superscript iii kit  (Thermo Fisher)


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    Name:
    SuperScript Plus Direct cDNA Labeling Module no purification
    Description:
    The SuperScript Plus Direct cDNA Labeling System is a microarray labeling kit that combines the superior performance of SuperScript III Reverse Transcriptase RT with the simplicity of direct labeling In place of an enzyme spike in the streamlined protocol incorporates balanced sets of novel array optimized nucleotides conjugated to Alexa Fluor 555 and 647 dyes This results in more reproducible array data with higher correlation coefficients than using standalone reagents The SuperScript Plus Direct cDNA Labeling System provides • Optimized all inclusive direct labeling with balanced aha Alexa Fluor nucleotides enabling higher numbers of array positives• Improved correlation coefficients for more accurate data Figure 1 • Low elution purification columns included for ease of useIn the SuperScript Plus cDNA Labeling method anchored oligo dT anneals to the mRNA template Figure 2 SuperScript III RT extends from the priming site incorporating dUTP labeled with Alexa Fluor dye directly to produce labeled cDNA Template RNA is degraded by base hydrolysis Free nucleotides and other contaminants are removed using low elution volume purification columns before hybridization to the microarray
    Catalog Number:
    L101504
    Price:
    None
    Category:
    Kits and Assays
    Applications:
    DNA & RNA Purification & Analysis|DNA Labeling|Nucleic Acid Labeling & Oligo Synthesis|Gene Expression Analysis & Genotyping
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    Structured Review

    Thermo Fisher superscript iii kit
    Effect of CTCF shRNA on K562 cells. ( A ) RT–PCR analysis of globin gene transcription in K562 cells after transduction with control or CTCF shRNA. Results of <t>three</t> <t>RNA</t> preparations are shown ±SEM. ( B ) ChIP performed on chromatin from K562
    The SuperScript Plus Direct cDNA Labeling System is a microarray labeling kit that combines the superior performance of SuperScript III Reverse Transcriptase RT with the simplicity of direct labeling In place of an enzyme spike in the streamlined protocol incorporates balanced sets of novel array optimized nucleotides conjugated to Alexa Fluor 555 and 647 dyes This results in more reproducible array data with higher correlation coefficients than using standalone reagents The SuperScript Plus Direct cDNA Labeling System provides • Optimized all inclusive direct labeling with balanced aha Alexa Fluor nucleotides enabling higher numbers of array positives• Improved correlation coefficients for more accurate data Figure 1 • Low elution purification columns included for ease of useIn the SuperScript Plus cDNA Labeling method anchored oligo dT anneals to the mRNA template Figure 2 SuperScript III RT extends from the priming site incorporating dUTP labeled with Alexa Fluor dye directly to produce labeled cDNA Template RNA is degraded by base hydrolysis Free nucleotides and other contaminants are removed using low elution volume purification columns before hybridization to the microarray
    https://www.bioz.com/result/superscript iii kit/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii kit - by Bioz Stars, 2021-04
    99/100 stars

    Images

    1) Product Images from "Cell type specificity of chromatin organization mediated by CTCF and cohesin"

    Article Title: Cell type specificity of chromatin organization mediated by CTCF and cohesin

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0912087107

    Effect of CTCF shRNA on K562 cells. ( A ) RT–PCR analysis of globin gene transcription in K562 cells after transduction with control or CTCF shRNA. Results of three RNA preparations are shown ±SEM. ( B ) ChIP performed on chromatin from K562
    Figure Legend Snippet: Effect of CTCF shRNA on K562 cells. ( A ) RT–PCR analysis of globin gene transcription in K562 cells after transduction with control or CTCF shRNA. Results of three RNA preparations are shown ±SEM. ( B ) ChIP performed on chromatin from K562

    Techniques Used: shRNA, Reverse Transcription Polymerase Chain Reaction, Transduction, Chromatin Immunoprecipitation

    2) Product Images from "Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III"

    Article Title: Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030212

    Low α-Amanitin Concentrations Inhibit U6 snRNA Maxigene Expression HeLa cells were transiently transfected with 1 μg U6–1 , U6–8 , and U6–9 maxigenes carrying a 9 bp insertion and treated simultaneously with 50 nM α-amanitin oleate for 20 h or left untreated. Expression of U6 maxigenes, LDHA, 28S rRNA, 5S rRNA and pre-tRNA Tyr was measured by gene-specific reverse transcription, followed by conventional PCR and agarose gel electrophoresis (A) or qPCR (B). qPCR values were normalized to 28S rRNA and expression levels are expressed relative to untreated controls. Error bars represent the SEM. The data represents the average of at least three independent experiments.
    Figure Legend Snippet: Low α-Amanitin Concentrations Inhibit U6 snRNA Maxigene Expression HeLa cells were transiently transfected with 1 μg U6–1 , U6–8 , and U6–9 maxigenes carrying a 9 bp insertion and treated simultaneously with 50 nM α-amanitin oleate for 20 h or left untreated. Expression of U6 maxigenes, LDHA, 28S rRNA, 5S rRNA and pre-tRNA Tyr was measured by gene-specific reverse transcription, followed by conventional PCR and agarose gel electrophoresis (A) or qPCR (B). qPCR values were normalized to 28S rRNA and expression levels are expressed relative to untreated controls. Error bars represent the SEM. The data represents the average of at least three independent experiments.

