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Promega superscript iii reverse transcriptase
miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in <t>three</t> hepatoma-derived cell lines transfected with JFH1 <t>RNA.</t> ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p
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1) Product Images from "MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway"

Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

Journal: eLife

doi: 10.7554/eLife.41159

miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in three hepatoma-derived cell lines transfected with JFH1 RNA. ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p
Figure Legend Snippet: miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in three hepatoma-derived cell lines transfected with JFH1 RNA. ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p

Techniques Used: Western Blot, Derivative Assay, Transfection, Expressing, Quantitative RT-PCR, Plasmid Preparation, Isolation

STAT3 inhibits the transcriptional activation of IRF1. ( A ) qRT-PCR analysis of IRF1, IRF3, NFKB1 and RELA in HepG2 cells first treated with mimics or siRNAs, and then transfected with or without JFH1 RNA for 24 hr. ( B ) Analysis of IRF1 and IRF3 protein expression in HepG2 cells treated with siRNAs and then JFH1 RNA. ( C ) qRT-PCR analysis of IRF1 and IFNs in HepG2 cells transfected with vectors expressing IRF1 or RFP (after 2 days). ( D ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with IRF1 or RFP plasmids for 2 days, and then treated with poly(I:C) for 3–24 hr. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with the indicated doses of IRF1 plasmids (0.05–1 μg/well in a 24-well-plate) for 2 days. ( F ) Analysis of IRF1, p-STAT1 and MDA5 in HepG2 cells transfected with plasmids expressing 7 HA-tagged transcription factors (after 2 days). HA-GFP was used as a negative control. ( G ) Analysis of IRF1 and p-STAT1 in HepG2 cells first transfected with STAT3 siRNA for 2 days, and then treated with IFN-β or IL-29 for 5–360 min. qRT-PCR data are from one experiment that was representative of three experiments (mean ± SEM of technical triplicates). *p
Figure Legend Snippet: STAT3 inhibits the transcriptional activation of IRF1. ( A ) qRT-PCR analysis of IRF1, IRF3, NFKB1 and RELA in HepG2 cells first treated with mimics or siRNAs, and then transfected with or without JFH1 RNA for 24 hr. ( B ) Analysis of IRF1 and IRF3 protein expression in HepG2 cells treated with siRNAs and then JFH1 RNA. ( C ) qRT-PCR analysis of IRF1 and IFNs in HepG2 cells transfected with vectors expressing IRF1 or RFP (after 2 days). ( D ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with IRF1 or RFP plasmids for 2 days, and then treated with poly(I:C) for 3–24 hr. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with the indicated doses of IRF1 plasmids (0.05–1 μg/well in a 24-well-plate) for 2 days. ( F ) Analysis of IRF1, p-STAT1 and MDA5 in HepG2 cells transfected with plasmids expressing 7 HA-tagged transcription factors (after 2 days). HA-GFP was used as a negative control. ( G ) Analysis of IRF1 and p-STAT1 in HepG2 cells first transfected with STAT3 siRNA for 2 days, and then treated with IFN-β or IL-29 for 5–360 min. qRT-PCR data are from one experiment that was representative of three experiments (mean ± SEM of technical triplicates). *p

Techniques Used: Activation Assay, Quantitative RT-PCR, Transfection, Expressing, Negative Control

The effect of miR-122 on HCV translation. ( A ) Structure of pSGR-JFH1/Gluc constructs. ( B ) Luciferase assays of the activity of Gluc reporter treated with miR-122 mimic or XRN1 siRNA. HepG2 cells were firstly treated with mimics or siRNAs for 48 hr and then transfected with SGR-JFH1/Gluc RNA for 24 hr. The activities of Gluc were measured and normalized to the level in miR-NC-treated cells. Data shown are mean +SD (n = 3). Gluc data are one experiment representative of three independent experiments (mean ± SEM of technical triplicates). ( C ) Comparing the effect of miR-122 on the translation of wildtype (JFH1) and mutant (JFH1-M) HCV.
Figure Legend Snippet: The effect of miR-122 on HCV translation. ( A ) Structure of pSGR-JFH1/Gluc constructs. ( B ) Luciferase assays of the activity of Gluc reporter treated with miR-122 mimic or XRN1 siRNA. HepG2 cells were firstly treated with mimics or siRNAs for 48 hr and then transfected with SGR-JFH1/Gluc RNA for 24 hr. The activities of Gluc were measured and normalized to the level in miR-NC-treated cells. Data shown are mean +SD (n = 3). Gluc data are one experiment representative of three independent experiments (mean ± SEM of technical triplicates). ( C ) Comparing the effect of miR-122 on the translation of wildtype (JFH1) and mutant (JFH1-M) HCV.

