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    Structured Review

    Thermo Fisher superscript iii reverse transcriptase enzyme
    Biophysical and cistromic characteristics of the Class1 FOXA1 mutants. a) Distribution of class1 mutations on the protein map of FOXA1. b) 3D-structure of FKHD (FOXA3) with visualization of all mutated residues collectively identified as the 3D-mutational hotspot in FOXA1 across cancers. c) <t>DNA-bound</t> 3D structure of FKHD with visualization of all residues shown through crystallography to make direct base-specific contacts with the DNA in FOXA2 and FOXA3 proteins. FKHD; Forkhead domain. d) Representative fluorescent images of nuclei expressing different FOXA1 variants fused to GFP at the C-termini. e, f) FRAP recovery kinetic plots (left) and representative time-lapse images (right) from pre-bleaching (‘Pre’) to 100% recovery (red timestamps) for (e) wing2-altered class1 and (f) truncated class2 mutants (i.e. A287fs and P375fs), respectively (n=6 nuclei/variants; quantified in Fig 2d ). White lines indicate the border between bleached and unbleached areas. g) Representative FRAP fluorescence recovery kinetics in the bleached area for indicated FOXA1 variants. t½ line indicates the time to 50% recovery. Colored dots show raw data; superimposed solid curves show a hyperbolic fit with 95% confidence intervals. h) SPT quantification of chromatin bound (slow and fast) and unbound (freely diffusing) particles of WT and class1 FOXA1 variants, and average chromatin dwell times (mean ± s.d.) for the bound fractions (n≥500 particles/variant). i) Diffusion constant histograms of single particles of WT or distinct class1 FOXA1 mutants. Particles were categorized into chromatin bound (slow and fast) or unbound fractions using cutoffs marked by dashed lines (n≥500 particles/variant imaged in 3–5 distinct nuclei). j) Left, mRNA expression (qPCR) of labeled FOXA1 variants in stable, isogenic HEK293 cells (n=3 technical replicates). Right, overlaps between FOXA1 WT and class1 mutant cistromes from these cells (n=2 biological replicates). k) Top de novo motifs identified from the <t>three</t> FOXA1 cistromes from HEK293 cells (HOMER: hypergeometric test). l) mRNA expression (qPCR) of labeled FOXA1 variants in stable, isogenic 22RV1 cells (n=3 technical replicates). For j and l , centers show mean values and lines mark s.e.m. m) Overlap between WT (n=2 biological replicates) and class1 (n=4 biological replicates) cistromes from stable 22RV1 overexpression models. n) Overlap between the FOXA1 WT and AR union cistromes generated from 22RV1 cells overexpressing WT (n=2 biological replicates) or class1 mutant (I176M or R216G; n=2 biological replicates each) FOXA1 variants. o) De novo motif results for the WT or class1 mutant FOXA1 binding sites from PCa cells (HOMER: hypergeometric test). p) Percent of WT or class1 binding sites with perfect match to the core FOXA1 motif (5’-T[G/A]TT[T/G]AC-3’) and q) the consensus FOXA1 motifs identified from these sites. r) Left, Percent of WT or class1 binding sites harboring known motifs of the labelled FOXA1 or AR cofactors; Right, Enrichment of the cofactor motifs in the two cistromes relative to the background (n=top 5000 peaks by score/variant, see Methods ). s) Genomic distribution of WT and class1 binding sites in PCa cells.
    Superscript Iii Reverse Transcriptase Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer"

