: Inhibition, Expressing, shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR

Knockdown Efficiencies of Chemically Synthetic siRNAs following IFN-α Stimulation (A and B) A549 cells were transfected with siControl, sip53 (A), or simyc (B), followed by treatment with recombinant IFNα at 10 4 U/mL. After 48-hr incubation, c- myc and p53 mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4).
Figure Legend Snippet: Knockdown Efficiencies of Chemically Synthetic siRNAs following IFN-α Stimulation (A and B) A549 cells were transfected with siControl, sip53 (A), or simyc (B), followed by treatment with recombinant IFNα at 10 4 U/mL. After 48-hr incubation, c- myc and p53 mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4).

Techniques Used: Transfection, Recombinant, Incubation, Quantitative RT-PCR

Inhibition of shRNA-Mediated Knockdown by IFN-α Stimulation (A and B) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 2 , 10 3 , or 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. (D and E) A549 cells were transfected with pHMU6-shLuc, -shmyc (D), or -shp53 (E), followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc (D) and p53 (E) mRNA levels in the cells were similarly determined. These experiments were repeated at least three times, and representative data are shown. All data are expressed as the means ± SD (n = 4). *p
Figure Legend Snippet: Inhibition of shRNA-Mediated Knockdown by IFN-α Stimulation (A and B) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 2 , 10 3 , or 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. (D and E) A549 cells were transfected with pHMU6-shLuc, -shmyc (D), or -shp53 (E), followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc (D) and p53 (E) mRNA levels in the cells were similarly determined. These experiments were repeated at least three times, and representative data are shown. All data are expressed as the means ± SD (n = 4). *p

Techniques Used: Inhibition, shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR, Western Blot

Dicer and Ago2 Expression Levels following IFN-α Stimulation H1299 and A549 cells were treated with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, dicer and Ago2 mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) TRBP protein levels in the cells were determined by western blotting analysis. The qRT-PCR data are expressed as the means ± SD (n = 4).
Figure Legend Snippet: Dicer and Ago2 Expression Levels following IFN-α Stimulation H1299 and A549 cells were treated with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, dicer and Ago2 mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) TRBP protein levels in the cells were determined by western blotting analysis. The qRT-PCR data are expressed as the means ± SD (n = 4).

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

Inhibition of Ad Vector-Expressing shRNA-Mediated Knockdown by IFN-α Stimulation (A) 4 × 10 8 IFU Ad-shLuc was intravenously administered to C57BL/6 mice. 3 hr after the administration, IFN-β mRNA levels in the spleen were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). (B–D) A549 cells were transduced with Ad-shp53, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, the copy numbers of sip53 in the cells were determined by qRT-PCR (B). After 48-hr incubation, p53 mRNA (C) and protein (D) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. Data are expressed as the means ± SD (n = 4). **p
Figure Legend Snippet: Inhibition of Ad Vector-Expressing shRNA-Mediated Knockdown by IFN-α Stimulation (A) 4 × 10 8 IFU Ad-shLuc was intravenously administered to C57BL/6 mice. 3 hr after the administration, IFN-β mRNA levels in the spleen were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). (B–D) A549 cells were transduced with Ad-shp53, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, the copy numbers of sip53 in the cells were determined by qRT-PCR (B). After 48-hr incubation, p53 mRNA (C) and protein (D) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. Data are expressed as the means ± SD (n = 4). **p

Techniques Used: Inhibition, Plasmid Preparation, Expressing, shRNA, Mouse Assay, Quantitative RT-PCR, Transduction, Recombinant, Incubation, Western Blot

Efficiencies of shRNA-Mediated Knockdown following Exposure to IFN-β, IFN-γ, and TNF-α (A) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-β at 10 2 or 10 3 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. (B) H1299 cells were similarly transfected, followed by treatment with recombinant IFN-γ and TNF-α at 5 × 10 3 U/mL and 100 ng/mL, respectively. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. All data are expressed as the means ± SD (n = 4). *p
Figure Legend Snippet: Efficiencies of shRNA-Mediated Knockdown following Exposure to IFN-β, IFN-γ, and TNF-α (A) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-β at 10 2 or 10 3 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. (B) H1299 cells were similarly transfected, followed by treatment with recombinant IFN-γ and TNF-α at 5 × 10 3 U/mL and 100 ng/mL, respectively. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. All data are expressed as the means ± SD (n = 4). *p

Techniques Used: shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR

Type I IFN Responses Following Introduction of shRNA-Expressing Vectors (A–D) A549 (A and C) and H1299 (B and D) cells were transfected with pHMU6-shLuc (A and B) or siControl (indicated as siRNA) (C and D). After 24-hr incubation, IFNβ, ISG54, and ISG56 mRNA levels in the cells were determined by qRT-PCR. The data are expressed as the means ± SD (n = 3). (E) 100 μg pHMU6-shLuc was intramuscularly administered to C57BL/6 mice. 3 hr after administration, the IFN-β mRNA levels in the mouse muscle were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). *p
Figure Legend Snippet: Type I IFN Responses Following Introduction of shRNA-Expressing Vectors (A–D) A549 (A and C) and H1299 (B and D) cells were transfected with pHMU6-shLuc (A and B) or siControl (indicated as siRNA) (C and D). After 24-hr incubation, IFNβ, ISG54, and ISG56 mRNA levels in the cells were determined by qRT-PCR. The data are expressed as the means ± SD (n = 3). (E) 100 μg pHMU6-shLuc was intramuscularly administered to C57BL/6 mice. 3 hr after administration, the IFN-β mRNA levels in the mouse muscle were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). *p

Techniques Used: shRNA, Expressing, Transfection, Incubation, Quantitative RT-PCR, Mouse Assay

Processing of shRNA to siRNA in IFN-α-Treated Cells (A and B) H1299 cells were transfected with pHMU6-shp53 or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, shRNA and siRNA copy numbers in the cells were determined by northern blotting analysis (A) and qRT-PCR (B). Data are expressed as the means ± SD (n = 4). *p
Figure Legend Snippet: Processing of shRNA to siRNA in IFN-α-Treated Cells (A and B) H1299 cells were transfected with pHMU6-shp53 or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, shRNA and siRNA copy numbers in the cells were determined by northern blotting analysis (A) and qRT-PCR (B). Data are expressed as the means ± SD (n = 4). *p

Techniques Used: shRNA, Transfection, Recombinant, Incubation, Northern Blot, Quantitative RT-PCR

2) Product Images from "Common microRNA–mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states"

Article Title: Common microRNA–mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.426