Techniques Used: Construct, Luciferase, Activity Assay, Transfection, Mutagenesis

miR-122 regulates IFN response by repressing STAT3 phosphorylation. ( A ) Analysis of p-STAT1 expression in HepG2 cells first transfected with mimics (NC or miR-122) for 2 days and then treated with IFN-β or IL-29 for 5–60 min. ( B ) qRT-PCR analysis of the five SOCS genes in HepG2 cells first treated with mimics for 2 days, and then transfected with JFH1 RNA for 24 hr. ( C ) qRT-PCR analysis of STAT3 mRNA in HepG2 cells, treated as in panel B. ( D ) Analysis of total and phosphorylated STAT3 in HepG2 cells treated with three independent siRNAs (si-1, si-2 and si-3) at a final concentration of 20 nM. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with STAT3 siRNA and then treated with JFH1 RNA or poly(I:C). ( F ) Analysis of the dose-dependent effects of cryptotanshinone (CTS) and S3I-201 on p-STAT3. HepG2 cells were treated with either CST or S3I-201 at the indicated concentrations for 24 hr. qRT-PCR data are from one experiment that was representative of two ( B ) or three ( C ) independent experiments (mean ± SEM of technical triplicates).
Figure Legend Snippet: miR-122 regulates IFN response by repressing STAT3 phosphorylation. ( A ) Analysis of p-STAT1 expression in HepG2 cells first transfected with mimics (NC or miR-122) for 2 days and then treated with IFN-β or IL-29 for 5–60 min. ( B ) qRT-PCR analysis of the five SOCS genes in HepG2 cells first treated with mimics for 2 days, and then transfected with JFH1 RNA for 24 hr. ( C ) qRT-PCR analysis of STAT3 mRNA in HepG2 cells, treated as in panel B. ( D ) Analysis of total and phosphorylated STAT3 in HepG2 cells treated with three independent siRNAs (si-1, si-2 and si-3) at a final concentration of 20 nM. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with STAT3 siRNA and then treated with JFH1 RNA or poly(I:C). ( F ) Analysis of the dose-dependent effects of cryptotanshinone (CTS) and S3I-201 on p-STAT3. HepG2 cells were treated with either CST or S3I-201 at the indicated concentrations for 24 hr. qRT-PCR data are from one experiment that was representative of two ( B ) or three ( C ) independent experiments (mean ± SEM of technical triplicates).

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Concentration Assay

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Amplification:

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Synthesized:

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Cytometry:

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Quantitative RT-PCR:

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SYBR Green Assay:

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Microarray:

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Incubation:

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Proliferation Assay:

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Expressing:

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Western Blot:

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Flow Cytometry:

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Cell Culture:

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Generated:

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Inhibition:

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Reverse Transcription Polymerase Chain Reaction:

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Multiplex Assay:

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Isolation:

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Article Title: Zinc-finger protein X-linked is a novel predictor of prognosis in patients with colorectal cancer
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Article Title: Altered Expression of Porcine Piwi Genes and piRNA during Development
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Polymerase Chain Reaction:

Article Title: Stress Response and Adaptation of Listeria monocytogenes 08-5923 Exposed to a Sublethal Dose of Carnocyclin A
Article Snippet: Total RNA was isolated and reverse transcribed into cDNA using SuperScript III reverse transcriptase (Promega, WI) according to the manufacturer's protocol. .. Quantifications of the transcripts were carried out (7500 Fast real-time PCR system; Applied Biosystems, CA) using a QuantiFast SYBR green PCR kit (Qiagen Inc.).

Article Title: Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide
Article Snippet: Superscript III Reverse Transcriptase and the MTS CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay were from Promega (Madison, WI), and the Versagene RNA purification kit from Gentra Systems (Minneapolis, MN). .. SYBR Green Master Mix and other PCR reagents were from Applied Biosystems (Foster City, CA).