    Techniques Used: Expressing, Transfection, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    Pol III Transcribes U6–1 Maxigene Primer extension analysis of RNA from HeLa cells cotransfected with GFP expression plasmid and U6–1 maxigene. (A) Design of the two U6–1 maxigenes with insertions at +66 and +87 bp (white boxes) to allow for maxigene-specific reverse transcription. Construct “U6–1 maxiT” harbors five thymidine residues directly downstream of the linker insertion; “U6–1 MaxiC” harbors the same primer binding site but lacks the T's. The cross-hatched box represents the reverse primer specific for the downstream insertion, yielding extension products of either 114 bp (U6–1 maxiT) or 109 bp (U6–1 maxiC). Grey filled box and black filled box represent reverse primers specific for upstream insertion with or without T residues, respectively, leading to extension products of 83 bp (U6–1 maxiT) or 79 bp (U6–1 maxiC). Primer extension of mRNA derived from cotransfected GFP plasmid yields a 158 bp product. Primer extension products were separated on a denaturing polyacrylamide gel and exposed on a PhosphorImager. (B) A representative gel is shown.
    Figure Legend Snippet: Pol III Transcribes U6–1 Maxigene Primer extension analysis of RNA from HeLa cells cotransfected with GFP expression plasmid and U6–1 maxigene. (A) Design of the two U6–1 maxigenes with insertions at +66 and +87 bp (white boxes) to allow for maxigene-specific reverse transcription. Construct “U6–1 maxiT” harbors five thymidine residues directly downstream of the linker insertion; “U6–1 MaxiC” harbors the same primer binding site but lacks the T's. The cross-hatched box represents the reverse primer specific for the downstream insertion, yielding extension products of either 114 bp (U6–1 maxiT) or 109 bp (U6–1 maxiC). Grey filled box and black filled box represent reverse primers specific for upstream insertion with or without T residues, respectively, leading to extension products of 83 bp (U6–1 maxiT) or 79 bp (U6–1 maxiC). Primer extension of mRNA derived from cotransfected GFP plasmid yields a 158 bp product. Primer extension products were separated on a denaturing polyacrylamide gel and exposed on a PhosphorImager. (B) A representative gel is shown.

    Techniques Used: Expressing, Plasmid Preparation, Construct, Binding Assay, Derivative Assay

    α-Amanitin Reduces Endogenous U6 snRNA Expression HeLa cells were transfected with a tRNA Arg maxigene and 16 h later treated with 10 μg/ml α-amanitin or left untreated. After 3 h, 250 μCi [ 32 P] orthophosphate was added to the medium for 6 h. Total RNA was collected and 5S rRNA along with tRNA Arg , U2 and U6 snRNAs were hybrid selected with biotinylated complementary oligos. Hybrid-selected RNA was separated on a denaturating polyacrylamide gel and exposed to PhosphorImager plates. The 5S rRNA bands served as a loading control. Three biological replicates were analyzed and a representative gel is shown.
    Figure Legend Snippet: α-Amanitin Reduces Endogenous U6 snRNA Expression HeLa cells were transfected with a tRNA Arg maxigene and 16 h later treated with 10 μg/ml α-amanitin or left untreated. After 3 h, 250 μCi [ 32 P] orthophosphate was added to the medium for 6 h. Total RNA was collected and 5S rRNA along with tRNA Arg , U2 and U6 snRNAs were hybrid selected with biotinylated complementary oligos. Hybrid-selected RNA was separated on a denaturating polyacrylamide gel and exposed to PhosphorImager plates. The 5S rRNA bands served as a loading control. Three biological replicates were analyzed and a representative gel is shown.

    Techniques Used: Expressing, Transfection

    3) Product Images from "The RNA-binding protein HuR contributes to neuroinflammation by promoting C-C chemokine receptor 6 (CCR6) expression on Th17 cells"

    Article Title: The RNA-binding protein HuR contributes to neuroinflammation by promoting C-C chemokine receptor 6 (CCR6) expression on Th17 cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.782771