Conjoint analysis of small RNA-seq and mRNA-seq for distinct piPSC lines. ( a ) Flow diagram showing the conjoint analysis procedure of small RNA-seq and mRNA-seq data. ( b ) Venn analysis between the predicted miRNA target mRNAs and the differentially expressed mRNAs with the same expression trend in piPS-L, piPS-F and piPS-LF. ( c ) The network showing the common miRNAs and their targets generated by conjoint analysis of small RNA-seq and mRNA-seq. ( d ) qRT-PCR validation of the expression levels of two miRNAs (miR-31863 and miR-370) with their target LIN28A in Dox-piPSCs. ( e ) qRT-PCR showing the expression level of miR-206 and its target OTX2 in Dox-piPSCs. ** P
Figure Legend Snippet: Conjoint analysis of small RNA-seq and mRNA-seq for distinct piPSC lines. ( a ) Flow diagram showing the conjoint analysis procedure of small RNA-seq and mRNA-seq data. ( b ) Venn analysis between the predicted miRNA target mRNAs and the differentially expressed mRNAs with the same expression trend in piPS-L, piPS-F and piPS-LF. ( c ) The network showing the common miRNAs and their targets generated by conjoint analysis of small RNA-seq and mRNA-seq. ( d ) qRT-PCR validation of the expression levels of two miRNAs (miR-31863 and miR-370) with their target LIN28A in Dox-piPSCs. ( e ) qRT-PCR showing the expression level of miR-206 and its target OTX2 in Dox-piPSCs. ** P

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

MiR-370 represses the expression level of LIN28A . ( a ) Images showing the morphology of Dox-piPSCs at undifferentiated (DOX+) and differentiated (DOX−) state. Scale bar, 100 μ m. ( b ) qRT-PCR analysis showing the expression level changes of LIN28A and miR-370 in Dox-piPSCs at undifferentiated and differentiated state. ( c ) Comparative analysis of Pri-miR-370 promoter of Sus scrofa (pig), homo sapiens (human) and Mus musculus (mouse).The conservative promoter region is predicted to be bound by 18 transcription factors ( ERF, CDX1, ELF5, ELK1, ELK3, ARNT, SRY, FLI1, ETS1, ETV1, SPIB, ETV5, ETV4, TCFL5, TFAP2B, TFAP2A, RUNX1 and RUNX3 ). ( d , g , j ) Colony images of piPS-LF, Dox-piPSCs and mESCs J1 transduced with vector control lentivirus and miR-370 lentivirus. Scale bar, 50 μ m. ( e , h , k ) qRT-PCR results showing the downregulation of LIN28A mRNA level in piPS-LF, Dox-piPSCs and mESCs J1 transduced with miR-370 lentivirus. The counterpart cells transduced with vector control lentivirus are used as control. ** P
Figure Legend Snippet: MiR-370 represses the expression level of LIN28A . ( a ) Images showing the morphology of Dox-piPSCs at undifferentiated (DOX+) and differentiated (DOX−) state. Scale bar, 100 μ m. ( b ) qRT-PCR analysis showing the expression level changes of LIN28A and miR-370 in Dox-piPSCs at undifferentiated and differentiated state. ( c ) Comparative analysis of Pri-miR-370 promoter of Sus scrofa (pig), homo sapiens (human) and Mus musculus (mouse).The conservative promoter region is predicted to be bound by 18 transcription factors ( ERF, CDX1, ELF5, ELK1, ELK3, ARNT, SRY, FLI1, ETS1, ETV1, SPIB, ETV5, ETV4, TCFL5, TFAP2B, TFAP2A, RUNX1 and RUNX3 ). ( d , g , j ) Colony images of piPS-LF, Dox-piPSCs and mESCs J1 transduced with vector control lentivirus and miR-370 lentivirus. Scale bar, 50 μ m. ( e , h , k ) qRT-PCR results showing the downregulation of LIN28A mRNA level in piPS-LF, Dox-piPSCs and mESCs J1 transduced with miR-370 lentivirus. The counterpart cells transduced with vector control lentivirus are used as control. ** P

Techniques Used: Expressing, Quantitative RT-PCR, Transduction, Plasmid Preparation

3) Product Images from "CTGF increases matrix metalloproteinases expression and subsequently promotes tumor metastasis in human osteosarcoma through down-regulating miR-519d"

Article Title: CTGF increases matrix metalloproteinases expression and subsequently promotes tumor metastasis in human osteosarcoma through down-regulating miR-519d

Journal: Oncotarget

doi:

CTGF increases cell migration by suppressing miR-519d expression (A) The miR-519d expression in indicated cells was examined by qRT-PCR. (B) MG-63 cells and U-2 OS cells were incubated with rCTGF (5-50 ng/ml) for 24 h, miR-519d expression examined by qRT-PCR. (C) Cells were transfected with miR-519d mimic or inhibitor for 24 h and cell migration examined by Transwell assay. Results are expressed as mean ± SEM. *, p
Figure Legend Snippet: CTGF increases cell migration by suppressing miR-519d expression (A) The miR-519d expression in indicated cells was examined by qRT-PCR. (B) MG-63 cells and U-2 OS cells were incubated with rCTGF (5-50 ng/ml) for 24 h, miR-519d expression examined by qRT-PCR. (C) Cells were transfected with miR-519d mimic or inhibitor for 24 h and cell migration examined by Transwell assay. Results are expressed as mean ± SEM. *, p

Techniques Used: Migration, Expressing, Quantitative RT-PCR, Incubation, Transfection, Transwell Assay

MEK/ERK pathway is involved in CTGF-induce migration and suppression of miRNA-519d (A-D) Cells were pretreated with PD98059 (10 μM) and U0126 (10 μM) for 30 min or transfected with MEK1 and ERK2 mutant for 24 h, the MMPs expression and cell migration was examined by q-PCR, ELISA, and Transwell assay. (E) MG-63 cells were incubated with rCTGF (50 ng/ml) for indicated time intervals, MEK and ERK phosphorylation examined by western blot. (F) Cells were pretreated with PD98059 (10 μM) and U0126 (10 μM) for 30 min or transfected with MEK1 and ERK2 mutant for 24 h followed, the miR-519d expression was examined by qRT-PCR. Results expressed as mean ± SEM. *, p
Figure Legend Snippet: MEK/ERK pathway is involved in CTGF-induce migration and suppression of miRNA-519d (A-D) Cells were pretreated with PD98059 (10 μM) and U0126 (10 μM) for 30 min or transfected with MEK1 and ERK2 mutant for 24 h, the MMPs expression and cell migration was examined by q-PCR, ELISA, and Transwell assay. (E) MG-63 cells were incubated with rCTGF (50 ng/ml) for indicated time intervals, MEK and ERK phosphorylation examined by western blot. (F) Cells were pretreated with PD98059 (10 μM) and U0126 (10 μM) for 30 min or transfected with MEK1 and ERK2 mutant for 24 h followed, the miR-519d expression was examined by qRT-PCR. Results expressed as mean ± SEM. *, p

Techniques Used: Migration, Transfection, Mutagenesis, Expressing, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transwell Assay, Incubation, Western Blot, Quantitative RT-PCR

Related Articles

Transduction:

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: qRT-PCR Analysis Cells were transfected with siRNAs or shRNA-expressing plasmids or were transduced with Ad vectors at the indicated titers. .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Real-time Polymerase Chain Reaction:

Article Title: CCL3 promotes angiogenesis by dysregulation of miR-374b/ VEGF-A axis in human osteosarcoma cells
Article Snippet: Paragraph title: Quantitative real-time PCR ... The cycling conditions were 10 min of polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA).