Article Title: Transcription profiling of a recently colonised pyrethroid resistant Anopheles gambiae strain from Ghana
Article Snippet: Extracted total RNA was reverse transcribed to cDNA using an oligo d(T)14 primer and Superscript III reverse transcriptase (Promega). .. PCR primers (Table ) were designed to amplify two P450 genes, CYP307A1 and CYP6M2 , along with the ribosomal gene S7 [GenBank: AY380336 ] [ ] which served as an internal standard to account for differences in initial cDNA and reaction efficiency.

Article Title: Emery–Dreifuss muscular dystrophy–linked genes and centronuclear myopathy–linked genes regulate myonuclear movement by distinct mechanisms
Article Snippet: Paragraph title: RNA isolation, construction of cDNA library, and reverse transcription PCR ... Purified RNA was incubated with SuperScript III reverse transcriptase at 42°C for 2 h, and then reactions were terminated at 85°C for 5 min. RT-PCR was set up after inactivation of reverse transcription using the GoTaq Flexi DNA Polymerase (M8291; Promega).

Article Title: Altered Expression of Porcine Piwi Genes and piRNA during Development
Article Snippet: Reverse transcription was carried out with 40 U/µl of SuperScript III reverse-transcriptase, 0.5 mM dNTP mix, 1×buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 ), 5 mM DTT and 20 U/µl of RNase Inhibitor (Promega) in a final reaction volume of 20 µl at 50°C for 1 hour. .. The reactions were stored at −20°C until PCR amplification.

Article Title: Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells
Article Snippet: .. Total RNA was isolated from cultured cells using Trizol (Invitrogen). cDNAs were synthesized using oligo (dT) and SuperScript III reverse transcriptase (Promega, Madison, WI, USA), then amplified using iQ SYBR Green PCR Master Mix (Bio-Rad Laboratories, Hercules, CA, USA) in conjunction with Mx3005P QPCR Systems (Agilent Technologies, Santa Clara, CA, USA). .. Primer sets were as follows: nSMase 2 qPCR primer P109289 (Bioneer); glyceraldehyde-3-phosphate dehydrogenase, 5′-TGGTATCGTGGAAGGACTCATGAC-3′ (forward) and 5′-ATGCCAGTGAGCTTCCCGTTCAGC -3′ (reverse).

Article Title: Resistance monitoring and cross-resistance role of CYP6CW1 between buprofezin and pymetrozine in field populations of Laodelphax striatellus (Fallén)
Article Snippet: The first-strand cDNA was synthesized from 2 µg of total RNA using an oligo(dT)15 primer and Superscript III reverse transcriptase (Promega). .. The PCR primer sequences and the expected size of each PCR product are shown in a previous study and in Table .

Article Title: Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25
Article Snippet: Paragraph title: Quantitative reverse-transcription PCR ... First-strand complementary DNA (cDNA) was synthesized from 5 μg of total RNA with SuperScript III reverse transcriptase and random primers (Promega, Madison, WI, USA).

Transfection:

Article Title: Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells
Article Snippet: Total RNA was isolated from cultured cells using Trizol (Invitrogen). cDNAs were synthesized using oligo (dT) and SuperScript III reverse transcriptase (Promega, Madison, WI, USA), then amplified using iQ SYBR Green PCR Master Mix (Bio-Rad Laboratories, Hercules, CA, USA) in conjunction with Mx3005P QPCR Systems (Agilent Technologies, Santa Clara, CA, USA). .. In transfections, cells were cultured in complete medium, seeded into 96 well plate at 0.5 – 1.0 × 104 cells per well using culture media without antibiotics overnight along with mixture of Lipofectamine (Invitrogen) and SCR or siRNAs (Bioneer) at 10 nM.

Purification:

Article Title: Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide
Article Snippet: .. Superscript III Reverse Transcriptase and the MTS CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay were from Promega (Madison, WI), and the Versagene RNA purification kit from Gentra Systems (Minneapolis, MN). .. The Western Lightning enhanced chemiluminescence kit (ECL) was from Perkin Elmer Life Sciences, Inc. (Boston, MA), the OxyBlot Protein Oxidation Detection kit from Chemicon International (Temecula, CA) and precast gels were from Pierce Biotechnology (Rockford, IL).