    Article Title: Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer

    Journal: Nature

    doi: 10.1038/s41586-019-1347-4

    Biophysical and cistromic characteristics of the Class1 FOXA1 mutants. a) Distribution of class1 mutations on the protein map of FOXA1. b) 3D-structure of FKHD (FOXA3) with visualization of all mutated residues collectively identified as the 3D-mutational hotspot in FOXA1 across cancers. c) DNA-bound 3D structure of FKHD with visualization of all residues shown through crystallography to make direct base-specific contacts with the DNA in FOXA2 and FOXA3 proteins. FKHD; Forkhead domain. d) Representative fluorescent images of nuclei expressing different FOXA1 variants fused to GFP at the C-termini. e, f) FRAP recovery kinetic plots (left) and representative time-lapse images (right) from pre-bleaching (‘Pre’) to 100% recovery (red timestamps) for (e) wing2-altered class1 and (f) truncated class2 mutants (i.e. A287fs and P375fs), respectively (n=6 nuclei/variants; quantified in Fig 2d ). White lines indicate the border between bleached and unbleached areas. g) Representative FRAP fluorescence recovery kinetics in the bleached area for indicated FOXA1 variants. t½ line indicates the time to 50% recovery. Colored dots show raw data; superimposed solid curves show a hyperbolic fit with 95% confidence intervals. h) SPT quantification of chromatin bound (slow and fast) and unbound (freely diffusing) particles of WT and class1 FOXA1 variants, and average chromatin dwell times (mean ± s.d.) for the bound fractions (n≥500 particles/variant). i) Diffusion constant histograms of single particles of WT or distinct class1 FOXA1 mutants. Particles were categorized into chromatin bound (slow and fast) or unbound fractions using cutoffs marked by dashed lines (n≥500 particles/variant imaged in 3–5 distinct nuclei). j) Left, mRNA expression (qPCR) of labeled FOXA1 variants in stable, isogenic HEK293 cells (n=3 technical replicates). Right, overlaps between FOXA1 WT and class1 mutant cistromes from these cells (n=2 biological replicates). k) Top de novo motifs identified from the three FOXA1 cistromes from HEK293 cells (HOMER: hypergeometric test). l) mRNA expression (qPCR) of labeled FOXA1 variants in stable, isogenic 22RV1 cells (n=3 technical replicates). For j and l , centers show mean values and lines mark s.e.m. m) Overlap between WT (n=2 biological replicates) and class1 (n=4 biological replicates) cistromes from stable 22RV1 overexpression models. n) Overlap between the FOXA1 WT and AR union cistromes generated from 22RV1 cells overexpressing WT (n=2 biological replicates) or class1 mutant (I176M or R216G; n=2 biological replicates each) FOXA1 variants. o) De novo motif results for the WT or class1 mutant FOXA1 binding sites from PCa cells (HOMER: hypergeometric test). p) Percent of WT or class1 binding sites with perfect match to the core FOXA1 motif (5’-T[G/A]TT[T/G]AC-3’) and q) the consensus FOXA1 motifs identified from these sites. r) Left, Percent of WT or class1 binding sites harboring known motifs of the labelled FOXA1 or AR cofactors; Right, Enrichment of the cofactor motifs in the two cistromes relative to the background (n=top 5000 peaks by score/variant, see Methods ). s) Genomic distribution of WT and class1 binding sites in PCa cells.
    Figure Legend Snippet: Biophysical and cistromic characteristics of the Class1 FOXA1 mutants. a) Distribution of class1 mutations on the protein map of FOXA1. b) 3D-structure of FKHD (FOXA3) with visualization of all mutated residues collectively identified as the 3D-mutational hotspot in FOXA1 across cancers. c) DNA-bound 3D structure of FKHD with visualization of all residues shown through crystallography to make direct base-specific contacts with the DNA in FOXA2 and FOXA3 proteins. FKHD; Forkhead domain. d) Representative fluorescent images of nuclei expressing different FOXA1 variants fused to GFP at the C-termini. e, f) FRAP recovery kinetic plots (left) and representative time-lapse images (right) from pre-bleaching (‘Pre’) to 100% recovery (red timestamps) for (e) wing2-altered class1 and (f) truncated class2 mutants (i.e. A287fs and P375fs), respectively (n=6 nuclei/variants; quantified in Fig 2d ). White lines indicate the border between bleached and unbleached areas. g) Representative FRAP fluorescence recovery kinetics in the bleached area for indicated FOXA1 variants. t½ line indicates the time to 50% recovery. Colored dots show raw data; superimposed solid curves show a hyperbolic fit with 95% confidence intervals. h) SPT quantification of chromatin bound (slow and fast) and unbound (freely diffusing) particles of WT and class1 FOXA1 variants, and average chromatin dwell times (mean ± s.d.) for the bound fractions (n≥500 particles/variant). i) Diffusion constant histograms of single particles of WT or distinct class1 FOXA1 mutants. Particles were categorized into chromatin bound (slow and fast) or unbound fractions using cutoffs marked by dashed lines (n≥500 particles/variant imaged in 3–5 distinct nuclei). j) Left, mRNA expression (qPCR) of labeled FOXA1 variants in stable, isogenic HEK293 cells (n=3 technical replicates). Right, overlaps between FOXA1 WT and class1 mutant cistromes from these cells (n=2 biological replicates). k) Top de novo motifs identified from the three FOXA1 cistromes from HEK293 cells (HOMER: hypergeometric test). l) mRNA expression (qPCR) of labeled FOXA1 variants in stable, isogenic 22RV1 cells (n=3 technical replicates). For j and l , centers show mean values and lines mark s.e.m. m) Overlap between WT (n=2 biological replicates) and class1 (n=4 biological replicates) cistromes from stable 22RV1 overexpression models. n) Overlap between the FOXA1 WT and AR union cistromes generated from 22RV1 cells overexpressing WT (n=2 biological replicates) or class1 mutant (I176M or R216G; n=2 biological replicates each) FOXA1 variants. o) De novo motif results for the WT or class1 mutant FOXA1 binding sites from PCa cells (HOMER: hypergeometric test). p) Percent of WT or class1 binding sites with perfect match to the core FOXA1 motif (5’-T[G/A]TT[T/G]AC-3’) and q) the consensus FOXA1 motifs identified from these sites. r) Left, Percent of WT or class1 binding sites harboring known motifs of the labelled FOXA1 or AR cofactors; Right, Enrichment of the cofactor motifs in the two cistromes relative to the background (n=top 5000 peaks by score/variant, see Methods ). s) Genomic distribution of WT and class1 binding sites in PCa cells.

