superscript iii reverse transcriptase  (Thermo Fisher)


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    Thermo Fisher superscript iii reverse transcriptase
    Overexpressing a heterogeneous c-di-GMP sequesterer, YcgR, reduces glp expression. (A and B) Cells of wild-type B. burgdorferi strain B31 A3 (WT) and B31 A3 carrying a flgB -driven ycgR gene from E. coli (WT/ P flgB -ycgR ) were cultivated in the BSK-II medium at 37°C, harvested at the logarithmic phase, and subjected to <t>RNA</t> isolation and qRT-PCR analysis for glpF (A) or recA (B) transcript levels. (C) Growth curve of WT and WT/ P flgB -YcgR in the BSK-glycerol medium. Values indicate the fold changes compared with the wild-type strain from <t>three</t> biological replicates (±standard deviations). *, P
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    superscript iii reverse transcriptase - by Bioz Stars, 2020-01
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    qrt-pcr

    Images

    1) Product Images from "Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi"

    Article Title: Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00243-18

    Overexpressing a heterogeneous c-di-GMP sequesterer, YcgR, reduces glp expression. (A and B) Cells of wild-type B. burgdorferi strain B31 A3 (WT) and B31 A3 carrying a flgB -driven ycgR gene from E. coli (WT/ P flgB -ycgR ) were cultivated in the BSK-II medium at 37°C, harvested at the logarithmic phase, and subjected to RNA isolation and qRT-PCR analysis for glpF (A) or recA (B) transcript levels. (C) Growth curve of WT and WT/ P flgB -YcgR in the BSK-glycerol medium. Values indicate the fold changes compared with the wild-type strain from three biological replicates (±standard deviations). *, P
    Figure Legend Snippet: Overexpressing a heterogeneous c-di-GMP sequesterer, YcgR, reduces glp expression. (A and B) Cells of wild-type B. burgdorferi strain B31 A3 (WT) and B31 A3 carrying a flgB -driven ycgR gene from E. coli (WT/ P flgB -ycgR ) were cultivated in the BSK-II medium at 37°C, harvested at the logarithmic phase, and subjected to RNA isolation and qRT-PCR analysis for glpF (A) or recA (B) transcript levels. (C) Growth curve of WT and WT/ P flgB -YcgR in the BSK-glycerol medium. Values indicate the fold changes compared with the wild-type strain from three biological replicates (±standard deviations). *, P

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    PlzA is a positive regulator for glp expression. (A) Wild-type Borrelia strain B31 A3 (WT), the plzA mutant (PlzA − ), and its complemented strain (PlzA com ) were cultivated in the BSK-II medium to mid-logarithmic phase at 37°C. RNAs were isolated from these cells and subjected to qRT-PCR analysis for glpF transcript levels. Values indicate the fold changes (with the mean value of the wild-type strain as 1.0) from three biological replicates (±standard deviations). (B) Growth defect of the plzA mutant with glycerol as the carbon source. Wild-type strain (WT), the rrp1 mutant (Rrp1 − ), and the plzA mutant (PlzA − ) were cultivated in the BSK-glycerol medium. *, P
    Figure Legend Snippet: PlzA is a positive regulator for glp expression. (A) Wild-type Borrelia strain B31 A3 (WT), the plzA mutant (PlzA − ), and its complemented strain (PlzA com ) were cultivated in the BSK-II medium to mid-logarithmic phase at 37°C. RNAs were isolated from these cells and subjected to qRT-PCR analysis for glpF transcript levels. Values indicate the fold changes (with the mean value of the wild-type strain as 1.0) from three biological replicates (±standard deviations). (B) Growth defect of the plzA mutant with glycerol as the carbon source. Wild-type strain (WT), the rrp1 mutant (Rrp1 − ), and the plzA mutant (PlzA − ) were cultivated in the BSK-glycerol medium. *, P