Article Title: miR-196 regulates axial patterning and pectoral appendage initiation
Article Snippet: .. Two micrograms total RNA from each sample was reverse transcribed into cDNA for qPCR using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech, cat:PT4445-1). .. A 1/70th aliquot of each microRNA cDNA reaction was used in a 20 µL qPCR amplification reaction and qPCR assayed in StepOnePlus (Applied Biosystem).

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: Real-time RT-PCR analysis was performed using Fast SYBR Green Master Mix (Life Technologies) and a StepOnePlus real-time PCR system (Life Technologies) as previously described. .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Article Title: Common microRNA–mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states
Article Snippet: Quantitative RT-PCR Total RNAs from Dox-piPSCs were reverse transcribed to cDNAs using Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instruction. .. For the quantitative RT-PCR, reactions were performed in triplicate using SYBR Green PCR Master Mix (TransGen Biotech, Peking, China), and detected with the CFX96 real-time PCR system (Bio-Rad, Hercules, USA).

Article Title: microRNA-495 Inhibits Chondrogenic Differentiation in Human Mesenchymal Stem Cells by Targeting Sox9
Article Snippet: Paragraph title: Quantitative real-time polymerase chain reaction ... For precursor-microRNA reverse transcription, an Mir-X™ microRNA First-Strand Synthesis kit (Clontech) was used following the manufacturer's instructions, and cDNA synthesized from microRNAs was quantified using SYBR qRT-PCR (Clontech).

Article Title: CTGF increases matrix metalloproteinases expression and subsequently promotes tumor metastasis in human osteosarcoma through down-regulating miR-519d
Article Snippet: Paragraph title: Quantitative real-time PCR ... The cycling conditions were 10 min of polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA).

Article Title: High-throughput sequencing reveals differential expression of miRNAs in prehierarchal follicles of laying and brooding geese
Article Snippet: .. Total RNA was extracted as described for the library preparation, and cDNA synthesis and qRT-PCR analyses were performed with Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (TaKaRa, Dalian, China) on a Real-Time PCR Detection System, respectively. ..

Article Title: Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression
Article Snippet: One microgram RNA was reverse transcribed to cDNAs using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, USA) according to the manufacturer’s procedure. .. For qRT-PCR, reactions were performed for 95°C 30 s at the first cycle, following another 40 cycles of 95°C 5 s and 60°C 30 s in triplicate using SYBR Green PCR Master Mix (TransGen Biotech, Peking, China), and detected by the CFX96 real-time PCR system (Bio-Rad, Hercules, USA).

RNA Extraction:

Article Title: miR-196 regulates axial patterning and pectoral appendage initiation
Article Snippet: From each experimental and control group, total RNA was isolated from 50 zebrafish embryos at 27 hpf using the RoboZol RNA extraction reagent for total RNA including microRNA (Amresco Cat:N580-100ML). .. Two micrograms total RNA from each sample was reverse transcribed into cDNA for qPCR using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech, cat:PT4445-1).

shRNA:

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: qRT-PCR Analysis Cells were transfected with siRNAs or shRNA-expressing plasmids or were transduced with Ad vectors at the indicated titers. .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Fluorescence:

Article Title: microRNA-495 Inhibits Chondrogenic Differentiation in Human Mesenchymal Stem Cells by Targeting Sox9
Article Snippet: SYBR fluorescence was detected during the annealing/extension phase, and all real-time PCR products had a final size of ∼100 bp. .. For precursor-microRNA reverse transcription, an Mir-X™ microRNA First-Strand Synthesis kit (Clontech) was used following the manufacturer's instructions, and cDNA synthesized from microRNAs was quantified using SYBR qRT-PCR (Clontech).

Synthesized:

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: Total RNA was isolated from the cells using ISOGEN (Nippon Gene). cDNA was synthesized using 500 ng total RNA with a Superscript VILO cDNA synthesis kit (Life Technologies). .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Article Title: microRNA-495 Inhibits Chondrogenic Differentiation in Human Mesenchymal Stem Cells by Targeting Sox9
Article Snippet: .. For precursor-microRNA reverse transcription, an Mir-X™ microRNA First-Strand Synthesis kit (Clontech) was used following the manufacturer's instructions, and cDNA synthesized from microRNAs was quantified using SYBR qRT-PCR (Clontech). .. The list of primer sets used for specific mature-microRNA amplification was obtained from Genolution (Genolution) and Bioneer.

Isolation:

Article Title: miR-196 regulates axial patterning and pectoral appendage initiation
Article Snippet: From each experimental and control group, total RNA was isolated from 50 zebrafish embryos at 27 hpf using the RoboZol RNA extraction reagent for total RNA including microRNA (Amresco Cat:N580-100ML). .. Two micrograms total RNA from each sample was reverse transcribed into cDNA for qPCR using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech, cat:PT4445-1).

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: Total RNA was isolated from the cells using ISOGEN (Nippon Gene). cDNA was synthesized using 500 ng total RNA with a Superscript VILO cDNA synthesis kit (Life Technologies). .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Sequencing:

Article Title: CCL3 promotes angiogenesis by dysregulation of miR-374b/ VEGF-A axis in human osteosarcoma cells
Article Snippet: Q-PCR assays were carried out in triplicate using a StepOnePlus sequence detection system. .. The cycling conditions were 10 min of polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA).

Article Title: CTGF increases matrix metalloproteinases expression and subsequently promotes tumor metastasis in human osteosarcoma through down-regulating miR-519d
Article Snippet: Q-PCR assays were carried out in triplicate using a StepOnePlus sequence detection system. .. The cycling conditions were 10 min of polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA).

Article Title: High-throughput sequencing reveals differential expression of miRNAs in prehierarchal follicles of laying and brooding geese
Article Snippet: Follicle samples were taken from the same individuals of the constructed libraries to verify the sequencing results, and tissues (including spleen, liver, intestine, heart, lung and breast muscle) were obtained from three individual laying geese to determine whether follicle-specific miRNAs existed. .. Total RNA was extracted as described for the library preparation, and cDNA synthesis and qRT-PCR analyses were performed with Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (TaKaRa, Dalian, China) on a Real-Time PCR Detection System, respectively.

Transfection:

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: qRT-PCR Analysis Cells were transfected with siRNAs or shRNA-expressing plasmids or were transduced with Ad vectors at the indicated titers. .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Quantitative RT-PCR:

Article Title: CCL3 promotes angiogenesis by dysregulation of miR-374b/ VEGF-A axis in human osteosarcoma cells
Article Snippet: .. The cycling conditions were 10 min of polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA). ..