    Techniques Used: Expressing, Fluorescence, Single-particle Tracking, Variant Assay, Diffusion-based Assay, Real-time Polymerase Chain Reaction, Labeling, Mutagenesis, Over Expression, Generated, Binding Assay

    Genomic characteristics of the three classes of FOXA1 alterations in prostate and breast cancer. a) Bi-allelic inactivation and b) copy-number variations of FOXA1 across mCRPC (n=371). c) FOXA1 expression in benign (n=51), primary (n=501), and metastatic (n=535) prostate RNA-seq libraries. d) Distribution and functional categorization of FOXA1 mutations (all cases in the aggregate cohort) on the protein map of FOXA1. TAD, trans-activating domain; RD, regulatory domain. e) Aggregate and class-specific distribution of FOXA1 mutations in advanced breast cancer (MSK-Impact cohort). f) Structural classification of FOXA1 locus rearrangements in breast cancer (TCGA and CCLE cell lines). g) Variant allele frequency of FOXA1 mutations by tumor stage, and h) clonality estimates of class1 and class2 mutations in tumor content corrected primary PCa (n=500) and mCRPC (n=370) specimens. i) Mutual exclusivity or co-occurrence of FOXA1 mutations (two-sided Fisher’s exact test). Mutations in AR, WNT, and PI3K were aggregated at the pathway level. ETS, ETS gene fusions; DRD, DNA repair defects and included alterations in BRCA½, ATM and CDK12; MMRD, mismatch repair deficiency (total n=371). j) Mutual exclusivity of ETS and/or SPOP (n=26) aberrations with FOXA1 (n=46) alterations distinguished by class in mCRPC (n=371). k) Co-occurrence of WNT (n=58) and DRD (n=107) pathway alterations with FOXA1 alteration classes in mCRPC (n=371). l) Stage and class-specific increase in FOXA1 expression levels in primary (n=500) and metastatic PCa (n=357). Right: two-sided t-test. Left: two-way ANOVA. For all boxplots: center shows median, box extends from Q1 to Q3, and whiskers span Q1/Q3±1.5xIQR.
    Figure Legend Snippet: Genomic characteristics of the three classes of FOXA1 alterations in prostate and breast cancer. a) Bi-allelic inactivation and b) copy-number variations of FOXA1 across mCRPC (n=371). c) FOXA1 expression in benign (n=51), primary (n=501), and metastatic (n=535) prostate RNA-seq libraries. d) Distribution and functional categorization of FOXA1 mutations (all cases in the aggregate cohort) on the protein map of FOXA1. TAD, trans-activating domain; RD, regulatory domain. e) Aggregate and class-specific distribution of FOXA1 mutations in advanced breast cancer (MSK-Impact cohort). f) Structural classification of FOXA1 locus rearrangements in breast cancer (TCGA and CCLE cell lines). g) Variant allele frequency of FOXA1 mutations by tumor stage, and h) clonality estimates of class1 and class2 mutations in tumor content corrected primary PCa (n=500) and mCRPC (n=370) specimens. i) Mutual exclusivity or co-occurrence of FOXA1 mutations (two-sided Fisher’s exact test). Mutations in AR, WNT, and PI3K were aggregated at the pathway level. ETS, ETS gene fusions; DRD, DNA repair defects and included alterations in BRCA½, ATM and CDK12; MMRD, mismatch repair deficiency (total n=371). j) Mutual exclusivity of ETS and/or SPOP (n=26) aberrations with FOXA1 (n=46) alterations distinguished by class in mCRPC (n=371). k) Co-occurrence of WNT (n=58) and DRD (n=107) pathway alterations with FOXA1 alteration classes in mCRPC (n=371). l) Stage and class-specific increase in FOXA1 expression levels in primary (n=500) and metastatic PCa (n=357). Right: two-sided t-test. Left: two-way ANOVA. For all boxplots: center shows median, box extends from Q1 to Q3, and whiskers span Q1/Q3±1.5xIQR.