Article Title: Emery–Dreifuss muscular dystrophy–linked genes and centronuclear myopathy–linked genes regulate myonuclear movement by distinct mechanisms
Article Snippet: .. Purified RNA was incubated with SuperScript III reverse transcriptase at 42°C for 2 h, and then reactions were terminated at 85°C for 5 min. RT-PCR was set up after inactivation of reverse transcription using the GoTaq Flexi DNA Polymerase (M8291; Promega). .. Primers were designed to amplify a ∼120–base pair sequence within each targeted mRNA and a 315–base pair sequence within RP49 as a control.

Sequencing:

Article Title: Emery–Dreifuss muscular dystrophy–linked genes and centronuclear myopathy–linked genes regulate myonuclear movement by distinct mechanisms
Article Snippet: Purified RNA was incubated with SuperScript III reverse transcriptase at 42°C for 2 h, and then reactions were terminated at 85°C for 5 min. RT-PCR was set up after inactivation of reverse transcription using the GoTaq Flexi DNA Polymerase (M8291; Promega). .. Primers were designed to amplify a ∼120–base pair sequence within each targeted mRNA and a 315–base pair sequence within RP49 as a control.

cDNA Library Assay:

Article Title: Emery–Dreifuss muscular dystrophy–linked genes and centronuclear myopathy–linked genes regulate myonuclear movement by distinct mechanisms
Article Snippet: Paragraph title: RNA isolation, construction of cDNA library, and reverse transcription PCR ... Purified RNA was incubated with SuperScript III reverse transcriptase at 42°C for 2 h, and then reactions were terminated at 85°C for 5 min. RT-PCR was set up after inactivation of reverse transcription using the GoTaq Flexi DNA Polymerase (M8291; Promega).

RNA Extraction:

Article Title: Overexpression of NDC80 is correlated with prognosis of pancreatic cancer and regulates cell proliferation
Article Snippet: Paragraph title: RNA extraction and quantitative real-time polymerase chain reaction ... Complementary DNA (cDNA) was generated using Superscript III Reverse Transcriptase (Promega, USA).

Software:

Article Title: Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25
Article Snippet: CD4+ LAG3+ , CD4+ LAG3− , CD127low CD25+ ICOS+ , CD127low CD25+ ICOS− and CD8+ T-cell populations were sorted by flow cytometry (BD Influx and BD ARIA III with FACSDiva software) from murine spleens and tumors and placed in RLT buffer (Qiagen, Hilden, Germany) containing 1% 2-mercaptoethanol, followed by their storage at −80 °C. .. First-strand complementary DNA (cDNA) was synthesized from 5 μg of total RNA with SuperScript III reverse transcriptase and random primers (Promega, Madison, WI, USA).

Real-time Polymerase Chain Reaction:

Article Title: Stress Response and Adaptation of Listeria monocytogenes 08-5923 Exposed to a Sublethal Dose of Carnocyclin A
Article Snippet: Total RNA was isolated and reverse transcribed into cDNA using SuperScript III reverse transcriptase (Promega, WI) according to the manufacturer's protocol. .. Quantifications of the transcripts were carried out (7500 Fast real-time PCR system; Applied Biosystems, CA) using a QuantiFast SYBR green PCR kit (Qiagen Inc.).

Article Title: Zinc-finger protein X-linked is a novel predictor of prognosis in patients with colorectal cancer
Article Snippet: Paragraph title: Quantitative real-time polymerase chain reaction ... The obtained RNA was used to synthesize cDNA by Superscript III Reverse Transcriptase (Promega, USA).

Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway
Article Snippet: .. RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002). .. Western blot Protein samples were prepared using RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM Sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2 mM EDTA) with 1X Protease Inhibitor Cocktail (Roche, 04693132001) and 1x PhosSTOP phosphatase inhibitor (Roche, 04906837001).

Article Title: Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells
Article Snippet: .. Total RNA was isolated from cultured cells using Trizol (Invitrogen). cDNAs were synthesized using oligo (dT) and SuperScript III reverse transcriptase (Promega, Madison, WI, USA), then amplified using iQ SYBR Green PCR Master Mix (Bio-Rad Laboratories, Hercules, CA, USA) in conjunction with Mx3005P QPCR Systems (Agilent Technologies, Santa Clara, CA, USA). .. Primer sets were as follows: nSMase 2 qPCR primer P109289 (Bioneer); glyceraldehyde-3-phosphate dehydrogenase, 5′-TGGTATCGTGGAAGGACTCATGAC-3′ (forward) and 5′-ATGCCAGTGAGCTTCCCGTTCAGC -3′ (reverse).

