superscript iii reverse transcriptase system  (Thermo Fisher)


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    Structured Review

    Thermo Fisher superscript iii reverse transcriptase system
    Superscript Iii Reverse Transcriptase System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase system/product/Thermo Fisher
    Average 97 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase system - by Bioz Stars, 2020-04
    97/100 stars

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    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I
    Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, Quantitative Real-Time PCR, and Traditional PCR ... First-strand cDNAs were synthesized from a 5-μg aliquot of the various RNAs using the Superscript III Reverse Transcriptase System (Invitrogen Canada, Burlington, Ontario, Canada).

    Transfection:

    Article Title: Mutations in MAB21L2 Result in Ocular Coloboma, Microcornea and Cataracts
    Article Snippet: RNA isolation, RT-PCR and mRNA injections To detect transcript levels in HLE-B3 cells, RNA from cells was isolated following transfection with expression plasmids containing sequences encoding for either wild type, or p.(Arg51Gly) or p.(Arg51_Phe52del) MAB21L2 alleles using TRIzol Reagent (Cat. #15596-026) (Life Technologies) 48-hours after transfection. .. Each sample was treated with DNase I (Cat. #18068) and reverse transcribed in triplicate using the SuperScript III Reverse Transcriptase system (Cat. #18080) (Life Technologies).

    Amplification:

    Article Title: Mutations in MAB21L2 Result in Ocular Coloboma, Microcornea and Cataracts
    Article Snippet: Each sample was treated with DNase I (Cat. #18068) and reverse transcribed in triplicate using the SuperScript III Reverse Transcriptase system (Cat. #18080) (Life Technologies). .. Samples were amplified for MAB21L2 using the following primers: MAB21L2-FLAG-F 5’- CAAAGACGATGACGACAAGG-3’ and MAB21L2-rt-R 5’- GGTAGAGCACCACCTCAAATTC-3’ (PCR product equal 305 bp).

    Synthesized:

    Article Title: The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I
    Article Snippet: .. First-strand cDNAs were synthesized from a 5-μg aliquot of the various RNAs using the Superscript III Reverse Transcriptase System (Invitrogen Canada, Burlington, Ontario, Canada). .. MA-10 Leydig cells were cultured in serum-free media containing vehicle, 100 n m bisindolylmaleimide I, 10 μ m H-89, 20 μ m KN-93, 1 μ m ML-7, 100 n m staurosporine, or 10 μ m PD98059 for 30 min before addition of 10 μ m FSK for 2 h before RNA isolation.

    Isolation:

    Article Title: Mutations in MAB21L2 Result in Ocular Coloboma, Microcornea and Cataracts
    Article Snippet: Paragraph title: RNA isolation, RT-PCR and mRNA injections ... Each sample was treated with DNase I (Cat. #18068) and reverse transcribed in triplicate using the SuperScript III Reverse Transcriptase system (Cat. #18080) (Life Technologies).

    Article Title: The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I
    Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, Quantitative Real-Time PCR, and Traditional PCR ... First-strand cDNAs were synthesized from a 5-μg aliquot of the various RNAs using the Superscript III Reverse Transcriptase System (Invitrogen Canada, Burlington, Ontario, Canada).

    Cell Culture:

    Article Title: The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I
    Article Snippet: First-strand cDNAs were synthesized from a 5-μg aliquot of the various RNAs using the Superscript III Reverse Transcriptase System (Invitrogen Canada, Burlington, Ontario, Canada). .. MA-10 Leydig cells were cultured in serum-free media containing vehicle, 100 n m bisindolylmaleimide I, 10 μ m H-89, 20 μ m KN-93, 1 μ m ML-7, 100 n m staurosporine, or 10 μ m PD98059 for 30 min before addition of 10 μ m FSK for 2 h before RNA isolation.