    Techniques Used: Expressing, RNA Sequencing Assay, Functional Assay, Variant Assay

    Transcriptional and genomic characteristics of Class3 FOXA1 rearrangements in prostate cancer. a) Dosage sensitivity of the FOXA1 gene. Expression of FOXA1 (RNA-seq) across mCRPC tumors (n=370) as a function of gene ploidy (as determined by absolute copy number at the FOXA1 locus (two-way ANOVA). b) Relative expression of FOXA1 (within the minimally amplified region) to TTC6 (outside the amplified region) in rearranged (n=50) (duplication or translocation) vs WT (n=320) FOXA1 loci (two-sided t-test). All boxplot center shows median, box marks Q1/Q3, whiskers span Q1/Q3±1.5xIQR. c) Association plot visualizing the relative enrichment of cases with both translocation and duplications within the FOXA1 locus (n=370). Over-abundance of cases with both events is quantified using Pearson-residuals. Significance of this association is based on the Chi-square test without continuity correction. Tloc, translocation; inv, inversion; del, deletion. d) FOXA1 locus visualization of linked-read (10X platform) whole genome-sequencing of the MDA-PCA-2B cell line. Alignments on the haplotype-resolved genome are shown in green and purple. Translocation and tandem-duplication calls are indicated in blue and red, respectively. e) Monoallelic expression of FOXA1 cell-lines with FOXMIND-ETV1 translocations in MDA-PCA-2b (n=6 biological replicates) and LNCaP (n=15 biological replicates). Phasing of FOXA1 SNPs to structural variants is based on linked-read sequencing (Methods). f) Biallelic expression of RNA from the FOXMIND locus assessed using three distinct SNPs in MDA-PCa-2b cells that harbor ETV1 translocation into the FOXA1 locus (n=7 biological replicates). g) mRNA (qPCR) expression of ETV1 and TTC6 upon sgRNA-mediated disruption of the FOXMIND or the MIPOL1-UTR enhancer in LNCaP cells, which also harbor ETV1 translocation into the FOXA1 locus (see Extended Data Fig. 9d for sgRNA binding sites). Distinct sgRNA pairs cutting at FOXMIND serve as biological replicates. Mean ± s.e.m are shown (n=3 technical replicates; two-way ANOVA and Tukey’s test).
    Figure Legend Snippet: Transcriptional and genomic characteristics of Class3 FOXA1 rearrangements in prostate cancer. a) Dosage sensitivity of the FOXA1 gene. Expression of FOXA1 (RNA-seq) across mCRPC tumors (n=370) as a function of gene ploidy (as determined by absolute copy number at the FOXA1 locus (two-way ANOVA). b) Relative expression of FOXA1 (within the minimally amplified region) to TTC6 (outside the amplified region) in rearranged (n=50) (duplication or translocation) vs WT (n=320) FOXA1 loci (two-sided t-test). All boxplot center shows median, box marks Q1/Q3, whiskers span Q1/Q3±1.5xIQR. c) Association plot visualizing the relative enrichment of cases with both translocation and duplications within the FOXA1 locus (n=370). Over-abundance of cases with both events is quantified using Pearson-residuals. Significance of this association is based on the Chi-square test without continuity correction. Tloc, translocation; inv, inversion; del, deletion. d) FOXA1 locus visualization of linked-read (10X platform) whole genome-sequencing of the MDA-PCA-2B cell line. Alignments on the haplotype-resolved genome are shown in green and purple. Translocation and tandem-duplication calls are indicated in blue and red, respectively. e) Monoallelic expression of FOXA1 cell-lines with FOXMIND-ETV1 translocations in MDA-PCA-2b (n=6 biological replicates) and LNCaP (n=15 biological replicates). Phasing of FOXA1 SNPs to structural variants is based on linked-read sequencing (Methods). f) Biallelic expression of RNA from the FOXMIND locus assessed using three distinct SNPs in MDA-PCa-2b cells that harbor ETV1 translocation into the FOXA1 locus (n=7 biological replicates). g) mRNA (qPCR) expression of ETV1 and TTC6 upon sgRNA-mediated disruption of the FOXMIND or the MIPOL1-UTR enhancer in LNCaP cells, which also harbor ETV1 translocation into the FOXA1 locus (see Extended Data Fig. 9d for sgRNA binding sites). Distinct sgRNA pairs cutting at FOXMIND serve as biological replicates. Mean ± s.e.m are shown (n=3 technical replicates; two-way ANOVA and Tukey’s test).