Article Title: miR-196 regulates axial patterning and pectoral appendage initiation
Article Snippet: .. Two micrograms total RNA from each sample was reverse transcribed into cDNA for qPCR using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech, cat:PT4445-1). .. A 1/70th aliquot of each microRNA cDNA reaction was used in a 20 µL qPCR amplification reaction and qPCR assayed in StepOnePlus (Applied Biosystem).

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc. .. Determination of Type I IFN Expression Levels In Vivo 100 μg shLuc-expressing plasmid (pHMU6-shLuc) and 4 × 108 IFU Ad-shLuc were intramuscularly and intravenously administered to mice, respectively.

Article Title: Common microRNA–mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states
Article Snippet: .. Quantitative RT-PCR Total RNAs from Dox-piPSCs were reverse transcribed to cDNAs using Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instruction. .. For the quantitative RT-PCR, reactions were performed in triplicate using SYBR Green PCR Master Mix (TransGen Biotech, Peking, China), and detected with the CFX96 real-time PCR system (Bio-Rad, Hercules, USA).

Article Title: microRNA-495 Inhibits Chondrogenic Differentiation in Human Mesenchymal Stem Cells by Targeting Sox9
Article Snippet: .. For precursor-microRNA reverse transcription, an Mir-X™ microRNA First-Strand Synthesis kit (Clontech) was used following the manufacturer's instructions, and cDNA synthesized from microRNAs was quantified using SYBR qRT-PCR (Clontech). .. The list of primer sets used for specific mature-microRNA amplification was obtained from Genolution (Genolution) and Bioneer.

Article Title: CTGF increases matrix metalloproteinases expression and subsequently promotes tumor metastasis in human osteosarcoma through down-regulating miR-519d
Article Snippet: .. The cycling conditions were 10 min of polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA). ..

Article Title: High-throughput sequencing reveals differential expression of miRNAs in prehierarchal follicles of laying and brooding geese
Article Snippet: .. Total RNA was extracted as described for the library preparation, and cDNA synthesis and qRT-PCR analyses were performed with Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (TaKaRa, Dalian, China) on a Real-Time PCR Detection System, respectively. ..

Article Title: Overexpression of miR-146a Might Regulate Polarization Transitions of BV-2 Cells Induced by High Glucose and Glucose Fluctuations
Article Snippet: .. Genomic DNA of miRNA was removed using Recombinant DNase I (RNase-free) (Takara, Japan). miRNA was reversed transcribed into cDNA using the Mir-X® miRNA FirstStrand Synthesis and SYBR® qRT-PCR (Takara, Japan). mRNA qRT-PCR was performed with the TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Japan). miRNA qRT-PCR was performed with Mir-X™ miRNA FirstStrand Synthesis and SYBR® qRT-PCR (Takara, Japan). .. Western Blot Total cell proteins were extracted and equal amounts of protein (20 μg/sample) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA).

Article Title: Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression
Article Snippet: .. One microgram RNA was reverse transcribed to cDNAs using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, USA) according to the manufacturer’s procedure. ..

Activation Assay:

Article Title: CCL3 promotes angiogenesis by dysregulation of miR-374b/ VEGF-A axis in human osteosarcoma cells
Article Snippet: .. The cycling conditions were 10 min of polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA). ..

Article Title: CTGF increases matrix metalloproteinases expression and subsequently promotes tumor metastasis in human osteosarcoma through down-regulating miR-519d
Article Snippet: .. The cycling conditions were 10 min of polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA). ..

SYBR Green Assay:

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: Real-time RT-PCR analysis was performed using Fast SYBR Green Master Mix (Life Technologies) and a StepOnePlus real-time PCR system (Life Technologies) as previously described. .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Article Title: Common microRNA–mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states
Article Snippet: Quantitative RT-PCR Total RNAs from Dox-piPSCs were reverse transcribed to cDNAs using Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instruction. .. For the quantitative RT-PCR, reactions were performed in triplicate using SYBR Green PCR Master Mix (TransGen Biotech, Peking, China), and detected with the CFX96 real-time PCR system (Bio-Rad, Hercules, USA).

Article Title: Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression
Article Snippet: One microgram RNA was reverse transcribed to cDNAs using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, USA) according to the manufacturer’s procedure. .. For qRT-PCR, reactions were performed for 95°C 30 s at the first cycle, following another 40 cycles of 95°C 5 s and 60°C 30 s in triplicate using SYBR Green PCR Master Mix (TransGen Biotech, Peking, China), and detected by the CFX96 real-time PCR system (Bio-Rad, Hercules, USA).

Polymerase Chain Reaction:

Article Title: CCL3 promotes angiogenesis by dysregulation of miR-374b/ VEGF-A axis in human osteosarcoma cells
Article Snippet: Quantitative real-time polymerase chain reaction (q-PCR) analysis was carried out using TaqMan® one-step PCR Master Mix (Applied Biosystems, Foster City, CA, USA). .. The cycling conditions were 10 min of polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA).

Article Title: Common microRNA–mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states
Article Snippet: Quantitative RT-PCR Total RNAs from Dox-piPSCs were reverse transcribed to cDNAs using Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instruction. .. For the quantitative RT-PCR, reactions were performed in triplicate using SYBR Green PCR Master Mix (TransGen Biotech, Peking, China), and detected with the CFX96 real-time PCR system (Bio-Rad, Hercules, USA).

Article Title: microRNA-495 Inhibits Chondrogenic Differentiation in Human Mesenchymal Stem Cells by Targeting Sox9
Article Snippet: The PCR procedure was initiated for 30 s at 95°C, followed by 40 thermal cycles of 5 s at 95°C and 20 s at 60°C. .. For precursor-microRNA reverse transcription, an Mir-X™ microRNA First-Strand Synthesis kit (Clontech) was used following the manufacturer's instructions, and cDNA synthesized from microRNAs was quantified using SYBR qRT-PCR (Clontech).

Article Title: CTGF increases matrix metalloproteinases expression and subsequently promotes tumor metastasis in human osteosarcoma through down-regulating miR-519d
Article Snippet: Quantitative real-time polymerase chain reaction (q-PCR) analysis was carried out using TaqMan® one-step PCR Master Mix (Applied Biosystems, Foster City, CA, USA). .. The cycling conditions were 10 min of polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. For miRNAs detection, reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech Laboratories, Inc., CA, USA).

Incubation:

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: After 5-hr incubation, the cells were treated with recombinant IFN-α and incubated for a total of 48 hr. .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Amplification:

Article Title: miR-196 regulates axial patterning and pectoral appendage initiation
Article Snippet: Two micrograms total RNA from each sample was reverse transcribed into cDNA for qPCR using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech, cat:PT4445-1). .. A 1/70th aliquot of each microRNA cDNA reaction was used in a 20 µL qPCR amplification reaction and qPCR assayed in StepOnePlus (Applied Biosystem).