    SYBR Green Assay:

    Article Title: The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I
    Article Snippet: First-strand cDNAs were synthesized from a 5-μg aliquot of the various RNAs using the Superscript III Reverse Transcriptase System (Invitrogen Canada, Burlington, Ontario, Canada). .. Quantitative real-time PCR was performed using a LightCycler 1.5 instrument and the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Laval, Canada) according to the manufacturer’s protocol.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Mutations in MAB21L2 Result in Ocular Coloboma, Microcornea and Cataracts
    Article Snippet: Paragraph title: RNA isolation, RT-PCR and mRNA injections ... Each sample was treated with DNase I (Cat. #18068) and reverse transcribed in triplicate using the SuperScript III Reverse Transcriptase system (Cat. #18080) (Life Technologies).

    Expressing:

    Article Title: Mutations in MAB21L2 Result in Ocular Coloboma, Microcornea and Cataracts
    Article Snippet: RNA isolation, RT-PCR and mRNA injections To detect transcript levels in HLE-B3 cells, RNA from cells was isolated following transfection with expression plasmids containing sequences encoding for either wild type, or p.(Arg51Gly) or p.(Arg51_Phe52del) MAB21L2 alleles using TRIzol Reagent (Cat. #15596-026) (Life Technologies) 48-hours after transfection. .. Each sample was treated with DNase I (Cat. #18068) and reverse transcribed in triplicate using the SuperScript III Reverse Transcriptase system (Cat. #18080) (Life Technologies).

    Polymerase Chain Reaction:

    Article Title: Mutations in MAB21L2 Result in Ocular Coloboma, Microcornea and Cataracts
    Article Snippet: Each sample was treated with DNase I (Cat. #18068) and reverse transcribed in triplicate using the SuperScript III Reverse Transcriptase system (Cat. #18080) (Life Technologies). .. Samples were amplified for MAB21L2 using the following primers: MAB21L2-FLAG-F 5’- CAAAGACGATGACGACAAGG-3’ and MAB21L2-rt-R 5’- GGTAGAGCACCACCTCAAATTC-3’ (PCR product equal 305 bp).

    Article Title: The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I
    Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, Quantitative Real-Time PCR, and Traditional PCR ... First-strand cDNAs were synthesized from a 5-μg aliquot of the various RNAs using the Superscript III Reverse Transcriptase System (Invitrogen Canada, Burlington, Ontario, Canada).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I
    Article Snippet: First-strand cDNAs were synthesized from a 5-μg aliquot of the various RNAs using the Superscript III Reverse Transcriptase System (Invitrogen Canada, Burlington, Ontario, Canada). .. PCRs were performed using the following specific primers: StAR forward, 5′-TTG GGC ATA CTC AAC AAC CA-3′, and reverse, 5′-CCT TGA CAT TTG GGT TCC AC-3′; Nur77 forward, 5′-GGC TTC TTC AAG CGC ACA GT-3′, and reverse, 5′-GCT GCT TGG GTT TTG AAG GTA G-3′; and SF-1 forward, 5′-TCC AGT ACG GCA AGG AAG AC-3′, and reverse, 5′-GGC TGT GGT TGT TCA GGA AT-3′.

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    Thermo Fisher superscript iii rt
    Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of <t>three</t> independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the <t>RNA-dependent-RNA</t> polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.
    Superscript Iii Rt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii rt/product/Thermo Fisher
    Average 99 stars, based on 3515 article reviews
    Price from $9.99 to $1999.99
    superscript iii rt - by Bioz Stars, 2020-04
    99/100 stars
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    Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.