    Techniques Used: Expressing, RNA Sequencing Assay, Amplification, Translocation Assay, Sequencing, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Binding Assay

    Related Articles

    Clone Assay:

    Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
    Article Snippet: For the cloning of SK-M and SK-V rescue constructs, RT-PCR was performed on total RNA extracts (TRIzol, Life Technologies) from Canton-S third instar larvae. .. The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA.

    Amplification:

    Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
    Article Snippet: The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA. .. For SK-M and SK-V cDNA amplification, PCR was performed using the reverse primer 5ʹ-TCAGCTAGAATGTGGAAACAGCAT-3ʹ, and either the forward primer 5ʹ-ATGTCAATTCA GAAGCTTAACGAC-3ʹ to target the SK-M isoform or the forward primer 5ʹ-ATGTCGCCGGCCTTCTGC-3ʹ to target the SK-V isoform.

    Article Title: Respiratory viruses in laryngeal croup of young children.
    Article Snippet: 13 The amplified product was detected by electrophoresis in 2% agarose gels after ethidium bromide staining. .. Viral RNA was reverse transcribed into cDNA with random hexamer primers (Roche, Mannheim, Germany) and Superscript III reverse transcriptase enzyme (Invitrogen, Carlsbad, Calif) following the manufacturer's procedures.

    Article Title: A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43.
    Article Snippet: .. Amplicon sequencing of HCoV-OC43 16 µl of extracted RNA were reversed transcribed into cDNA using random hexamers primers (Life Technologies) and 400U of SuperScript III reverse transcriptase enzyme (Life Technologies) during 2 h at 45°C. .. Obtained cDNA were amplified with the 2X PCR Precision Master Mix (Applied Biological Materials Inc) and multiple primer pairs divided into 12 groups (see primers sequence and groups in Supplementary data).

    Positive Control:

    Article Title: Respiratory viruses in laryngeal croup of young children.
    Article Snippet: SARS-corona-virus RNA (Frankfurt-1 strain) was included as a positive control in each run. .. Viral RNA was reverse transcribed into cDNA with random hexamer primers (Roche, Mannheim, Germany) and Superscript III reverse transcriptase enzyme (Invitrogen, Carlsbad, Calif) following the manufacturer's procedures.

    Synthesized:

    Article Title: Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer
    Article Snippet: RNA was quantified using the NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific) and 1ug of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript III Reverse Transcriptase enzyme (ThermoFisher Scientific) following manufacturer’s instructions. .. Gene expression was calculated relative to GAPDH and HPRT1 (loading control) using the delta-delta Ct method and normalized to the control group for graphing. quantitative PCR (qPCR) primers were designed using the Primer3Plus tool ( http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi ) and synthesized by Integrated DNA Technologies.

    Construct:

    Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
    Article Snippet: For the cloning of SK-M and SK-V rescue constructs, RT-PCR was performed on total RNA extracts (TRIzol, Life Technologies) from Canton-S third instar larvae. .. The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA.

    Electrophoresis:

    Article Title: Respiratory viruses in laryngeal croup of young children.
    Article Snippet: 13 The amplified product was detected by electrophoresis in 2% agarose gels after ethidium bromide staining. .. Viral RNA was reverse transcribed into cDNA with random hexamer primers (Roche, Mannheim, Germany) and Superscript III reverse transcriptase enzyme (Invitrogen, Carlsbad, Calif) following the manufacturer's procedures.

    Random Hexamer Labeling:

    Article Title: Respiratory viruses in laryngeal croup of young children.
    Article Snippet: .. Viral RNA was reverse transcribed into cDNA with random hexamer primers (Roche, Mannheim, Germany) and Superscript III reverse transcriptase enzyme (Invitrogen, Carlsbad, Calif) following the manufacturer's procedures. .. For the detection of influenza A and B viruses and RSV, cDNA was amplified in a real-time multiplex PCR 9 with minor modifications.

    Expressing:

    Article Title: Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy
    Article Snippet: Samples with a 260/280 ratio between 1.8 and 2.2 were considered acceptable; 500 ng of total mRNA was used to produce cDNA by reverse transcription using SuperScript III reverse transcriptase enzyme (Invitrogen) primed with 50 pmol of random hexamers (Sigma-Aldrich). qPCR was performed using the QuantiTech SYBR Green kit (QIAGEN) and the LightCycler 1.5 (Roche Diagnostics). .. Using LightCycler 1.5 software, data were analyzed and normalized to the expression of β-actin.