Article Title: Overexpression of NDC80 is correlated with prognosis of pancreatic cancer and regulates cell proliferation
Article Snippet: Paragraph title: RNA extraction and quantitative real-time polymerase chain reaction ... Complementary DNA (cDNA) was generated using Superscript III Reverse Transcriptase (Promega, USA).

Article Title: Resistance monitoring and cross-resistance role of CYP6CW1 between buprofezin and pymetrozine in field populations of Laodelphax striatellus (Fallén)
Article Snippet: The first-strand cDNA was synthesized from 2 µg of total RNA using an oligo(dT)15 primer and Superscript III reverse transcriptase (Promega). .. The relative expression levels of the 47 P450 genes in the YN and field populations were determined by qPCR with ADP ribosylation factor (ARF) as a reference .

Article Title: Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25
Article Snippet: First-strand complementary DNA (cDNA) was synthesized from 5 μg of total RNA with SuperScript III reverse transcriptase and random primers (Promega, Madison, WI, USA). .. Expression of il-2, il-10 and egr-2 genes was analyzed by means of TaqMan Gene Expression Assays using the Universal Master Mix II (with UNG) (both from Applied Biosystems, Foster City, CA, USA), on a StepOnePlus Real-Time PCR System (Applied Biosystems).

Negative Control:

Article Title: Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells
Article Snippet: Inhibition and siRNA interference In inhibition studies, cells were plated into 96 well plates at 0.3 – 1.0 ×104 cells per well overnight, preincubated for 1 hr with neutral sphingomyelinase 2 (nSMase 2) inhibitor GW4869 at 10 μΜ (Sigma-Aldrich), then treated with PPD at specified concentrations for 48 h. Two nSMase 2 siRNAs were purchased from Bioneer (Daejeon, Korea): siRNA 1141725 sense: GCU ACU UCG AGU ACA UCC U and antisense AGG AUG UAC UCG AAG UAG C and siRNA 1141726 sense: CAC GAA CGG CCU GUA CGA U and antisense: AUG GUA CAG GCC GUU CGU G. Scrambled siRNA (SCR) (Bioneer) was used as negative control: sense CCU ACG CCA CCA AUU UCG U and antisense: ACG AAA UUG GUG GCG UAG G. Real-time PCR was performed to quantify mRNA expressions of nSMase 2 after knockdown with siRNA 1141725 and 1141726 (Additional file ). .. Total RNA was isolated from cultured cells using Trizol (Invitrogen). cDNAs were synthesized using oligo (dT) and SuperScript III reverse transcriptase (Promega, Madison, WI, USA), then amplified using iQ SYBR Green PCR Master Mix (Bio-Rad Laboratories, Hercules, CA, USA) in conjunction with Mx3005P QPCR Systems (Agilent Technologies, Santa Clara, CA, USA).

Agarose Gel Electrophoresis:

Article Title: Emery–Dreifuss muscular dystrophy–linked genes and centronuclear myopathy–linked genes regulate myonuclear movement by distinct mechanisms
Article Snippet: Purified RNA was incubated with SuperScript III reverse transcriptase at 42°C for 2 h, and then reactions were terminated at 85°C for 5 min. RT-PCR was set up after inactivation of reverse transcription using the GoTaq Flexi DNA Polymerase (M8291; Promega). .. PCR products were run on a 2% agarose gel and visualized with ethidium bromide.

Concentration Assay:

Article Title: Emery–Dreifuss muscular dystrophy–linked genes and centronuclear myopathy–linked genes regulate myonuclear movement by distinct mechanisms
Article Snippet: RNA integrity and concentration were determined using the NanoDrop2000 system (Thermo Fisher Scientific). .. Purified RNA was incubated with SuperScript III reverse transcriptase at 42°C for 2 h, and then reactions were terminated at 85°C for 5 min. RT-PCR was set up after inactivation of reverse transcription using the GoTaq Flexi DNA Polymerase (M8291; Promega).