    Journal: Viruses

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

    doi: 10.3390/v10070368

    Figure Lengend Snippet: Construction and characterization of synZIKV-R2A reporter virus genomes. ( A ) Schematic representation of the synZIKV-R2A reporter virus genomes. For both strains the R2A reporter cassette (light red) was inserted into the wild-type pFK-synZIKV plasmids via MLuI/KpnI restriction sites. The NotI/NruI sites flanking the RLuc gene allow for the exchange of the reporter gene. ( B ) Immunofluorescence analysis of VeroE6 cells transfected with synZIKV-R2A in vitro transcripts. Cells were grown on coverslips, fixed 72 h and 96 h after transfection and stained with E-specific antibody (green). Nuclear DNA was counterstained with DAPI (grey). Scale bar = 15 μm. ( C ) Replication kinetics of the synZIKV-R2A reporter viruses in VeroE6 cells. After electroporation (EPO) cells were harvested at given time points and RLuc activity was determined. Values were normalized to the 4 h-value reflecting transfection efficiency. Mean ± SEM of three independent experiments is shown. Replication deficient mutants containing two mutations affecting the active site of the RNA-dependent-RNA polymerase in NS5 (GAA) served as negative controls. ( D ) VeroE6 cells were transfected with synZIKV-R2A RNAs, cell culture supernatants were collected 72 h post- transfection (P0) and passaged three times by infection of VeroE6 cells (P1-P3) in 72 h intervals. Culture supernatants obtained from each passage were used to inoculate Huh7 cells. In the case of supernatant obtained directly from transfected VeroE6 cells (P0), Huh7 cells were inoculated with undiluted (undil) or 1:10 diluted supernatant. After 72 h cells were harvested and RLuc activity in cell lysates was determined. Mean ± SEM from two independent experiments is shown. ( E ) Virus titres as determined by plaque assay for each synZIKV-R2A passage; values are mean ± SEM of two independent experiments. ( F ) Stability of the reporter gene. SynZIKV-R2A viruses released into culture supernatants were harvested after each passage as described in panel D, RNA was isolated and the region encompassing the RLuc coding sequence was amplified by using random hexamer primers for reverse transcription and specific primers for subsequent PCR. The ~1350 bp long DNA fragment in the P0 virus sample corresponds to the reporter gene, while the ~250 bp long fragment corresponds to the WT sequence. ( G ) Antiviral assay using synZIKV-R2A viruses. VeroE6 cells were inoculated with a 1:10 dilution of a P0 stock and one hour later the medium was replaced with DMEM containing the indicated amount of 2′CMC. RLuc activity was measured in cell lysates 72 h post-infection. Mean ± SEM from two independent experiments is shown.

    Article Snippet: Viral RNA was isolated from cell lysates using the NucleoSpin RNA II kit (Machery-Nagel, Düren, Germany) and reverse transcribed using SuperScript III RT (Thermo Fisher Scientific Waltham, MA, USA). cDNA was amplified by PCR and amplicons were sequenced by Sanger sequencing (GATC Biotech, Constance, Germany) using primers spanning the complete ZIKV genome.

    Techniques: Immunofluorescence, Transfection, In Vitro, Staining, Electroporation, Activity Assay, Cell Culture, Infection, Plaque Assay, Isolation, Sequencing, Amplification, Random Hexamer Labeling, Polymerase Chain Reaction, Antiviral Assay

    Quantification of transcript amounts of jhp0832 in H. pylori strains J99, J99-mut and the J99 mutants with point mutations within the GCGC motifs. qPCR results are represented in the right panel, three different biological replicates were performed. Statistics: One-way ANOVA, ** P

    Journal: Nucleic Acids Research

    Article Title: The core genome m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in orchestrating gene expression in Helicobacter pylori

    doi: 10.1093/nar/gky1307

    Figure Lengend Snippet: Quantification of transcript amounts of jhp0832 in H. pylori strains J99, J99-mut and the J99 mutants with point mutations within the GCGC motifs. qPCR results are represented in the right panel, three different biological replicates were performed. Statistics: One-way ANOVA, ** P

    Article Snippet: Quantitative PCR (qPCR) One μg of RNA was used for cDNA synthesis using the SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Darmstadt, Germany) as described before ( ). qPCR was performed with gene specific primers ( ) and SYBR Green Master Mix (Qiagen, Hilden, Germany).