    Article Title: Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer
    Article Snippet: RNA was quantified using the NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific) and 1ug of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript III Reverse Transcriptase enzyme (ThermoFisher Scientific) following manufacturer’s instructions. .. Gene expression was calculated relative to GAPDH and HPRT1 (loading control) using the delta-delta Ct method and normalized to the control group for graphing. quantitative PCR (qPCR) primers were designed using the Primer3Plus tool ( http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi ) and synthesized by Integrated DNA Technologies.

    Real-time Polymerase Chain Reaction:

    Article Title: Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy
    Article Snippet: .. Samples with a 260/280 ratio between 1.8 and 2.2 were considered acceptable; 500 ng of total mRNA was used to produce cDNA by reverse transcription using SuperScript III reverse transcriptase enzyme (Invitrogen) primed with 50 pmol of random hexamers (Sigma-Aldrich). qPCR was performed using the QuantiTech SYBR Green kit (QIAGEN) and the LightCycler 1.5 (Roche Diagnostics). .. Each reaction tube contained 2 μl cDNA sample, 10 μl SYBR Green QuantiTech Reagent (QIAGEN), 1.25 μ m primer pair (Sigma-Aldrich), and RNase free water (Invitrogen) to a final volume of 20 μl.

    Article Title: Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer
    Article Snippet: Paragraph title: RNA extraction and quantitative polymerase chain reaction (qPCR) ... RNA was quantified using the NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific) and 1ug of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript III Reverse Transcriptase enzyme (ThermoFisher Scientific) following manufacturer’s instructions.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
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    Article Title: Respiratory viruses in laryngeal croup of young children.
    Article Snippet: RT-PCR was performed with a primer pair that amplifies a 251 bp fragment of the polymerase gene that is well conserved between coronavirus 229E, coronavirus OC43, coronavirus NL-63, and the SARS-coronavirus. .. Viral RNA was reverse transcribed into cDNA with random hexamer primers (Roche, Mannheim, Germany) and Superscript III reverse transcriptase enzyme (Invitrogen, Carlsbad, Calif) following the manufacturer's procedures.

    Generated:

    Article Title: Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model
    Article Snippet: .. Viral cDNA was generated from the extracted total RNA using SuperScript III reverse transcriptase enzyme (Invitrogen, Carlsbad, CA), PCR nucleotides (New England Biolabs, MA), and a Gag-specific reverse primer ( ). .. The amounts of SHIV DNA and RNA per million CD4+ T cells in blood and necropsy tissues were estimated by amplifying cDNA and genomic DNA with the primers and probes described in , using digital droplet PCR (ddPCR) as described previously ( ).

    Sequencing:

    Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
    Article Snippet: The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA. .. The complete sequence for the newly cloned SK-V isoform was deposited in GenBank (accession number MH001552).

    Article Title: A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43.
    Article Snippet: .. Amplicon sequencing of HCoV-OC43 16 µl of extracted RNA were reversed transcribed into cDNA using random hexamers primers (Life Technologies) and 400U of SuperScript III reverse transcriptase enzyme (Life Technologies) during 2 h at 45°C. .. Obtained cDNA were amplified with the 2X PCR Precision Master Mix (Applied Biological Materials Inc) and multiple primer pairs divided into 12 groups (see primers sequence and groups in Supplementary data).

    Magnetic Cell Separation:

    Article Title: Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model
    Article Snippet: CD4+ T cells were enriched from PBMCs and tissue mononuclear cells using a negative-selection magnetic-activated cell sorting (MACS) system per the manufacturer’s instructions (Miltenyi Biotec, Germany). .. Viral cDNA was generated from the extracted total RNA using SuperScript III reverse transcriptase enzyme (Invitrogen, Carlsbad, CA), PCR nucleotides (New England Biolabs, MA), and a Gag-specific reverse primer ( ).

    Isolation:

    Article Title: Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model
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    RNA Extraction:

    Article Title: Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy
    Article Snippet: Paragraph title: RNA extraction and qPCR. ... Samples with a 260/280 ratio between 1.8 and 2.2 were considered acceptable; 500 ng of total mRNA was used to produce cDNA by reverse transcription using SuperScript III reverse transcriptase enzyme (Invitrogen) primed with 50 pmol of random hexamers (Sigma-Aldrich). qPCR was performed using the QuantiTech SYBR Green kit (QIAGEN) and the LightCycler 1.5 (Roche Diagnostics).