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    Promega superscript iii reverse transcriptase
    miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in <t>three</t> hepatoma-derived cell lines transfected with JFH1 <t>RNA.</t> ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p
    Superscript Iii Reverse Transcriptase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega superscript iii reaction mix
    Cell density-dependent change in the levels of piRNAs and their precursors. ( A ) The expression levels of <t>piR-1,</t> piR-2, and 5 S rRNA in BmN4 cells with different densities were analyzed by Northern blot. ( B ) The Northern blot bands were quantified and shown as relative abundances to signal intensities from the cells with 6.0 × 10 3 cells/cm 2 starting density (set as 1). Each data set represents the average of <t>three</t> independent experiments with bars showing the SD. The asterisks indicate significant difference (P
    Superscript Iii Reaction Mix, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reaction mix/product/Promega
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    93
    Promega superscript iii reverse transcriptase platinum taq dna polymerase
    Cell density-dependent change in the levels of piRNAs and their precursors. ( A ) The expression levels of <t>piR-1,</t> piR-2, and 5 S rRNA in BmN4 cells with different densities were analyzed by Northern blot. ( B ) The Northern blot bands were quantified and shown as relative abundances to signal intensities from the cells with 6.0 × 10 3 cells/cm 2 starting density (set as 1). Each data set represents the average of <t>three</t> independent experiments with bars showing the SD. The asterisks indicate significant difference (P
    Superscript Iii Reverse Transcriptase Platinum Taq Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase platinum taq dna polymerase/product/Promega
    Average 93 stars, based on 1 article reviews
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    miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in three hepatoma-derived cell lines transfected with JFH1 RNA. ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p

    Journal: eLife

    Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

    doi: 10.7554/eLife.41159

    Figure Lengend Snippet: miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in three hepatoma-derived cell lines transfected with JFH1 RNA. ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p

    Article Snippet: RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002).

    Techniques: Western Blot, Derivative Assay, Transfection, Expressing, Quantitative RT-PCR, Plasmid Preparation, Isolation

    STAT3 inhibits the transcriptional activation of IRF1. ( A ) qRT-PCR analysis of IRF1, IRF3, NFKB1 and RELA in HepG2 cells first treated with mimics or siRNAs, and then transfected with or without JFH1 RNA for 24 hr. ( B ) Analysis of IRF1 and IRF3 protein expression in HepG2 cells treated with siRNAs and then JFH1 RNA. ( C ) qRT-PCR analysis of IRF1 and IFNs in HepG2 cells transfected with vectors expressing IRF1 or RFP (after 2 days). ( D ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with IRF1 or RFP plasmids for 2 days, and then treated with poly(I:C) for 3–24 hr. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with the indicated doses of IRF1 plasmids (0.05–1 μg/well in a 24-well-plate) for 2 days. ( F ) Analysis of IRF1, p-STAT1 and MDA5 in HepG2 cells transfected with plasmids expressing 7 HA-tagged transcription factors (after 2 days). HA-GFP was used as a negative control. ( G ) Analysis of IRF1 and p-STAT1 in HepG2 cells first transfected with STAT3 siRNA for 2 days, and then treated with IFN-β or IL-29 for 5–360 min. qRT-PCR data are from one experiment that was representative of three experiments (mean ± SEM of technical triplicates). *p

    Journal: eLife

    Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

    doi: 10.7554/eLife.41159

    Figure Lengend Snippet: STAT3 inhibits the transcriptional activation of IRF1. ( A ) qRT-PCR analysis of IRF1, IRF3, NFKB1 and RELA in HepG2 cells first treated with mimics or siRNAs, and then transfected with or without JFH1 RNA for 24 hr. ( B ) Analysis of IRF1 and IRF3 protein expression in HepG2 cells treated with siRNAs and then JFH1 RNA. ( C ) qRT-PCR analysis of IRF1 and IFNs in HepG2 cells transfected with vectors expressing IRF1 or RFP (after 2 days). ( D ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with IRF1 or RFP plasmids for 2 days, and then treated with poly(I:C) for 3–24 hr. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with the indicated doses of IRF1 plasmids (0.05–1 μg/well in a 24-well-plate) for 2 days. ( F ) Analysis of IRF1, p-STAT1 and MDA5 in HepG2 cells transfected with plasmids expressing 7 HA-tagged transcription factors (after 2 days). HA-GFP was used as a negative control. ( G ) Analysis of IRF1 and p-STAT1 in HepG2 cells first transfected with STAT3 siRNA for 2 days, and then treated with IFN-β or IL-29 for 5–360 min. qRT-PCR data are from one experiment that was representative of three experiments (mean ± SEM of technical triplicates). *p

    Article Snippet: RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002).

    Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Expressing, Negative Control

    The effect of miR-122 on HCV translation. ( A ) Structure of pSGR-JFH1/Gluc constructs. ( B ) Luciferase assays of the activity of Gluc reporter treated with miR-122 mimic or XRN1 siRNA. HepG2 cells were firstly treated with mimics or siRNAs for 48 hr and then transfected with SGR-JFH1/Gluc RNA for 24 hr. The activities of Gluc were measured and normalized to the level in miR-NC-treated cells. Data shown are mean +SD (n = 3). Gluc data are one experiment representative of three independent experiments (mean ± SEM of technical triplicates). ( C ) Comparing the effect of miR-122 on the translation of wildtype (JFH1) and mutant (JFH1-M) HCV.

    Journal: eLife

    Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

    doi: 10.7554/eLife.41159

    Figure Lengend Snippet: The effect of miR-122 on HCV translation. ( A ) Structure of pSGR-JFH1/Gluc constructs. ( B ) Luciferase assays of the activity of Gluc reporter treated with miR-122 mimic or XRN1 siRNA. HepG2 cells were firstly treated with mimics or siRNAs for 48 hr and then transfected with SGR-JFH1/Gluc RNA for 24 hr. The activities of Gluc were measured and normalized to the level in miR-NC-treated cells. Data shown are mean +SD (n = 3). Gluc data are one experiment representative of three independent experiments (mean ± SEM of technical triplicates). ( C ) Comparing the effect of miR-122 on the translation of wildtype (JFH1) and mutant (JFH1-M) HCV.

    Article Snippet: RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002).

    Techniques: Construct, Luciferase, Activity Assay, Transfection, Mutagenesis

    miR-122 regulates IFN response by repressing STAT3 phosphorylation. ( A ) Analysis of p-STAT1 expression in HepG2 cells first transfected with mimics (NC or miR-122) for 2 days and then treated with IFN-β or IL-29 for 5–60 min. ( B ) qRT-PCR analysis of the five SOCS genes in HepG2 cells first treated with mimics for 2 days, and then transfected with JFH1 RNA for 24 hr. ( C ) qRT-PCR analysis of STAT3 mRNA in HepG2 cells, treated as in panel B. ( D ) Analysis of total and phosphorylated STAT3 in HepG2 cells treated with three independent siRNAs (si-1, si-2 and si-3) at a final concentration of 20 nM. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with STAT3 siRNA and then treated with JFH1 RNA or poly(I:C). ( F ) Analysis of the dose-dependent effects of cryptotanshinone (CTS) and S3I-201 on p-STAT3. HepG2 cells were treated with either CST or S3I-201 at the indicated concentrations for 24 hr. qRT-PCR data are from one experiment that was representative of two ( B ) or three ( C ) independent experiments (mean ± SEM of technical triplicates).

    Journal: eLife

    Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

    doi: 10.7554/eLife.41159

    Figure Lengend Snippet: miR-122 regulates IFN response by repressing STAT3 phosphorylation. ( A ) Analysis of p-STAT1 expression in HepG2 cells first transfected with mimics (NC or miR-122) for 2 days and then treated with IFN-β or IL-29 for 5–60 min. ( B ) qRT-PCR analysis of the five SOCS genes in HepG2 cells first treated with mimics for 2 days, and then transfected with JFH1 RNA for 24 hr. ( C ) qRT-PCR analysis of STAT3 mRNA in HepG2 cells, treated as in panel B. ( D ) Analysis of total and phosphorylated STAT3 in HepG2 cells treated with three independent siRNAs (si-1, si-2 and si-3) at a final concentration of 20 nM. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with STAT3 siRNA and then treated with JFH1 RNA or poly(I:C). ( F ) Analysis of the dose-dependent effects of cryptotanshinone (CTS) and S3I-201 on p-STAT3. HepG2 cells were treated with either CST or S3I-201 at the indicated concentrations for 24 hr. qRT-PCR data are from one experiment that was representative of two ( B ) or three ( C ) independent experiments (mean ± SEM of technical triplicates).