Article Title: microRNA-495 Inhibits Chondrogenic Differentiation in Human Mesenchymal Stem Cells by Targeting Sox9
Article Snippet: For precursor-microRNA reverse transcription, an Mir-X™ microRNA First-Strand Synthesis kit (Clontech) was used following the manufacturer's instructions, and cDNA synthesized from microRNAs was quantified using SYBR qRT-PCR (Clontech). .. The list of primer sets used for specific mature-microRNA amplification was obtained from Genolution (Genolution) and Bioneer.

Construct:

Article Title: High-throughput sequencing reveals differential expression of miRNAs in prehierarchal follicles of laying and brooding geese
Article Snippet: Follicle samples were taken from the same individuals of the constructed libraries to verify the sequencing results, and tissues (including spleen, liver, intestine, heart, lung and breast muscle) were obtained from three individual laying geese to determine whether follicle-specific miRNAs existed. .. Total RNA was extracted as described for the library preparation, and cDNA synthesis and qRT-PCR analyses were performed with Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (TaKaRa, Dalian, China) on a Real-Time PCR Detection System, respectively.

Expressing:

Article Title: miR-196 regulates axial patterning and pectoral appendage initiation
Article Snippet: Two micrograms total RNA from each sample was reverse transcribed into cDNA for qPCR using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech, cat:PT4445-1). .. The relative expression of miR-196a and miR196b were normalized to the U6 spliceosomal RNA provided in the Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR kit (Clontech).

Article Title: Common microRNA–mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states
Article Snippet: Quantitative RT-PCR Total RNAs from Dox-piPSCs were reverse transcribed to cDNAs using Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instruction. .. Relative expression levels of target genes and deferentially expressed miRNAs were normalized to either U6 (for miRNAs) or β -Actin expression (for mRNAs).

Reverse Transcription Polymerase Chain Reaction:

Article Title: miR-196 regulates axial patterning and pectoral appendage initiation
Article Snippet: Paragraph title: RT-PCR and qPCR Analysis ... Two micrograms total RNA from each sample was reverse transcribed into cDNA for qPCR using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech, cat:PT4445-1).

Article Title: Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression
Article Snippet: Paragraph title: Reverse transcription polymerase chain reaction ... One microgram RNA was reverse transcribed to cDNAs using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) and Mir-X™ miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech, USA) according to the manufacturer’s procedure.

Chloramphenicol Acetyltransferase Assay:

Article Title: miR-196 regulates axial patterning and pectoral appendage initiation
Article Snippet: .. Two micrograms total RNA from each sample was reverse transcribed into cDNA for qPCR using Mir-X™ miRNA First-Strand Synthesis and SYBR® qRT-PCR (Clontech, cat:PT4445-1). .. A 1/70th aliquot of each microRNA cDNA reaction was used in a 20 µL qPCR amplification reaction and qPCR assayed in StepOnePlus (Applied Biosystem).

Recombinant:

Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA
Article Snippet: After 5-hr incubation, the cells were treated with recombinant IFN-α and incubated for a total of 48 hr. .. Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

Article Title: Overexpression of miR-146a Might Regulate Polarization Transitions of BV-2 Cells Induced by High Glucose and Glucose Fluctuations
Article Snippet: .. Genomic DNA of miRNA was removed using Recombinant DNase I (RNase-free) (Takara, Japan). miRNA was reversed transcribed into cDNA using the Mir-X® miRNA FirstStrand Synthesis and SYBR® qRT-PCR (Takara, Japan). mRNA qRT-PCR was performed with the TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Japan). miRNA qRT-PCR was performed with Mir-X™ miRNA FirstStrand Synthesis and SYBR® qRT-PCR (Takara, Japan). .. Western Blot Total cell proteins were extracted and equal amounts of protein (20 μg/sample) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA).

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    TaKaRa nucleospin rna extraction kit
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    IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by <t>qRT-PCR.</t> All data are expressed as the means ± SD (n = 4). *p
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    circ-DONSON recruits the NURF complex to activate SOX4 transcription. a ChIP assay showed that SNF2L, BPTF and RBBP4 were enriched on SOX4 promoter. b ChIP assay showed that circ-DONSON silencing attenuated the enrichment of SNF2L, BPTF and RBBP4 on SOX4 promoter. c DNA-FISH verified that circ-DONSON silencing abrogated the colocalization between SNF2L and SOX4 promoter in BGC-823 cells. Scale bar: 5 μm. d , e Depletion of SNF2L, BPTF or RBBP4 impaired the enrichment of active markers H3K27ac and H3K4me3 on SOX4 promoter. f Luciferase reporter assay showed that overexpression of SNF2L, BPTF or RBBP4 increased the luciferase activity while circ-DONSON silencing abrogated it. The SOX4 promoter region was constructed into the pGL3 luciferase vector. g , h <t>qRT-PCR</t> and western blotting analyses of SOX4 expression after knockdown of SNF2L, BPTF or RBBP4. i qRT-PCR analysis indicated that SOX4 expression was negatively correlated with SNF2L in GC tissues. ** P
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    IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Inhibition, Expressing, shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR

    Knockdown Efficiencies of Chemically Synthetic siRNAs following IFN-α Stimulation (A and B) A549 cells were transfected with siControl, sip53 (A), or simyc (B), followed by treatment with recombinant IFNα at 10 4 U/mL. After 48-hr incubation, c- myc and p53 mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Knockdown Efficiencies of Chemically Synthetic siRNAs following IFN-α Stimulation (A and B) A549 cells were transfected with siControl, sip53 (A), or simyc (B), followed by treatment with recombinant IFNα at 10 4 U/mL. After 48-hr incubation, c- myc and p53 mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4).

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Transfection, Recombinant, Incubation, Quantitative RT-PCR

    Inhibition of shRNA-Mediated Knockdown by IFN-α Stimulation (A and B) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 2 , 10 3 , or 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. (D and E) A549 cells were transfected with pHMU6-shLuc, -shmyc (D), or -shp53 (E), followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc (D) and p53 (E) mRNA levels in the cells were similarly determined. These experiments were repeated at least three times, and representative data are shown. All data are expressed as the means ± SD (n = 4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Inhibition of shRNA-Mediated Knockdown by IFN-α Stimulation (A and B) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 2 , 10 3 , or 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. (D and E) A549 cells were transfected with pHMU6-shLuc, -shmyc (D), or -shp53 (E), followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc (D) and p53 (E) mRNA levels in the cells were similarly determined. These experiments were repeated at least three times, and representative data are shown. All data are expressed as the means ± SD (n = 4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Inhibition, shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR, Western Blot

    Dicer and Ago2 Expression Levels following IFN-α Stimulation H1299 and A549 cells were treated with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, dicer and Ago2 mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) TRBP protein levels in the cells were determined by western blotting analysis. The qRT-PCR data are expressed as the means ± SD (n = 4).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Dicer and Ago2 Expression Levels following IFN-α Stimulation H1299 and A549 cells were treated with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, dicer and Ago2 mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) TRBP protein levels in the cells were determined by western blotting analysis. The qRT-PCR data are expressed as the means ± SD (n = 4).