    Article Title: Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer
    Article Snippet: Paragraph title: RNA extraction and quantitative polymerase chain reaction (qPCR) ... RNA was quantified using the NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific) and 1ug of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript III Reverse Transcriptase enzyme (ThermoFisher Scientific) following manufacturer’s instructions.

    Purification:

    Article Title: A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43.
    Article Snippet: Amplicon sequencing of HCoV-OC43 16 µl of extracted RNA were reversed transcribed into cDNA using random hexamers primers (Life Technologies) and 400U of SuperScript III reverse transcriptase enzyme (Life Technologies) during 2 h at 45°C. .. PCR conditions were 40 cycles of annealing at 60°C for 20 s and extension at 72°C for 60 s. Amplified products were purified with Nu-cleoMag NGS Clean-up and Size Select (Macherey-Nagel, Hoerdt, France).

    Polymerase Chain Reaction:

    Article Title: Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model
    Article Snippet: .. Viral cDNA was generated from the extracted total RNA using SuperScript III reverse transcriptase enzyme (Invitrogen, Carlsbad, CA), PCR nucleotides (New England Biolabs, MA), and a Gag-specific reverse primer ( ). .. The amounts of SHIV DNA and RNA per million CD4+ T cells in blood and necropsy tissues were estimated by amplifying cDNA and genomic DNA with the primers and probes described in , using digital droplet PCR (ddPCR) as described previously ( ).

    Article Title: Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer
    Article Snippet: RNA was quantified using the NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific) and 1ug of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript III Reverse Transcriptase enzyme (ThermoFisher Scientific) following manufacturer’s instructions. .. 20ng of cDNA was inputted per polymerase chain reaction (PCR) using the FAST SYBR Green Universal Master Mix (ThermoFisher Scientific) and every sample was quantified in triplicates.

    Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
    Article Snippet: The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA. .. For SK-M and SK-V cDNA amplification, PCR was performed using the reverse primer 5ʹ-TCAGCTAGAATGTGGAAACAGCAT-3ʹ, and either the forward primer 5ʹ-ATGTCAATTCA GAAGCTTAACGAC-3ʹ to target the SK-M isoform or the forward primer 5ʹ-ATGTCGCCGGCCTTCTGC-3ʹ to target the SK-V isoform.

    Article Title: Respiratory viruses in laryngeal croup of young children.
    Article Snippet: Viral RNA was reverse transcribed into cDNA with random hexamer primers (Roche, Mannheim, Germany) and Superscript III reverse transcriptase enzyme (Invitrogen, Carlsbad, Calif) following the manufacturer's procedures. .. For the detection of influenza A and B viruses and RSV, cDNA was amplified in a real-time multiplex PCR 9 with minor modifications.

    Article Title: A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43.
    Article Snippet: Amplicon sequencing of HCoV-OC43 16 µl of extracted RNA were reversed transcribed into cDNA using random hexamers primers (Life Technologies) and 400U of SuperScript III reverse transcriptase enzyme (Life Technologies) during 2 h at 45°C. .. Obtained cDNA were amplified with the 2X PCR Precision Master Mix (Applied Biological Materials Inc) and multiple primer pairs divided into 12 groups (see primers sequence and groups in Supplementary data).

    FACS:

    Article Title: Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model
    Article Snippet: CD4+ T cells were enriched from PBMCs and tissue mononuclear cells using a negative-selection magnetic-activated cell sorting (MACS) system per the manufacturer’s instructions (Miltenyi Biotec, Germany). .. Viral cDNA was generated from the extracted total RNA using SuperScript III reverse transcriptase enzyme (Invitrogen, Carlsbad, CA), PCR nucleotides (New England Biolabs, MA), and a Gag-specific reverse primer ( ).

    Plasmid Preparation:

    Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
    Article Snippet: The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA. .. The SK-M and SK-V PCR products were subsequently cloned into the TOPO-XL vector (Life Technologies) and fully sequenced.

    Software:

    Article Title: Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy
    Article Snippet: Samples with a 260/280 ratio between 1.8 and 2.2 were considered acceptable; 500 ng of total mRNA was used to produce cDNA by reverse transcription using SuperScript III reverse transcriptase enzyme (Invitrogen) primed with 50 pmol of random hexamers (Sigma-Aldrich). qPCR was performed using the QuantiTech SYBR Green kit (QIAGEN) and the LightCycler 1.5 (Roche Diagnostics). .. Using LightCycler 1.5 software, data were analyzed and normalized to the expression of β-actin.