    Techniques: Real-time Polymerase Chain Reaction

    SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining mRNA. D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, RNA was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P

    Journal: The FASEB Journal

    Article Title: Bcr-Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic-driven mechanism of protein up-regulation

    doi: 10.1096/fj.201701016R

    Figure Lengend Snippet: SGMS1 is transcriptionally up-regulated in K562 cells. A , B ) SGMS1 ) were used to quantify hnRNA expression, normalized to β-actin and expressed as MNE. Primer locations are indicated on the SGMS1 locus (white boxes represent untranslated exons, black boxes represent translated exons, and lines represent introns). Negative control for cDNA synthesis (without superscript III) in K562 showed no amplification, thus verifying the absence of genomic DNA. Results from 3 independent experiments are shown. C ) K562 and HL-60 cells were treated with vehicle (H 2 O) or actinomycin D (5 μg/ml) over a 2-h time course. SGMS1 ) to quantify the percent of remaining mRNA. D ) K562 cells were treated with imatinib (1 μM) for 8 h. Cells were harvested and lysates were prepared for Western blot analysis. Total STAT5 and pSTAT5 levels were measured in control and treated cells. E ) In control and imatinib-treated cells, RNA was extracted to measure hnRNA levels of SGMS1. Intron VII and exon 8–specific RT-PCR primers were used. Results represent 3 independent experiments. ND, not detectable; SSIII, superscript III. * P

    Article Snippet: Dephosphorylated and uncapped mRNA was added to the GeneRacer RNA Oligo (provided in the kit) and incubated with T4 RNA ligase at 37°C for 1 h. The uncapped, full-length mRNA that was ligated to the GeneRacer RNA oligo (provided in the kit) was used for reverse transcription of mRNA into cDNA using Superscript III reverse transcriptase and random primers according to the standard protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Negative Control, Amplification, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Overexpression of wild-type or inactive ClpP proteins has varied effects on Chlamydia species. C. trachomatis serovar L2 was transformed with aTc-inducible shuttle vectors encoding either wild-type or active-site mutants of each ClpP paralog with a 6×His tag at the C terminus. HEp2 cells were infected with each transformant, and expression was induced at 10 h postinfection (hpi). (A) ClpP1 wild-type and S92A mutant overexpression assay. Overexpression of inactive ClpP1 has a negative impact on the bacteria. (B) ClpP2 wild-type and S98A mutant overexpression assay. Parameters were the same as those described above. Overexpression does not appear to negatively affect Chlamydia species. Samples were stained for major outer membrane protein (MOMP; green), 6×His-tagged ClpP protein of interest (red), and DNA (blue). Representative images of three independent experiments are presented. Scale bars are equal to 10 μm. Images were acquired on a Zeiss LSM 800 laser scanning confocal microscope with a 60× objective and a 3× digital magnification. (C) Inclusion-forming unit (IFU) assay measuring the effect of increasing levels of ClpP protein induction on chlamydial growth. Values are averages of the results from three independent experiments and are reported as a percentage of the respective uninduced sample. *, P

    Journal: Journal of Bacteriology

    Article Title: Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

    doi: 10.1128/JB.00635-18

    Figure Lengend Snippet: Overexpression of wild-type or inactive ClpP proteins has varied effects on Chlamydia species. C. trachomatis serovar L2 was transformed with aTc-inducible shuttle vectors encoding either wild-type or active-site mutants of each ClpP paralog with a 6×His tag at the C terminus. HEp2 cells were infected with each transformant, and expression was induced at 10 h postinfection (hpi). (A) ClpP1 wild-type and S92A mutant overexpression assay. Overexpression of inactive ClpP1 has a negative impact on the bacteria. (B) ClpP2 wild-type and S98A mutant overexpression assay. Parameters were the same as those described above. Overexpression does not appear to negatively affect Chlamydia species. Samples were stained for major outer membrane protein (MOMP; green), 6×His-tagged ClpP protein of interest (red), and DNA (blue). Representative images of three independent experiments are presented. Scale bars are equal to 10 μm. Images were acquired on a Zeiss LSM 800 laser scanning confocal microscope with a 60× objective and a 3× digital magnification. (C) Inclusion-forming unit (IFU) assay measuring the effect of increasing levels of ClpP protein induction on chlamydial growth. Values are averages of the results from three independent experiments and are reported as a percentage of the respective uninduced sample. *, P

    Article Snippet: DNA was removed from total RNA by rigorous DNA-free treatment (Thermo Fisher) before 1 µg was reverse transcribed with Superscript III reverse transcriptase (RT; Thermo Fisher).

    Techniques: Over Expression, Transformation Assay, Infection, Expressing, Mutagenesis, Staining, Microscopy