    Techniques Used: Expressing, Mutagenesis, Isolation, Quantitative RT-PCR

    Overexpressing plzA in wild-type B. burgdorferi represses glp expression, which can be rescued by cooverexpression of plzA and rrp1 . Cells of wild-type strain B31 5A4NP1 (WT), WT overexpressing plzA (WT/ P flgB -plzA ), and WT cooverexpressing plzA and rrp1 (WT/ P flgB -plzA+P flaB -rrp1 ), were cultivated at 37°C in the BSK-II medium, harvested at mid-logarithmic phase, and subjected to RNA extraction and qRT-PCR analysis. (A and B) The levels of glpF (A) and plzA (B) expression were reported as fold changes, with the mean value of wild-type strain as 1.0. All data were calculated from three biological replicates (±standard deviations). *, P
    Figure Legend Snippet: Overexpressing plzA in wild-type B. burgdorferi represses glp expression, which can be rescued by cooverexpression of plzA and rrp1 . Cells of wild-type strain B31 5A4NP1 (WT), WT overexpressing plzA (WT/ P flgB -plzA ), and WT cooverexpressing plzA and rrp1 (WT/ P flgB -plzA+P flaB -rrp1 ), were cultivated at 37°C in the BSK-II medium, harvested at mid-logarithmic phase, and subjected to RNA extraction and qRT-PCR analysis. (A and B) The levels of glpF (A) and plzA (B) expression were reported as fold changes, with the mean value of wild-type strain as 1.0. All data were calculated from three biological replicates (±standard deviations). *, P

    Techniques Used: Expressing, RNA Extraction, Quantitative RT-PCR

    Regulation of glp expression by c-di-GMP. (A) Wild-type strain 5A4NP1 (WT), the rrp1 mutant (Rrp1 − ), and the isogenic complementation strain (Rrp1 com ) were cultivated in regular BSK-II (BSK-glucose) medium at 37°C and harvested at different growth phases. E, mid-logarithmic phase (5 × 10 6 cells per ml); M, late-logarithmic phase (2 × 10 7 cells per ml); S, stationary phase (5 × 10 8 cells per ml). (B) The same set of strains was also cultivated in either BSK-glucose or BSK-glycerol medium to mid-logarithmic phase. (C) Wild-type B. burgdorferi strain B31 A3 (WT) and the isogenic pdeB overexpression strain (WT/ P flgB -pdeB ) cells were cultivated in the BSK-II medium to mid-logarithmic phase at 37°C. RNA was isolated from all of the cultures and subjected to qRT-PCR analysis for glpF expression. In panels A to C, values represent the average numbers of glpF transcripts per 100 copies of flaB from three biological replicates. Error bars indicate standard deviations. (D) Growth curve of WT and WT/ P flgB -pdeB in the BSK-glycerol medium. *, P
    Figure Legend Snippet: Regulation of glp expression by c-di-GMP. (A) Wild-type strain 5A4NP1 (WT), the rrp1 mutant (Rrp1 − ), and the isogenic complementation strain (Rrp1 com ) were cultivated in regular BSK-II (BSK-glucose) medium at 37°C and harvested at different growth phases. E, mid-logarithmic phase (5 × 10 6 cells per ml); M, late-logarithmic phase (2 × 10 7 cells per ml); S, stationary phase (5 × 10 8 cells per ml). (B) The same set of strains was also cultivated in either BSK-glucose or BSK-glycerol medium to mid-logarithmic phase. (C) Wild-type B. burgdorferi strain B31 A3 (WT) and the isogenic pdeB overexpression strain (WT/ P flgB -pdeB ) cells were cultivated in the BSK-II medium to mid-logarithmic phase at 37°C. RNA was isolated from all of the cultures and subjected to qRT-PCR analysis for glpF expression. In panels A to C, values represent the average numbers of glpF transcripts per 100 copies of flaB from three biological replicates. Error bars indicate standard deviations. (D) Growth curve of WT and WT/ P flgB -pdeB in the BSK-glycerol medium. *, P

    Techniques Used: Expressing, Mutagenesis, Over Expression, Isolation, Quantitative RT-PCR

    2) Product Images from "Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein"

    Article Title: Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52386-0

    The relative expression level and cellular stability between A and B group isoforms. ( A ) Comparison of endogenous expression levels between CCM2 isoform pairs among various tissues. The relative mRNA expression levels of paired-CCM2 isoforms (2 −∆CT ) were presented with bar plots, in which light grey bars represent A group isoforms, dark grey bars represent their respective counterparts, B group isoforms. For experimental design, the left three panels represent expression levels between CCM2 isoform pairs with primer set, CCM2-A100 and CCM2-B200; the right three panels for CCM2 isoform pairs with primer set, CCM2-A101 and CCM2-B201. For tissue location and cell lines, upper two panels represent the expression levels of paired-CCM2 isoforms among major tissues (see Fig. 2A ), middle two panels for various brain tissues, and lower two panels for multiple cell lines (see Suppl. Fig. 1 ) Middle and lower panels are further described in supplemental Fig. 1 . One-way ANOVA was also performed for the comparison between A and B groups of isoforms among different tissues and cells; it was found there is a very significant difference (P
    Figure Legend Snippet: The relative expression level and cellular stability between A and B group isoforms. ( A ) Comparison of endogenous expression levels between CCM2 isoform pairs among various tissues. The relative mRNA expression levels of paired-CCM2 isoforms (2 −∆CT ) were presented with bar plots, in which light grey bars represent A group isoforms, dark grey bars represent their respective counterparts, B group isoforms. For experimental design, the left three panels represent expression levels between CCM2 isoform pairs with primer set, CCM2-A100 and CCM2-B200; the right three panels for CCM2 isoform pairs with primer set, CCM2-A101 and CCM2-B201. For tissue location and cell lines, upper two panels represent the expression levels of paired-CCM2 isoforms among major tissues (see Fig. 2A ), middle two panels for various brain tissues, and lower two panels for multiple cell lines (see Suppl. Fig. 1 ) Middle and lower panels are further described in supplemental Fig. 1 . One-way ANOVA was also performed for the comparison between A and B groups of isoforms among different tissues and cells; it was found there is a very significant difference (P