    Article Snippet: RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Concentration Assay

    Cell density-dependent change in the levels of piRNAs and their precursors. ( A ) The expression levels of piR-1, piR-2, and 5 S rRNA in BmN4 cells with different densities were analyzed by Northern blot. ( B ) The Northern blot bands were quantified and shown as relative abundances to signal intensities from the cells with 6.0 × 10 3 cells/cm 2 starting density (set as 1). Each data set represents the average of three independent experiments with bars showing the SD. The asterisks indicate significant difference (P

    Journal: Scientific Reports

    Article Title: Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells

    doi: 10.1038/s41598-017-04429-7

    Figure Lengend Snippet: Cell density-dependent change in the levels of piRNAs and their precursors. ( A ) The expression levels of piR-1, piR-2, and 5 S rRNA in BmN4 cells with different densities were analyzed by Northern blot. ( B ) The Northern blot bands were quantified and shown as relative abundances to signal intensities from the cells with 6.0 × 10 3 cells/cm 2 starting density (set as 1). Each data set represents the average of three independent experiments with bars showing the SD. The asterisks indicate significant difference (P

    Article Snippet: For piRNA-1 (piR-1) and piRNA-2 (piR-2), to reverse-transcribe mature piRNA, 100 ng of the DNase-treated BmN4 total RNA was incubated with 50 nM of stem-loop RT primer (piR-1; 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTTCGA-3′, piR-2; 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCGCAG-3′), 1 × SuperScript III reaction mix (0.25 mM each dNTP, 1 × First-Strand Buffer, 5 mM DTT, and 200 U Superscript III reverse transcriptase), and 1 U of RNase inhibitor (Promega) for 30 min at 16 °C, 30 min at 42 °C, and then 15 min at 70 °C.

    Techniques: Expressing, Northern Blot

    Cell density-dependent change in the transposon levels. ( A ) Siwi mRNA from BmN4 cells treated with dsRNA targeting Renilla luciferase (Rluc, negative control) or Siwi was analyzed by qRT-PCR. Rp49 was used as an internal control, and the level in the Rluc dsRNA-treated cells was set as 1. The average of three independent experiments with bars showing the SD is shown. ( B ) The levels of Siwi and β-actin (control) in the Rluc- or Siwi-depleted cells were analyzed by Western blot. ( C ) The levels of piR-1 and let-7 miRNA (control) in the Rluc- or Siwi-depleted cells were analyzed by Northern blots. ( D , E ) The expression levels of Yamato and Kimono transposons in the Rluc- or Siwi-depleted cells ( D ) and the cells with the indicated starting densities ( E ) were quantified by qRT- PCR. The expression levels in the Rluc-depleted cells or the cells with 6.0 × 10 3 cells/cm 2 starting density were set as 1. Each data set represents the average of three independent experiments with bars showing the SD. The asterisks indicate significant difference (P

    Journal: Scientific Reports

    Article Title: Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells

    doi: 10.1038/s41598-017-04429-7

    Figure Lengend Snippet: Cell density-dependent change in the transposon levels. ( A ) Siwi mRNA from BmN4 cells treated with dsRNA targeting Renilla luciferase (Rluc, negative control) or Siwi was analyzed by qRT-PCR. Rp49 was used as an internal control, and the level in the Rluc dsRNA-treated cells was set as 1. The average of three independent experiments with bars showing the SD is shown. ( B ) The levels of Siwi and β-actin (control) in the Rluc- or Siwi-depleted cells were analyzed by Western blot. ( C ) The levels of piR-1 and let-7 miRNA (control) in the Rluc- or Siwi-depleted cells were analyzed by Northern blots. ( D , E ) The expression levels of Yamato and Kimono transposons in the Rluc- or Siwi-depleted cells ( D ) and the cells with the indicated starting densities ( E ) were quantified by qRT- PCR. The expression levels in the Rluc-depleted cells or the cells with 6.0 × 10 3 cells/cm 2 starting density were set as 1. Each data set represents the average of three independent experiments with bars showing the SD. The asterisks indicate significant difference (P

    Article Snippet: For piRNA-1 (piR-1) and piRNA-2 (piR-2), to reverse-transcribe mature piRNA, 100 ng of the DNase-treated BmN4 total RNA was incubated with 50 nM of stem-loop RT primer (piR-1; 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTTCGA-3′, piR-2; 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCGCAG-3′), 1 × SuperScript III reaction mix (0.25 mM each dNTP, 1 × First-Strand Buffer, 5 mM DTT, and 200 U Superscript III reverse transcriptase), and 1 U of RNase inhibitor (Promega) for 30 min at 16 °C, 30 min at 42 °C, and then 15 min at 70 °C.

    Techniques: Luciferase, Negative Control, Quantitative RT-PCR, Western Blot, Northern Blot, Expressing