    HuR binds to and stabilizes Ccr6 mRNA. Naive CD4 + T cells were isolated from spleens of WT mice and stimulated under Th17 cell–polarizing conditions. A , RIP analysis was performed to detect Ccr6 mRNA enrichment after IP using anti-HuR (3A2) or isotype-matched antibody (IgG1) from Th17 cell cytoplasmic extracts. Il23r mRNA was assayed as negative control. B , WT and HuR KO Th17 polarized cells were either left untreated or treated with actinomycin D ( Act D ; 3 μg/ml) and harvested 1, 3, and 5 h later. Ccr6 mRNA levels were determined by qRT-PCR analysis. C , WT and HuR KO Th17 cell lysates were size-fractionated through sucrose gradients, and RNA was isolated from each of 12 fractions, reverse-transcribed, and measured by qRT-PCR analysis. The relative distribution of Ccr6 mRNA along the gradient was calculated and plotted as a percentage of the total Ccr6 mRNA in the gradient. D , the distribution of actin mRNA, encoding a housekeeping protein, was included as a negative control. E , overexpression of HuR by HuR plasmid (0.2 μg) transfection increased the luciferase activity of vector containing Ccr6 mRNA ORF and 3′-UTR (0.2 μg) in HeLa cells in comparison with empty vector control groups. One of three independent experiments is shown in A–D . Data in E represent one of two individual experiments. **, p
    Figure Legend Snippet: HuR binds to and stabilizes Ccr6 mRNA. Naive CD4 + T cells were isolated from spleens of WT mice and stimulated under Th17 cell–polarizing conditions. A , RIP analysis was performed to detect Ccr6 mRNA enrichment after IP using anti-HuR (3A2) or isotype-matched antibody (IgG1) from Th17 cell cytoplasmic extracts. Il23r mRNA was assayed as negative control. B , WT and HuR KO Th17 polarized cells were either left untreated or treated with actinomycin D ( Act D ; 3 μg/ml) and harvested 1, 3, and 5 h later. Ccr6 mRNA levels were determined by qRT-PCR analysis. C , WT and HuR KO Th17 cell lysates were size-fractionated through sucrose gradients, and RNA was isolated from each of 12 fractions, reverse-transcribed, and measured by qRT-PCR analysis. The relative distribution of Ccr6 mRNA along the gradient was calculated and plotted as a percentage of the total Ccr6 mRNA in the gradient. D , the distribution of actin mRNA, encoding a housekeeping protein, was included as a negative control. E , overexpression of HuR by HuR plasmid (0.2 μg) transfection increased the luciferase activity of vector containing Ccr6 mRNA ORF and 3′-UTR (0.2 μg) in HeLa cells in comparison with empty vector control groups. One of three independent experiments is shown in A–D . Data in E represent one of two individual experiments. **, p

    Techniques Used: Isolation, Mouse Assay, Negative Control, Activated Clotting Time Assay, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Transfection, Luciferase, Activity Assay

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    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing
    Article Snippet: RLM-RT-PCR analysis and northern blotting The mapping of the 5′ ends of specific transcripts was done using RNA ligase-mediated RT-PCR as described previously (Kime et al ., ). .. Unless otherwise stated, cDNA was synthesized using SuperScript® RT III (Invitrogen) with random hexamers (100 nM) and 200 ng of RNA template according to the instruction of the vendor of the reverse transcriptase. .. The PCR reaction was carried out using GoTaq® DNA polymerase (Promega) according to the vendor's instruction using cDNA diluted with RNase-free water as the template.

    Article Title: Translational repression of pre-formed cytokine-encoding mRNA prevents chronic activation of memory T cells
    Article Snippet: Representative flow cytometry gating strategies are found in Fig S9. .. Total RNA was extracted using Trizol (Invitrogen) and cDNA was synthesized with SuperScript III (Invitrogen), unless differently specified. .. MiR were extracted from FACS-sorted CD8+ CD44hi WT and IFN-γ-ARE-Del OTI T cells using Total RNA Purification Plus Kit (Norgen Biotek), and cDNA was synthesized with miScript II RT kit (Qiagen).

    Random Hexamer Labeling:

    Article Title: Trans-Synaptic Spread of Tau Pathology In Vivo
    Article Snippet: RNA extracted from LCM isolated cells was analyzed using a 2100 Bioanalyzer (Agilent, Technologies, Santa Clara, CA). .. 2.5 ng of RNA was employed for first strand cDNA synthesis, in a 20 µl reaction volume using a 1∶1 ratio of random hexamer and oligo-dT primers and Superscript® III RT enzyme (Life Technologies Inc., Carlsbad, CA). cDNA products were diluted with 70 µl RNAse free water, prior to quantitative PCR of the human tau transgene and the housekeeping gene β-actin. .. PCRs were performed by monitoring in real time the increase in fluorescence of the SYBR Green dye, using a Bio-Rad iQ5 detector system (Bio-Rad, Hercules, CA).

    Real-time Polymerase Chain Reaction:

    Article Title: Trans-Synaptic Spread of Tau Pathology In Vivo
    Article Snippet: RNA extracted from LCM isolated cells was analyzed using a 2100 Bioanalyzer (Agilent, Technologies, Santa Clara, CA). .. 2.5 ng of RNA was employed for first strand cDNA synthesis, in a 20 µl reaction volume using a 1∶1 ratio of random hexamer and oligo-dT primers and Superscript® III RT enzyme (Life Technologies Inc., Carlsbad, CA). cDNA products were diluted with 70 µl RNAse free water, prior to quantitative PCR of the human tau transgene and the housekeeping gene β-actin. .. PCRs were performed by monitoring in real time the increase in fluorescence of the SYBR Green dye, using a Bio-Rad iQ5 detector system (Bio-Rad, Hercules, CA).