    Techniques Used: Expressing

    Molecular interactions defined with yeast two-hybrid system. ( A ) Interactions between various NPXY-motif containing protein fragments and CCM2 PTB-less isoforms (CCM2-116, CCM2-107, and CCM2-212). CCM2-101 serves as positive control, while CCM2-1209 as negative control. pGAD-T with p53 is a system control. ( B ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and a CCM2 PTB-less isoform (CCM2-116). CCM2-102 and full-length CCM2 PTB domain serves as positive control, while CCM2-1209 as negative control. ( C ) Interactions between protein fragments containing various number of NPXY-motifs and CCM2 PTB-less isoforms (CCM2-107, and CCM2-212). CCM2-206 serves as positive control, while CCM2-1209 as negative control. ( D ) Interactions between various NPXY-motif containing protein fragments and CCM2 exons (6, 6A, and 6B). CCM2-PTB serves as positive control, while pGAD as negative control. pGAD-T with p53 is a system control. ( E ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and CCM2 exons (6, 6A, and 6B). Large-T serves as system control. ( F ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and CCM2 exons (6, 6A, and 6B) and duplicate forms (2 × 6 and 2 × 6A). Large-T serves as system control. ( G ) Interactions between CCM3 protein and CCM2 exons (6, 6A, and 6B). Large-T with p53 serves as system control. ( H ) Competition assays between CCM3 protein with either CCM1-HK5 (containing 1 st NPXY motif) (upper panel) or CCM1-THK (containing 2 nd and 3 rd NPXY motif) (lower panel) binding to CCM2 exons (6, 6A, and 6B). β -galactosidase activity of each transformant was measured, normalized, and converted to relative β -galactosidase activity (RBGA). The normalized data were represented with means and standard deviations (M ± SD) generated from at least three independent assays (n = 3). RBGA +++ , ++ : significantly higher than that observed in any negative controls (P
    Figure Legend Snippet: Molecular interactions defined with yeast two-hybrid system. ( A ) Interactions between various NPXY-motif containing protein fragments and CCM2 PTB-less isoforms (CCM2-116, CCM2-107, and CCM2-212). CCM2-101 serves as positive control, while CCM2-1209 as negative control. pGAD-T with p53 is a system control. ( B ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and a CCM2 PTB-less isoform (CCM2-116). CCM2-102 and full-length CCM2 PTB domain serves as positive control, while CCM2-1209 as negative control. ( C ) Interactions between protein fragments containing various number of NPXY-motifs and CCM2 PTB-less isoforms (CCM2-107, and CCM2-212). CCM2-206 serves as positive control, while CCM2-1209 as negative control. ( D ) Interactions between various NPXY-motif containing protein fragments and CCM2 exons (6, 6A, and 6B). CCM2-PTB serves as positive control, while pGAD as negative control. pGAD-T with p53 is a system control. ( E ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and CCM2 exons (6, 6A, and 6B). Large-T serves as system control. ( F ) Interactions between wild type (W) and mutated (M) three NPXY motifs of CCM1 (K2, K5, and K8) and CCM2 exons (6, 6A, and 6B) and duplicate forms (2 × 6 and 2 × 6A). Large-T serves as system control. ( G ) Interactions between CCM3 protein and CCM2 exons (6, 6A, and 6B). Large-T with p53 serves as system control. ( H ) Competition assays between CCM3 protein with either CCM1-HK5 (containing 1 st NPXY motif) (upper panel) or CCM1-THK (containing 2 nd and 3 rd NPXY motif) (lower panel) binding to CCM2 exons (6, 6A, and 6B). β -galactosidase activity of each transformant was measured, normalized, and converted to relative β -galactosidase activity (RBGA). The normalized data were represented with means and standard deviations (M ± SD) generated from at least three independent assays (n = 3). RBGA +++ , ++ : significantly higher than that observed in any negative controls (P