    SYBR Green Assay:

    Article Title: Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy
    Article Snippet: .. Samples with a 260/280 ratio between 1.8 and 2.2 were considered acceptable; 500 ng of total mRNA was used to produce cDNA by reverse transcription using SuperScript III reverse transcriptase enzyme (Invitrogen) primed with 50 pmol of random hexamers (Sigma-Aldrich). qPCR was performed using the QuantiTech SYBR Green kit (QIAGEN) and the LightCycler 1.5 (Roche Diagnostics). .. Each reaction tube contained 2 μl cDNA sample, 10 μl SYBR Green QuantiTech Reagent (QIAGEN), 1.25 μ m primer pair (Sigma-Aldrich), and RNase free water (Invitrogen) to a final volume of 20 μl.

    Article Title: Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer
    Article Snippet: RNA was quantified using the NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific) and 1ug of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript III Reverse Transcriptase enzyme (ThermoFisher Scientific) following manufacturer’s instructions. .. 20ng of cDNA was inputted per polymerase chain reaction (PCR) using the FAST SYBR Green Universal Master Mix (ThermoFisher Scientific) and every sample was quantified in triplicates.

    Multiplex Assay:

    Article Title: Respiratory viruses in laryngeal croup of young children.
    Article Snippet: Viral RNA was reverse transcribed into cDNA with random hexamer primers (Roche, Mannheim, Germany) and Superscript III reverse transcriptase enzyme (Invitrogen, Carlsbad, Calif) following the manufacturer's procedures. .. For the detection of influenza A and B viruses and RSV, cDNA was amplified in a real-time multiplex PCR 9 with minor modifications.

    Next-Generation Sequencing:

    Article Title: A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43.
    Article Snippet: Amplicon sequencing of HCoV-OC43 16 µl of extracted RNA were reversed transcribed into cDNA using random hexamers primers (Life Technologies) and 400U of SuperScript III reverse transcriptase enzyme (Life Technologies) during 2 h at 45°C. .. PCR conditions were 40 cycles of annealing at 60°C for 20 s and extension at 72°C for 60 s. Amplified products were purified with Nu-cleoMag NGS Clean-up and Size Select (Macherey-Nagel, Hoerdt, France).

    Spectrophotometry:

    Article Title: Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy
    Article Snippet: Quantity and quality of RNA were measured using a Nanodrop Spectrophotometer (Thermo Fisher Scientific). .. Samples with a 260/280 ratio between 1.8 and 2.2 were considered acceptable; 500 ng of total mRNA was used to produce cDNA by reverse transcription using SuperScript III reverse transcriptase enzyme (Invitrogen) primed with 50 pmol of random hexamers (Sigma-Aldrich). qPCR was performed using the QuantiTech SYBR Green kit (QIAGEN) and the LightCycler 1.5 (Roche Diagnostics).

    Article Title: Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer
    Article Snippet: .. RNA was quantified using the NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific) and 1ug of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript III Reverse Transcriptase enzyme (ThermoFisher Scientific) following manufacturer’s instructions. .. 20ng of cDNA was inputted per polymerase chain reaction (PCR) using the FAST SYBR Green Universal Master Mix (ThermoFisher Scientific) and every sample was quantified in triplicates.

    Cell Counting:

    Article Title: Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model
    Article Snippet: Viral cDNA was generated from the extracted total RNA using SuperScript III reverse transcriptase enzyme (Invitrogen, Carlsbad, CA), PCR nucleotides (New England Biolabs, MA), and a Gag-specific reverse primer ( ). .. Therefore, an input CD4+ T cell count of 10,000 was defined as the threshold cell count (TCC) for the analysis, and samples having input numbers of cells less than the TCC were not analyzed.

    Staining:

    Article Title: Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Human Immunodeficiency Virus-Infected Infant Rhesus Macaque Model
    Article Snippet: Enriched CD4+ T cells were stained with the fluorescently conjugated antibodies listed in in the supplemental material and sorted for naive, memory, and Tfh CD4+ T cell subsets ( ). .. Viral cDNA was generated from the extracted total RNA using SuperScript III reverse transcriptase enzyme (Invitrogen, Carlsbad, CA), PCR nucleotides (New England Biolabs, MA), and a Gag-specific reverse primer ( ).

    Article Title: Respiratory viruses in laryngeal croup of young children.
    Article Snippet: 13 The amplified product was detected by electrophoresis in 2% agarose gels after ethidium bromide staining. .. Viral RNA was reverse transcribed into cDNA with random hexamer primers (Roche, Mannheim, Germany) and Superscript III reverse transcriptase enzyme (Invitrogen, Carlsbad, Calif) following the manufacturer's procedures.

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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
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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