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Expressing, Recombinant, Incubation, Quantitative RT-PCR, Western Blot

    Inhibition of Ad Vector-Expressing shRNA-Mediated Knockdown by IFN-α Stimulation (A) 4 × 10 8 IFU Ad-shLuc was intravenously administered to C57BL/6 mice. 3 hr after the administration, IFN-β mRNA levels in the spleen were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). (B–D) A549 cells were transduced with Ad-shp53, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, the copy numbers of sip53 in the cells were determined by qRT-PCR (B). After 48-hr incubation, p53 mRNA (C) and protein (D) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. Data are expressed as the means ± SD (n = 4). **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Inhibition of Ad Vector-Expressing shRNA-Mediated Knockdown by IFN-α Stimulation (A) 4 × 10 8 IFU Ad-shLuc was intravenously administered to C57BL/6 mice. 3 hr after the administration, IFN-β mRNA levels in the spleen were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). (B–D) A549 cells were transduced with Ad-shp53, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, the copy numbers of sip53 in the cells were determined by qRT-PCR (B). After 48-hr incubation, p53 mRNA (C) and protein (D) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. Data are expressed as the means ± SD (n = 4). **p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Inhibition, Plasmid Preparation, Expressing, shRNA, Mouse Assay, Quantitative RT-PCR, Transduction, Recombinant, Incubation, Western Blot

    Efficiencies of shRNA-Mediated Knockdown following Exposure to IFN-β, IFN-γ, and TNF-α (A) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-β at 10 2 or 10 3 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. (B) H1299 cells were similarly transfected, followed by treatment with recombinant IFN-γ and TNF-α at 5 × 10 3 U/mL and 100 ng/mL, respectively. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. All data are expressed as the means ± SD (n = 4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Efficiencies of shRNA-Mediated Knockdown following Exposure to IFN-β, IFN-γ, and TNF-α (A) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-β at 10 2 or 10 3 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. (B) H1299 cells were similarly transfected, followed by treatment with recombinant IFN-γ and TNF-α at 5 × 10 3 U/mL and 100 ng/mL, respectively. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. All data are expressed as the means ± SD (n = 4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR

    Type I IFN Responses Following Introduction of shRNA-Expressing Vectors (A–D) A549 (A and C) and H1299 (B and D) cells were transfected with pHMU6-shLuc (A and B) or siControl (indicated as siRNA) (C and D). After 24-hr incubation, IFNβ, ISG54, and ISG56 mRNA levels in the cells were determined by qRT-PCR. The data are expressed as the means ± SD (n = 3). (E) 100 μg pHMU6-shLuc was intramuscularly administered to C57BL/6 mice. 3 hr after administration, the IFN-β mRNA levels in the mouse muscle were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Type I IFN Responses Following Introduction of shRNA-Expressing Vectors (A–D) A549 (A and C) and H1299 (B and D) cells were transfected with pHMU6-shLuc (A and B) or siControl (indicated as siRNA) (C and D). After 24-hr incubation, IFNβ, ISG54, and ISG56 mRNA levels in the cells were determined by qRT-PCR. The data are expressed as the means ± SD (n = 3). (E) 100 μg pHMU6-shLuc was intramuscularly administered to C57BL/6 mice. 3 hr after administration, the IFN-β mRNA levels in the mouse muscle were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: shRNA, Expressing, Transfection, Incubation, Quantitative RT-PCR, Mouse Assay

    Processing of shRNA to siRNA in IFN-α-Treated Cells (A and B) H1299 cells were transfected with pHMU6-shp53 or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, shRNA and siRNA copy numbers in the cells were determined by northern blotting analysis (A) and qRT-PCR (B). Data are expressed as the means ± SD (n = 4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Processing of shRNA to siRNA in IFN-α-Treated Cells (A and B) H1299 cells were transfected with pHMU6-shp53 or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, shRNA and siRNA copy numbers in the cells were determined by northern blotting analysis (A) and qRT-PCR (B). Data are expressed as the means ± SD (n = 4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: shRNA, Transfection, Recombinant, Incubation, Northern Blot, Quantitative RT-PCR

    circ-DONSON recruits the NURF complex to activate SOX4 transcription. a ChIP assay showed that SNF2L, BPTF and RBBP4 were enriched on SOX4 promoter. b ChIP assay showed that circ-DONSON silencing attenuated the enrichment of SNF2L, BPTF and RBBP4 on SOX4 promoter. c DNA-FISH verified that circ-DONSON silencing abrogated the colocalization between SNF2L and SOX4 promoter in BGC-823 cells. Scale bar: 5 μm. d , e Depletion of SNF2L, BPTF or RBBP4 impaired the enrichment of active markers H3K27ac and H3K4me3 on SOX4 promoter. f Luciferase reporter assay showed that overexpression of SNF2L, BPTF or RBBP4 increased the luciferase activity while circ-DONSON silencing abrogated it. The SOX4 promoter region was constructed into the pGL3 luciferase vector. g , h qRT-PCR and western blotting analyses of SOX4 expression after knockdown of SNF2L, BPTF or RBBP4. i qRT-PCR analysis indicated that SOX4 expression was negatively correlated with SNF2L in GC tissues. ** P

    Journal: Molecular Cancer

    Article Title: Circular RNA circ-DONSON facilitates gastric cancer growth and invasion via NURF complex dependent activation of transcription factor SOX4

    doi: 10.1186/s12943-019-1006-2

    Figure Lengend Snippet: circ-DONSON recruits the NURF complex to activate SOX4 transcription. a ChIP assay showed that SNF2L, BPTF and RBBP4 were enriched on SOX4 promoter. b ChIP assay showed that circ-DONSON silencing attenuated the enrichment of SNF2L, BPTF and RBBP4 on SOX4 promoter. c DNA-FISH verified that circ-DONSON silencing abrogated the colocalization between SNF2L and SOX4 promoter in BGC-823 cells. Scale bar: 5 μm. d , e Depletion of SNF2L, BPTF or RBBP4 impaired the enrichment of active markers H3K27ac and H3K4me3 on SOX4 promoter. f Luciferase reporter assay showed that overexpression of SNF2L, BPTF or RBBP4 increased the luciferase activity while circ-DONSON silencing abrogated it. The SOX4 promoter region was constructed into the pGL3 luciferase vector. g , h qRT-PCR and western blotting analyses of SOX4 expression after knockdown of SNF2L, BPTF or RBBP4. i qRT-PCR analysis indicated that SOX4 expression was negatively correlated with SNF2L in GC tissues. ** P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs were isolated using TRIzol and inversely transcribed into cDNA using M-MLV and the SYBR Green Master Mix kit (Takara, Otsu, Japan). qPCR was completed as described previously [ ].