    Techniques Used: Positive Control, Negative Control, Binding Assay, Activity Assay, Generated

    Genomic structure, conservation, and variability among alterative start-codon exons and promotors of CCM2. ( A ) The complex promoter regions of human CCM2 locus were defined with bioinformatics (promoter prediction software from top to bottom: Cister, promotor2.0, Softberry, MEME, CTCFBSDB, BDGP/NNPP and Genscan as indicated by different colors). Symbols on top of DNA templates are on positive strand, below are on negative strand. The single promoter for the original bonafide start-codon exon, exon 1, simply lies immediately upstream of the transcription start site for exon 1, as P0. The promoter region for exon 1A is much more complicated. Although a seemingly weak promoter, P1 lies immediately upstream of its transcription-start site; in addition, there are three relatively strong promoters (P2-P4) upstream adjacent to P1 promoter. Three exons (exon 1B, 1D, 1E) with the transcription start site driven by these three promoters (P2-P4), respectively, usually skip exon 1A (coding exon), presumably to down-regulate the transcription level of group B CCM2 isoforms. Genomic structure of 5′ region of the human CCM2 locus is schematically summarized in this map. Noncoding region within a transcription-start exon labeled as white box while coding region within the exon labeled as black box. ( B ) Multiple-alignment between two alterative start codon exons (exon 1 and exon 1A) across species reveals a vertebrate-specific exon 1 and a mammalian-specific exon 1A and their evolutional relationship. Exon 1A is evolutionarily evolved from exon1 with its C-terminus homolog to the N-terminus of exon1. ( C ) Phylogenetic relationships between exon 1A and exon 1 among CCM2 isoforms across species based on neighbor joining (NJ) method which hypothesizes a stochastic process in different lineages during evolution.
    Figure Legend Snippet: Genomic structure, conservation, and variability among alterative start-codon exons and promotors of CCM2. ( A ) The complex promoter regions of human CCM2 locus were defined with bioinformatics (promoter prediction software from top to bottom: Cister, promotor2.0, Softberry, MEME, CTCFBSDB, BDGP/NNPP and Genscan as indicated by different colors). Symbols on top of DNA templates are on positive strand, below are on negative strand. The single promoter for the original bonafide start-codon exon, exon 1, simply lies immediately upstream of the transcription start site for exon 1, as P0. The promoter region for exon 1A is much more complicated. Although a seemingly weak promoter, P1 lies immediately upstream of its transcription-start site; in addition, there are three relatively strong promoters (P2-P4) upstream adjacent to P1 promoter. Three exons (exon 1B, 1D, 1E) with the transcription start site driven by these three promoters (P2-P4), respectively, usually skip exon 1A (coding exon), presumably to down-regulate the transcription level of group B CCM2 isoforms. Genomic structure of 5′ region of the human CCM2 locus is schematically summarized in this map. Noncoding region within a transcription-start exon labeled as white box while coding region within the exon labeled as black box. ( B ) Multiple-alignment between two alterative start codon exons (exon 1 and exon 1A) across species reveals a vertebrate-specific exon 1 and a mammalian-specific exon 1A and their evolutional relationship. Exon 1A is evolutionarily evolved from exon1 with its C-terminus homolog to the N-terminus of exon1. ( C ) Phylogenetic relationships between exon 1A and exon 1 among CCM2 isoforms across species based on neighbor joining (NJ) method which hypothesizes a stochastic process in different lineages during evolution.

    Techniques Used: Software, Labeling

    3) Product Images from "RNA-binding protein FXR1 is presented in rat brain in amyloid form"

    Article Title: RNA-binding protein FXR1 is presented in rat brain in amyloid form