    Article Title: Premature Senescence and Increased TGF? Signaling in the Absence of Tgif1
    Article Snippet: DNA and RNA analysis Genomic DNA for genotype analysis was purified from ear punch (at P21) and genotype was determined by PCR, as previously described. .. RNA was isolated and purified using Absolutely RNA kit (Stratagene). cDNA was generated using Superscript III (Invitrogen), and analyzed in triplicate by real time PCR using a BioRad MyIQ cycler and Sensimix Plus SYBRgreen plus FITC mix (Bioline), with intron spanning primer pairs , selected using Primer3 ( http://frodo.wi.mit.edu/ ). .. Expression was normalized to Rpl4 and Actin using the delta Ct method, and is shown as mean plus standard deviation of triplicates.

    Article Title: Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection
    Article Snippet: Assay for infectivity of cell culture grown HCV To quantify virus infectivity in cells expressing ISGs, IFI6 or IFI27, a predetermined titer (moi of ~1) of HCVcc (clone JFH1) were incubated with cells for 5 hours at 37°C. .. Cells were rinsed, lysed, and RNA was isolated using the RNAeasy Miniprep kit (Qiagen). cDNA was generated using a Superscript III kit (Invitrogen), followed by real-time PCR to quantify viral RNA titer . ..

    Article Title: Role of the CCAAT-Binding Protein NFY in SCA17 Pathogenesis
    Article Snippet: The RNA was DNase treated, quantified, and reverse-transcribed to cDNA using the SuperScript™ III reverse transcriptase (Invitrogen). .. Using ABI StepOne™ Real-Time PCR System (Applied Biosystems), real-time quantitative PCR was performed on a cDNA amount equivalent to 12.5 ng total RNA with TaqMan fluorogenic probes Hs99999174_ml for HSPA5 and 4326321E for HPRT1 (endogenous control; Applied Biosystems). ..

    Isolation:

    Article Title: Premature Senescence and Increased TGF? Signaling in the Absence of Tgif1
    Article Snippet: DNA and RNA analysis Genomic DNA for genotype analysis was purified from ear punch (at P21) and genotype was determined by PCR, as previously described. .. RNA was isolated and purified using Absolutely RNA kit (Stratagene). cDNA was generated using Superscript III (Invitrogen), and analyzed in triplicate by real time PCR using a BioRad MyIQ cycler and Sensimix Plus SYBRgreen plus FITC mix (Bioline), with intron spanning primer pairs , selected using Primer3 ( http://frodo.wi.mit.edu/ ). .. Expression was normalized to Rpl4 and Actin using the delta Ct method, and is shown as mean plus standard deviation of triplicates.

    Article Title: Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection
    Article Snippet: Assay for infectivity of cell culture grown HCV To quantify virus infectivity in cells expressing ISGs, IFI6 or IFI27, a predetermined titer (moi of ~1) of HCVcc (clone JFH1) were incubated with cells for 5 hours at 37°C. .. Cells were rinsed, lysed, and RNA was isolated using the RNAeasy Miniprep kit (Qiagen). cDNA was generated using a Superscript III kit (Invitrogen), followed by real-time PCR to quantify viral RNA titer . ..

    Purification:

    Article Title: Premature Senescence and Increased TGF? Signaling in the Absence of Tgif1
    Article Snippet: DNA and RNA analysis Genomic DNA for genotype analysis was purified from ear punch (at P21) and genotype was determined by PCR, as previously described. .. RNA was isolated and purified using Absolutely RNA kit (Stratagene). cDNA was generated using Superscript III (Invitrogen), and analyzed in triplicate by real time PCR using a BioRad MyIQ cycler and Sensimix Plus SYBRgreen plus FITC mix (Bioline), with intron spanning primer pairs , selected using Primer3 ( http://frodo.wi.mit.edu/ ). .. Expression was normalized to Rpl4 and Actin using the delta Ct method, and is shown as mean plus standard deviation of triplicates.

    Generated:

    Article Title: Premature Senescence and Increased TGF? Signaling in the Absence of Tgif1
    Article Snippet: DNA and RNA analysis Genomic DNA for genotype analysis was purified from ear punch (at P21) and genotype was determined by PCR, as previously described. .. RNA was isolated and purified using Absolutely RNA kit (Stratagene). cDNA was generated using Superscript III (Invitrogen), and analyzed in triplicate by real time PCR using a BioRad MyIQ cycler and Sensimix Plus SYBRgreen plus FITC mix (Bioline), with intron spanning primer pairs , selected using Primer3 ( http://frodo.wi.mit.edu/ ). .. Expression was normalized to Rpl4 and Actin using the delta Ct method, and is shown as mean plus standard deviation of triplicates.

    Article Title: Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection
    Article Snippet: Assay for infectivity of cell culture grown HCV To quantify virus infectivity in cells expressing ISGs, IFI6 or IFI27, a predetermined titer (moi of ~1) of HCVcc (clone JFH1) were incubated with cells for 5 hours at 37°C. .. Cells were rinsed, lysed, and RNA was isolated using the RNAeasy Miniprep kit (Qiagen). cDNA was generated using a Superscript III kit (Invitrogen), followed by real-time PCR to quantify viral RNA titer . ..