    Techniques: Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization, Luciferase, Reporter Assay, Over Expression, Activity Assay, Construct, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing

    The NURF complex modulates GC progression. a BPTF and RBBP4 were upregulated in GC tissues according to TCGA database. b qRT-PCR analysis of expression levels of BPTF, RBBP4 and SNF2L in 142 GC tissues and adjacent normal tissues. c Relative expression of BPTF, RBBP4 and SNF2L in GC cell lines by qRT-PCR analysis. d - f CCK8, colony formation and EdU assays showed that knockdown of BPTF, RBBP4 or SNF2L suppressed proliferation of BGC-823 and AGS cells. g Knockdown of BPTF, RBBP4 or SNF2L promoted apoptosis of BGC-823 and AGS cells. h , i Transwell assay showed that knockdown of BPTF, RBBP4 or SNF2L inhibits migration and invasion of BGC-823 and AGS cells. ** P

    Journal: Molecular Cancer

    Article Title: Circular RNA circ-DONSON facilitates gastric cancer growth and invasion via NURF complex dependent activation of transcription factor SOX4

    doi: 10.1186/s12943-019-1006-2

    Figure Lengend Snippet: The NURF complex modulates GC progression. a BPTF and RBBP4 were upregulated in GC tissues according to TCGA database. b qRT-PCR analysis of expression levels of BPTF, RBBP4 and SNF2L in 142 GC tissues and adjacent normal tissues. c Relative expression of BPTF, RBBP4 and SNF2L in GC cell lines by qRT-PCR analysis. d - f CCK8, colony formation and EdU assays showed that knockdown of BPTF, RBBP4 or SNF2L suppressed proliferation of BGC-823 and AGS cells. g Knockdown of BPTF, RBBP4 or SNF2L promoted apoptosis of BGC-823 and AGS cells. h , i Transwell assay showed that knockdown of BPTF, RBBP4 or SNF2L inhibits migration and invasion of BGC-823 and AGS cells. ** P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs were isolated using TRIzol and inversely transcribed into cDNA using M-MLV and the SYBR Green Master Mix kit (Takara, Otsu, Japan). qPCR was completed as described previously [ ].

    Techniques: Quantitative RT-PCR, Expressing, Transwell Assay, Migration

    Effects of circ-DONSON on GC growth in vivo. a Tumor volumes were determined every 5 days. b 30 days after injection, the tumor weights were measured. The representative images of tumor tissues in each group were presented in the right. n = 4 for each group. c qRT-PCR analysis of circ-DONSON in tumor tissues. d , e IHC analysis of SOX4 and Ki67 expression in tumor tissues of each group. Scale bar: 50 μm. ** P

    Journal: Molecular Cancer

    Article Title: Circular RNA circ-DONSON facilitates gastric cancer growth and invasion via NURF complex dependent activation of transcription factor SOX4

    doi: 10.1186/s12943-019-1006-2

    Figure Lengend Snippet: Effects of circ-DONSON on GC growth in vivo. a Tumor volumes were determined every 5 days. b 30 days after injection, the tumor weights were measured. The representative images of tumor tissues in each group were presented in the right. n = 4 for each group. c qRT-PCR analysis of circ-DONSON in tumor tissues. d , e IHC analysis of SOX4 and Ki67 expression in tumor tissues of each group. Scale bar: 50 μm. ** P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs were isolated using TRIzol and inversely transcribed into cDNA using M-MLV and the SYBR Green Master Mix kit (Takara, Otsu, Japan). qPCR was completed as described previously [ ].

    Techniques: In Vivo, Injection, Quantitative RT-PCR, Immunohistochemistry, Expressing

    circ-DONSON is upregulated in GC tissues and positively correlated with poor prognosis. a Heatmap according to differentially expressed circRNAs between GC tissues and adjacent normal tissues in GSE83521 dataset. b Expression intensity of circ-DONSON in GC tissues and adjacent normal tissues based on GSE83521 dataset. c qRT-PCR analysis of relative circ-DONSON expression levels in 142GC tissues and their adjacent normal tissues. d Northern blotting analysis of circ-DONSON expression in GC tissues and paired normal tissues. e In situ hybridization (ISH) was used to analyze circ-DONSON expression in GC tissues and paired normal tissues. Scale bar: 50 μm. f Increased expression of circ-DONSON was observed in GC cell lines compared to GES-1 cells. g Relative expression of circ-DONSON in GC with different TNM stage. h Expression levels of circ-DONSON in GC patients with or without lymph node metastasis. i , j Kaplan-Meier plots of the overall survival and disease-free survival of GC patients with high ( n = 68) and low ( n = 74) levels of circ-DONSON. * P

    Journal: Molecular Cancer

    Article Title: Circular RNA circ-DONSON facilitates gastric cancer growth and invasion via NURF complex dependent activation of transcription factor SOX4

    doi: 10.1186/s12943-019-1006-2

    Figure Lengend Snippet: circ-DONSON is upregulated in GC tissues and positively correlated with poor prognosis. a Heatmap according to differentially expressed circRNAs between GC tissues and adjacent normal tissues in GSE83521 dataset. b Expression intensity of circ-DONSON in GC tissues and adjacent normal tissues based on GSE83521 dataset. c qRT-PCR analysis of relative circ-DONSON expression levels in 142GC tissues and their adjacent normal tissues. d Northern blotting analysis of circ-DONSON expression in GC tissues and paired normal tissues. e In situ hybridization (ISH) was used to analyze circ-DONSON expression in GC tissues and paired normal tissues. Scale bar: 50 μm. f Increased expression of circ-DONSON was observed in GC cell lines compared to GES-1 cells. g Relative expression of circ-DONSON in GC with different TNM stage. h Expression levels of circ-DONSON in GC patients with or without lymph node metastasis. i , j Kaplan-Meier plots of the overall survival and disease-free survival of GC patients with high ( n = 68) and low ( n = 74) levels of circ-DONSON. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs were isolated using TRIzol and inversely transcribed into cDNA using M-MLV and the SYBR Green Master Mix kit (Takara, Otsu, Japan). qPCR was completed as described previously [ ].

    Techniques: Expressing, Quantitative RT-PCR, Northern Blot, In Situ Hybridization

    circ-DONSON silencing suppresses GC cell proliferation, migration and invasion, and induces apoptosis. a qRT-PCR was performed to confirm the relative expression of circ-DONSON in BGC-823 and AGS cells transfected with two independent shRNAs targeting circ-DONSON. b - e CCK8, EdU, colony formation and Ki67 staining assays was used to analyze proliferation of BGC-823 and AGS cells after circ-DONSON silencing. f Annexin/PI staining followed by FACS analysis indicated that circ-DONSON knockdown induced apoptosis. g , h Transwell assay illustrated that circ-DONSON knockdown suppressed the migration and invasion of BGC-823 and AGS cells. i Western blotting analysis of N-cadherin and E-cadherin expression in BGC-823 and AGS cells after circ-DONSON depletion. * P

    Journal: Molecular Cancer

    Article Title: Circular RNA circ-DONSON facilitates gastric cancer growth and invasion via NURF complex dependent activation of transcription factor SOX4

    doi: 10.1186/s12943-019-1006-2

    Figure Lengend Snippet: circ-DONSON silencing suppresses GC cell proliferation, migration and invasion, and induces apoptosis. a qRT-PCR was performed to confirm the relative expression of circ-DONSON in BGC-823 and AGS cells transfected with two independent shRNAs targeting circ-DONSON. b - e CCK8, EdU, colony formation and Ki67 staining assays was used to analyze proliferation of BGC-823 and AGS cells after circ-DONSON silencing. f Annexin/PI staining followed by FACS analysis indicated that circ-DONSON knockdown induced apoptosis. g , h Transwell assay illustrated that circ-DONSON knockdown suppressed the migration and invasion of BGC-823 and AGS cells. i Western blotting analysis of N-cadherin and E-cadherin expression in BGC-823 and AGS cells after circ-DONSON depletion. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs were isolated using TRIzol and inversely transcribed into cDNA using M-MLV and the SYBR Green Master Mix kit (Takara, Otsu, Japan). qPCR was completed as described previously [ ].

    Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, Staining, FACS, Transwell Assay, Western Blot

    circ-DONSON associates with the NURF complex by directly interacting with SNF2L subunit. a qRT-PCR analysis showed that circ-DONSON was mainly localized in the nucleus of GC cells. b biotin-labeled linear circ-DONSON was used for incubation with BGC-823 cell lysates, followed by silver staining and mass spectrum identification. SNF2L was identified as a candidate for interaction with circ-DONSON. c RIP assay using anti-SNF2L showed that SNF2L precipitated circ-DONSON in BGC-823 and AGS cell lysates. d Pulldown assay confirmed that biotin-labeled linear circ-DONSON interacted with MYC-SNF2L. e RNA-FISH assay verified the colocalization between circ-DONSON and SNF2L in BGC-823 cells. Scale bar: 5 μm. f Domain mapping assay indicated that the region of 650–948 bp in circ-DONSON was essential for the interaction with SNF2L. g RNA-EMSA assay confirmed the interaction of circ-DONSON (650–948 bp) with SNF2L. h Pulldown assay using probes targeting circ-DONSON indicated that circ-DONSON interacted with the NURF complex in BGC-823 cells

    Journal: Molecular Cancer

    Article Title: Circular RNA circ-DONSON facilitates gastric cancer growth and invasion via NURF complex dependent activation of transcription factor SOX4

    doi: 10.1186/s12943-019-1006-2

    Figure Lengend Snippet: circ-DONSON associates with the NURF complex by directly interacting with SNF2L subunit. a qRT-PCR analysis showed that circ-DONSON was mainly localized in the nucleus of GC cells. b biotin-labeled linear circ-DONSON was used for incubation with BGC-823 cell lysates, followed by silver staining and mass spectrum identification. SNF2L was identified as a candidate for interaction with circ-DONSON. c RIP assay using anti-SNF2L showed that SNF2L precipitated circ-DONSON in BGC-823 and AGS cell lysates. d Pulldown assay confirmed that biotin-labeled linear circ-DONSON interacted with MYC-SNF2L. e RNA-FISH assay verified the colocalization between circ-DONSON and SNF2L in BGC-823 cells. Scale bar: 5 μm. f Domain mapping assay indicated that the region of 650–948 bp in circ-DONSON was essential for the interaction with SNF2L. g RNA-EMSA assay confirmed the interaction of circ-DONSON (650–948 bp) with SNF2L. h Pulldown assay using probes targeting circ-DONSON indicated that circ-DONSON interacted with the NURF complex in BGC-823 cells

    Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs were isolated using TRIzol and inversely transcribed into cDNA using M-MLV and the SYBR Green Master Mix kit (Takara, Otsu, Japan). qPCR was completed as described previously [ ].

    Techniques: Quantitative RT-PCR, Labeling, Incubation, Silver Staining, Fluorescence In Situ Hybridization, Mapping Assay

    circ-DONSON regulates GC cell malignant behaviors through activating SOX4. a qRT-PCR analysis of indicated gene expression in BGC-823 and AGS cells after circ-DONSON depletion. b Western blotting result showed that circ-DONSON silencing suppressed SOX4 expression in BGC-823 and AGS cells. c ChIP assay was performed to measure the association of circ-DONSON with SOX4 promoter. d Pulldown assay showed that biotin-labeled SOX4 promoter region precipitated circ-DONSON in BGC-823 and AGS cell lysates. e DNA-FISH assay indicated the co-localization between circ-DONSON and SOX4 promoter in BGC-823 and AGS cells. Scale bar: 5 μm. f ChIP assay showed that circ-DONSON silencing led to decreased enrichment of active marker H3K27ac on SOX4 promoter in BGC-823 and AGS cells. g SOX4 promoter was more resistant to DNaseI digestion after circ-DONSON knockdown. h - j CCK8, EdU and colony formation assays were performed to detect cell proliferation. k Restoration of SOX4 reduced the apoptosis of BGC-823 and AGS cells induced by circ-DONSON silencing. l , m Restoration of SOX4 rescued the abilities of migration and invasion in circ-DONSON knocked down BGC-823 and AGS cells. ** P

    Journal: Molecular Cancer

    Article Title: Circular RNA circ-DONSON facilitates gastric cancer growth and invasion via NURF complex dependent activation of transcription factor SOX4

    doi: 10.1186/s12943-019-1006-2

    Figure Lengend Snippet: circ-DONSON regulates GC cell malignant behaviors through activating SOX4. a qRT-PCR analysis of indicated gene expression in BGC-823 and AGS cells after circ-DONSON depletion. b Western blotting result showed that circ-DONSON silencing suppressed SOX4 expression in BGC-823 and AGS cells. c ChIP assay was performed to measure the association of circ-DONSON with SOX4 promoter. d Pulldown assay showed that biotin-labeled SOX4 promoter region precipitated circ-DONSON in BGC-823 and AGS cell lysates. e DNA-FISH assay indicated the co-localization between circ-DONSON and SOX4 promoter in BGC-823 and AGS cells. Scale bar: 5 μm. f ChIP assay showed that circ-DONSON silencing led to decreased enrichment of active marker H3K27ac on SOX4 promoter in BGC-823 and AGS cells. g SOX4 promoter was more resistant to DNaseI digestion after circ-DONSON knockdown. h - j CCK8, EdU and colony formation assays were performed to detect cell proliferation. k Restoration of SOX4 reduced the apoptosis of BGC-823 and AGS cells induced by circ-DONSON silencing. l , m Restoration of SOX4 rescued the abilities of migration and invasion in circ-DONSON knocked down BGC-823 and AGS cells. ** P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) Total RNAs were isolated using TRIzol and inversely transcribed into cDNA using M-MLV and the SYBR Green Master Mix kit (Takara, Otsu, Japan). qPCR was completed as described previously [ ].

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Chromatin Immunoprecipitation, Labeling, Fluorescence In Situ Hybridization, Marker, Migration