    Expressing:

    Article Title: TroA of Streptococcus suis Is Required for Manganese Acquisition and Full Virulence ▿
    Article Snippet: RNA quantity and quality were checked with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA) and a Bioanalyzer system (Agilent, Amstelveen, The Netherlands). .. Fifty ng of RNA (RNA integrity number [RIN] of > 7) was used to prepare cDNA using random hexamers (Promega) and Superscript III (Invitrogen) according the manufacturers' instructions. troA expression levels were subsequently measured using troA -specific primers ( ). .. In the PCR, 20-times-diluted cDNA was added to 1× Power SYBR green master mix (Applied Biosystems, Nieuwe Kerk aan de IJssel, The Netherlands) containing 0.625 μM (each) forward and reverse primer.

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  • 97
    Thermo Fisher superscript iii first strand synthesis kit
    Effect of perilipin 2 siRNA on lipolysis product-induced perilipin 2 expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media control or lipolysis products for 3 hours. A. Perilipin 2 siRNA reduced perilipin 2 gene expression in THP-1 cells treated with media and lipolysis products. Gene expression was measured by <t>qRT-PCR,</t> and normalized to GAPDH expression. Fold change was determined relative to the scramble siRNA media control group, n=9 separate samples per treatment group obtained from <t>three</t> experimental runs with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. B. Perilipin 2 siRNA reduced lipolysis product-induced perilipin 2 protein expression. i. Representative image of western blot. ii. Densitometry analysis of western blot, protein expression determined relative to β-actin, n=3 separate samples per treatment group obtained from one experimental run with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. All data shown is mean +/− standard deviation. Statistical differences are indicated by **(p
    Superscript Iii First Strand Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii first strand synthesis kit/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis kit - by Bioz Stars, 2021-04
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    99
    Thermo Fisher superscript iii platinum two step qrt pcr kit
    Up-regulation of Rrp2 in B. burgdorferi induces the expression of rpoS . Gene expression in OY159 was analyzed by SDS-PAGE (A), immunoblot (B, C), and <t>qRT-PCR</t> analyses (D). In (A) and (B), spirochetes grown in BSK-II media containing varying concentrations of IPTG were harvested when bacterial growth reached early stationary phase (∼10 8 cells per ml). In (C) and (D), spirochetes were grown in BSK-II medium. When bacterial growth reached mid-log phase (∼10 7 cells per ml), various amounts of IPTG were added into culture. Cells were collected at 9 h post-induction. In (A) and (C), concentrations of IPTG are indicated above the image. The arrow indicates OspC in (A). Specific antibodies, denoted as α- used in the immunoblot (B, C), are indicated on the left. In (D), data were collected from <t>three</t> independent experiments, and the bars represent the mean measurements ± standard deviation. The mean values between induced groups (100-, 200-, or 500 µM IPTG) and the uninduced group (0 µM IPTG) were compared using the Student’s t test and are significantly different (p
    Superscript Iii Platinum Two Step Qrt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii platinum two step qrt pcr kit/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii platinum two step qrt pcr kit - by Bioz Stars, 2021-04
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    Effect of perilipin 2 siRNA on lipolysis product-induced perilipin 2 expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media control or lipolysis products for 3 hours. A. Perilipin 2 siRNA reduced perilipin 2 gene expression in THP-1 cells treated with media and lipolysis products. Gene expression was measured by qRT-PCR, and normalized to GAPDH expression. Fold change was determined relative to the scramble siRNA media control group, n=9 separate samples per treatment group obtained from three experimental runs with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. B. Perilipin 2 siRNA reduced lipolysis product-induced perilipin 2 protein expression. i. Representative image of western blot. ii. Densitometry analysis of western blot, protein expression determined relative to β-actin, n=3 separate samples per treatment group obtained from one experimental run with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. All data shown is mean +/− standard deviation. Statistical differences are indicated by **(p

    Journal: Food & function

    Article Title: Inhibition of perilipin 2 expression reduces pro-inflammatory gene expression and increases lipid droplet size

    doi: 10.1039/c8fo01420e

    Figure Lengend Snippet: Effect of perilipin 2 siRNA on lipolysis product-induced perilipin 2 expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media control or lipolysis products for 3 hours. A. Perilipin 2 siRNA reduced perilipin 2 gene expression in THP-1 cells treated with media and lipolysis products. Gene expression was measured by qRT-PCR, and normalized to GAPDH expression. Fold change was determined relative to the scramble siRNA media control group, n=9 separate samples per treatment group obtained from three experimental runs with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. B. Perilipin 2 siRNA reduced lipolysis product-induced perilipin 2 protein expression. i. Representative image of western blot. ii. Densitometry analysis of western blot, protein expression determined relative to β-actin, n=3 separate samples per treatment group obtained from one experimental run with triplicate cultures. Analysis by two-way ANOVA indicated an interaction of the two factors. All data shown is mean +/− standard deviation. Statistical differences are indicated by **(p

    Article Snippet: RNA from each sample was reverse transcribed using Superscript III First Strand Synthesis Kit (Life Technologies) according to manufacturer’s instructions. qRT-PCR was performed with FastStart Universal SYBR Green Master (Roche) to quantify the gene expression.

    Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Standard Deviation

    Effects of perilipin 2 siRNA on TGRL lipolysis product-induced gene expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media or TGRL lipolysis products for 3 hours. Gene expression for A. SGK1, B. MAFF, C. IL-8, and D. CCL3. Gene expression was analyzed by qRT-PCR, normalized to GAPDH. Fold change is relative to scramble siRNA media control group. All data shown is mean +/− standard deviation, n=9 separate samples per treatment group obtained from three experimental runs with triplicate cultures. For each gene, analysis by two-way ANOVA indicated an interaction of the two factors. Tukey’s post hoc test was used to determine differences between groups. Statistical differences are indicated by **(p

    Journal: Food & function

    Article Title: Inhibition of perilipin 2 expression reduces pro-inflammatory gene expression and increases lipid droplet size

    doi: 10.1039/c8fo01420e

    Figure Lengend Snippet: Effects of perilipin 2 siRNA on TGRL lipolysis product-induced gene expression. THP-1 monocytes were pre-incubated with perilipin 2 siRNA or scramble siRNA prior to incubation with media or TGRL lipolysis products for 3 hours. Gene expression for A. SGK1, B. MAFF, C. IL-8, and D. CCL3. Gene expression was analyzed by qRT-PCR, normalized to GAPDH. Fold change is relative to scramble siRNA media control group. All data shown is mean +/− standard deviation, n=9 separate samples per treatment group obtained from three experimental runs with triplicate cultures. For each gene, analysis by two-way ANOVA indicated an interaction of the two factors. Tukey’s post hoc test was used to determine differences between groups. Statistical differences are indicated by **(p

    Article Snippet: RNA from each sample was reverse transcribed using Superscript III First Strand Synthesis Kit (Life Technologies) according to manufacturer’s instructions. qRT-PCR was performed with FastStart Universal SYBR Green Master (Roche) to quantify the gene expression.

    Techniques: Expressing, Incubation, Quantitative RT-PCR, Standard Deviation

    a Schematic representation of HIV-1 Gag protein domains. The four major domains of Gag (MA, CA, p7, and p6) are depicted including the two linker sequences p1 and p2. HIV-1 Gag interacts with the ESCRT complex proteins Tsg101 and Alix to regulate viral budding. The sequence of subtype B NL4-3 gag p6 is presented and the sequence motifs PTAP and YPXnL, which serve as the binding motif for Tsg101 and Alix, respectively, are highlighted using the square boxes. Con_C represents the subtype C gag p6 consensus amino acid sequence. The dashes represent sequence identity and the dots sequence deletion. b A comparative analysis of the PTAP sequence duplication in subtypes B and C. In subtype B, a partial PTAP duplication consisting of three amino-acid residues (APP) is common. In contrast in subtype C, a sequence duplication of 14 amino acids is common. A 14 amino acid sequence duplication of subtype C derived from the primary clinical isolate T004 is presented. The amino acid sequences, the original and the duplicated sequences, in Gag and Gag-Pol are depicted. The arrows represent the length of sequence duplication and the direction of polymerization by the reverse transcriptase while synthesizing the cDNA from the viral RNA. The core PTAP motifs are highlighted using the square boxes. The sequences flanking the 3 or 14 aa residues are shown in gray

    Journal: BMC Infectious Diseases

    Article Title: The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa

    doi: 10.1186/s12879-017-2184-4

    Figure Lengend Snippet: a Schematic representation of HIV-1 Gag protein domains. The four major domains of Gag (MA, CA, p7, and p6) are depicted including the two linker sequences p1 and p2. HIV-1 Gag interacts with the ESCRT complex proteins Tsg101 and Alix to regulate viral budding. The sequence of subtype B NL4-3 gag p6 is presented and the sequence motifs PTAP and YPXnL, which serve as the binding motif for Tsg101 and Alix, respectively, are highlighted using the square boxes. Con_C represents the subtype C gag p6 consensus amino acid sequence. The dashes represent sequence identity and the dots sequence deletion. b A comparative analysis of the PTAP sequence duplication in subtypes B and C. In subtype B, a partial PTAP duplication consisting of three amino-acid residues (APP) is common. In contrast in subtype C, a sequence duplication of 14 amino acids is common. A 14 amino acid sequence duplication of subtype C derived from the primary clinical isolate T004 is presented. The amino acid sequences, the original and the duplicated sequences, in Gag and Gag-Pol are depicted. The arrows represent the length of sequence duplication and the direction of polymerization by the reverse transcriptase while synthesizing the cDNA from the viral RNA. The core PTAP motifs are highlighted using the square boxes. The sequences flanking the 3 or 14 aa residues are shown in gray

    Article Snippet: The complementary DNA (cDNA) was synthesized using random hexamers and a commercial kit (SuperScript® III Reverse Transcriptase, Cat. No: 18080–051, Invitrogen, Carlsbad, California, USA).

    Techniques: Sequencing, Binding Assay, Derivative Assay

    amn expression in the MB. A , amn X8 flies express GAL4 in the MB α/β neurons. Dissected brains were used for immunostaining (green, mCD8::GFP). B , Schematic representation of the amn transcript with the localization of a MB enhancer sequence and of DPM enhancer-sensitive sequence. C , D , qPCR analyses of amn expression levels. Total RNA extracted from female heads were reverse-transcribed and further quantified by PCR. Expression relative to reference is expressed as a ratio (2 −Δ C p , where C p is the crossing point). C , 238Y/amn-RNAi1 and 238Y/amn-RNAi2 show 33% and 50% decrease in amn mRNA compared with the 238Y /+ control. Each bar corresponds to six measures from three independent experiments ( F (2,17) = 13.16, *** p = 0.0005, n = 6). D , VT30559/amn-RNAi1 and VT30559/amn-RNAi2 show 18% and 30% decrease in amn mRNA compared with the VT30559 /+ control. ( F (2,23) = 8.522, ** p = 0.002, n = 8). Each bar corresponds to eight measures from four independent experiments. Error bars indicate SEM.

    Journal: The Journal of Neuroscience

    Article Title: Amnesiac Is Required in the Adult Mushroom Body for Memory Formation

    doi: 10.1523/JNEUROSCI.0876-18.2018

    Figure Lengend Snippet: amn expression in the MB. A , amn X8 flies express GAL4 in the MB α/β neurons. Dissected brains were used for immunostaining (green, mCD8::GFP). B , Schematic representation of the amn transcript with the localization of a MB enhancer sequence and of DPM enhancer-sensitive sequence. C , D , qPCR analyses of amn expression levels. Total RNA extracted from female heads were reverse-transcribed and further quantified by PCR. Expression relative to reference is expressed as a ratio (2 −Δ C p , where C p is the crossing point). C , 238Y/amn-RNAi1 and 238Y/amn-RNAi2 show 33% and 50% decrease in amn mRNA compared with the 238Y /+ control. Each bar corresponds to six measures from three independent experiments ( F (2,17) = 13.16, *** p = 0.0005, n = 6). D , VT30559/amn-RNAi1 and VT30559/amn-RNAi2 show 18% and 30% decrease in amn mRNA compared with the VT30559 /+ control. ( F (2,23) = 8.522, ** p = 0.002, n = 8). Each bar corresponds to eight measures from four independent experiments. Error bars indicate SEM.

    Article Snippet: Samples were cleaned with the RNA Minielute Cleanup kit (Qiagen), and reverse-transcribed with oligo(dT)20 primers using the SuperScript III First-Strand kit (Life Technologies) according to the manufacturer's instructions.

    Techniques: Expressing, Immunostaining, Sequencing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Up-regulation of Rrp2 in B. burgdorferi induces the expression of rpoS . Gene expression in OY159 was analyzed by SDS-PAGE (A), immunoblot (B, C), and qRT-PCR analyses (D). In (A) and (B), spirochetes grown in BSK-II media containing varying concentrations of IPTG were harvested when bacterial growth reached early stationary phase (∼10 8 cells per ml). In (C) and (D), spirochetes were grown in BSK-II medium. When bacterial growth reached mid-log phase (∼10 7 cells per ml), various amounts of IPTG were added into culture. Cells were collected at 9 h post-induction. In (A) and (C), concentrations of IPTG are indicated above the image. The arrow indicates OspC in (A). Specific antibodies, denoted as α- used in the immunoblot (B, C), are indicated on the left. In (D), data were collected from three independent experiments, and the bars represent the mean measurements ± standard deviation. The mean values between induced groups (100-, 200-, or 500 µM IPTG) and the uninduced group (0 µM IPTG) were compared using the Student’s t test and are significantly different (p

    Journal: PLoS ONE

    Article Title: Synthesis of RpoS Is Dependent on a Putative Enhancer Binding Protein Rrp2 in Borrelia burgdorferi

    doi: 10.1371/journal.pone.0096917

    Figure Lengend Snippet: Up-regulation of Rrp2 in B. burgdorferi induces the expression of rpoS . Gene expression in OY159 was analyzed by SDS-PAGE (A), immunoblot (B, C), and qRT-PCR analyses (D). In (A) and (B), spirochetes grown in BSK-II media containing varying concentrations of IPTG were harvested when bacterial growth reached early stationary phase (∼10 8 cells per ml). In (C) and (D), spirochetes were grown in BSK-II medium. When bacterial growth reached mid-log phase (∼10 7 cells per ml), various amounts of IPTG were added into culture. Cells were collected at 9 h post-induction. In (A) and (C), concentrations of IPTG are indicated above the image. The arrow indicates OspC in (A). Specific antibodies, denoted as α- used in the immunoblot (B, C), are indicated on the left. In (D), data were collected from three independent experiments, and the bars represent the mean measurements ± standard deviation. The mean values between induced groups (100-, 200-, or 500 µM IPTG) and the uninduced group (0 µM IPTG) were compared using the Student’s t test and are significantly different (p

    Article Snippet: After RNA was further purified using RNeasy Mini Kit (Qiagen), 1 µg of RNA was used to synthesize cDNA using the SuperScript III Platinum Two-step qRT-PCR kit according to the manufacturer’s protocol (Invitrogen). qRT-PCR was employed to examine gene expression, using the relative quantification method (ΔΔC T) as described – , .

    Techniques: Expressing, SDS Page, Quantitative RT-PCR, Standard Deviation

    Gene expression in B. burgdorferi strain OY173. (A) Construction of an IPTG-inducible rrp2 -FLAG expression shuttle plasmid. The plasmid pRrp2-FLAG pRrp2 was introduced into strain OY01 ( rrp2 [G239C]), yielding OY173. SDS-PAGE (B), immunoblot (C, D), and qRT-PCR analyses (E) were performed to analyze gene expression. In (B) and (C), spirochetes grown in BSK-II medium containing varying concentrations of IPTG were harvested when bacterial growth reached early stationary phase (∼10 8 cells per ml). In (D) and (E), spirochetes were grown in BSK-II medium. When bacterial growth reached mid-log phase (∼10 7 cells per ml), various amounts of IPTG were added into culture. Cells were collected at 9 h post-induction. In (B) and (D), concentrations of IPTG are indicated above the image. The arrow indicates OspC in (B). Specific antibodies, denoted as α- used in the immunoblot (C, D), are indicated on the left. In (E), the bars represent the mean measurements ± standard deviation. The mean values between induced groups (100-, 200-, or 500 µM IPTG) and the uninduced group (0 µM IPTG) were compared using the Student’s t test and are significantly different (p

    Journal: PLoS ONE

    Article Title: Synthesis of RpoS Is Dependent on a Putative Enhancer Binding Protein Rrp2 in Borrelia burgdorferi

    doi: 10.1371/journal.pone.0096917

    Figure Lengend Snippet: Gene expression in B. burgdorferi strain OY173. (A) Construction of an IPTG-inducible rrp2 -FLAG expression shuttle plasmid. The plasmid pRrp2-FLAG pRrp2 was introduced into strain OY01 ( rrp2 [G239C]), yielding OY173. SDS-PAGE (B), immunoblot (C, D), and qRT-PCR analyses (E) were performed to analyze gene expression. In (B) and (C), spirochetes grown in BSK-II medium containing varying concentrations of IPTG were harvested when bacterial growth reached early stationary phase (∼10 8 cells per ml). In (D) and (E), spirochetes were grown in BSK-II medium. When bacterial growth reached mid-log phase (∼10 7 cells per ml), various amounts of IPTG were added into culture. Cells were collected at 9 h post-induction. In (B) and (D), concentrations of IPTG are indicated above the image. The arrow indicates OspC in (B). Specific antibodies, denoted as α- used in the immunoblot (C, D), are indicated on the left. In (E), the bars represent the mean measurements ± standard deviation. The mean values between induced groups (100-, 200-, or 500 µM IPTG) and the uninduced group (0 µM IPTG) were compared using the Student’s t test and are significantly different (p

    Article Snippet: After RNA was further purified using RNeasy Mini Kit (Qiagen), 1 µg of RNA was used to synthesize cDNA using the SuperScript III Platinum Two-step qRT-PCR kit according to the manufacturer’s protocol (Invitrogen). qRT-PCR was employed to examine gene expression, using the relative quantification method (ΔΔC T) as described – , .

    Techniques: Expressing, Plasmid Preparation, SDS Page, Quantitative RT-PCR, Standard Deviation

    Generation of an rrp2 conditional lethal mutant in B. burgdorferi . (A) Schematic representation of the bb0764 – bb0761 genes in the B. burgdorferi chromosome and the insertion of PflgB-kan cassette into rrp2 by homologous recombination. Arrows indicate the approximate positions of the oligonucleotide primers used for subsequent analyses. (B) Analyses of the wild-type 297 and the rrp2 conditional lethal mutant OY179 by PCR. The specific primer pairs are indicated on the right. Lanes WT, 297; lanes M1, M2, and M3, three clones of OY179.

    Journal: PLoS ONE

    Article Title: Synthesis of RpoS Is Dependent on a Putative Enhancer Binding Protein Rrp2 in Borrelia burgdorferi

    doi: 10.1371/journal.pone.0096917

    Figure Lengend Snippet: Generation of an rrp2 conditional lethal mutant in B. burgdorferi . (A) Schematic representation of the bb0764 – bb0761 genes in the B. burgdorferi chromosome and the insertion of PflgB-kan cassette into rrp2 by homologous recombination. Arrows indicate the approximate positions of the oligonucleotide primers used for subsequent analyses. (B) Analyses of the wild-type 297 and the rrp2 conditional lethal mutant OY179 by PCR. The specific primer pairs are indicated on the right. Lanes WT, 297; lanes M1, M2, and M3, three clones of OY179.

    Article Snippet: After RNA was further purified using RNeasy Mini Kit (Qiagen), 1 µg of RNA was used to synthesize cDNA using the SuperScript III Platinum Two-step qRT-PCR kit according to the manufacturer’s protocol (Invitrogen). qRT-PCR was employed to examine gene expression, using the relative quantification method (ΔΔC T) as described – , .

    Techniques: Mutagenesis, Homologous Recombination, Polymerase Chain Reaction, Clone Assay