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55528-6

    Amyloid aggregation of FXR1 protein depends on its N-terminal region. ( a ) The FXR1N(1-379)-YFP protein forms visible aggregates in yeast cells, whereas FXR1C(380-568)-YFP is evenly distributed in cytoplasm. Scale bar, 10 µm. Three independently obtained transformants expressing the FXR1N(1-379)-YFP and FXR1C(380-568)-YFP proteins were included in the analysis. About one hundred cells of each transformant were analyzed. ( b ) Protein lysates expressing the FXR1N(1-379)-YFP and FXR1C(380-568)-YFP proteins were centrifuged, separated into the soluble and insoluble fractions and analyzed by immunoblotting. The FXR1N(1-379)-YFP protein forms insoluble aggregates, whereas the FXR1C(380-568)-YFP is present in soluble form. P – pellet fraction; S - supernatant fraction. ( c , d ) - Relative intensities of bands corresponding to monomers and aggregates of FXR1N(1-379)-YFP and FXR1C(380-568)-YFP represented as mean ± SEM, for three independent protein samples.
    Figure Legend Snippet: Amyloid aggregation of FXR1 protein depends on its N-terminal region. ( a ) The FXR1N(1-379)-YFP protein forms visible aggregates in yeast cells, whereas FXR1C(380-568)-YFP is evenly distributed in cytoplasm. Scale bar, 10 µm. Three independently obtained transformants expressing the FXR1N(1-379)-YFP and FXR1C(380-568)-YFP proteins were included in the analysis. About one hundred cells of each transformant were analyzed. ( b ) Protein lysates expressing the FXR1N(1-379)-YFP and FXR1C(380-568)-YFP proteins were centrifuged, separated into the soluble and insoluble fractions and analyzed by immunoblotting. The FXR1N(1-379)-YFP protein forms insoluble aggregates, whereas the FXR1C(380-568)-YFP is present in soluble form. P – pellet fraction; S - supernatant fraction. ( c , d ) - Relative intensities of bands corresponding to monomers and aggregates of FXR1N(1-379)-YFP and FXR1C(380-568)-YFP represented as mean ± SEM, for three independent protein samples.

    Techniques Used: Expressing

    FXR1 forms SDS-resistant oligomers and aggregates in rat brain. ( a ) FXR1 is present in brain in fraction of oligomers and insoluble aggregates. Total protein lysate was divided into fractions of monomers less than 100 kDa, oligomers larger than 100 kDa, and insoluble aggregates. The fractions were subjected to SDD-PAGE and analyzed by immunoblotting with anti-FXR1 antibodies. ( b ) Relative intensity of bands corresponding to FXR1 monomers, oligomers and insoluble aggregates is represented as mean ± SEM for three independent brain samples. ( c ) A large portion of FXR1 in rat brain forms SDS-resistant aggregates. Total rat brain lysate was treated with1% SDS at RT, subjected to SDD-AGE, and analyzed by immunoblotting with anti-FXR1 antibodies. ( d ) Relative intensity of bands corresponding to FXR1 monomers and SDS-resistant aggregates is represented as mean ± SEM for three independent brain samples.
    Figure Legend Snippet: FXR1 forms SDS-resistant oligomers and aggregates in rat brain. ( a ) FXR1 is present in brain in fraction of oligomers and insoluble aggregates. Total protein lysate was divided into fractions of monomers less than 100 kDa, oligomers larger than 100 kDa, and insoluble aggregates. The fractions were subjected to SDD-PAGE and analyzed by immunoblotting with anti-FXR1 antibodies. ( b ) Relative intensity of bands corresponding to FXR1 monomers, oligomers and insoluble aggregates is represented as mean ± SEM for three independent brain samples. ( c ) A large portion of FXR1 in rat brain forms SDS-resistant aggregates. Total rat brain lysate was treated with1% SDS at RT, subjected to SDD-AGE, and analyzed by immunoblotting with anti-FXR1 antibodies. ( d ) Relative intensity of bands corresponding to FXR1 monomers and SDS-resistant aggregates is represented as mean ± SEM for three independent brain samples.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    4) Product Images from "A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana"

    Article Title: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008147

    U178G/U179G replicates but fails to exit local leaves following rub-inoculation. Total RNA was collected from: (A) inoculated leaves, (B) upper systemically infected leaves, or (C) petioles of inoculated leaves of 10 plants inoculated with U178G/U179G or wild type PSTVd (WT, one plant, positive control). Mock inoculation (M) was a negative control. (A) RNA blot assay indicates U178G/U179G replication in rub-inoculated leaves. (B) RNA blot assay indicates U178G/U179G is unable to traffic to upper leaves following rub inoculation. (C) RT-PCR indicates U178G/U179G is not present in petioles and fails to exit inoculated leaves. In A and B, the region of the blot corresponding to circular progeny genomes is shown. Loading controls were ribosomal RNA (rRNA) (A and B) and RT-PCR of actin mRNA (C), detected by ethidium bromide staining. Images are representative of 10 (A and B) and three (C) independent experiments.
    Figure Legend Snippet: U178G/U179G replicates but fails to exit local leaves following rub-inoculation. Total RNA was collected from: (A) inoculated leaves, (B) upper systemically infected leaves, or (C) petioles of inoculated leaves of 10 plants inoculated with U178G/U179G or wild type PSTVd (WT, one plant, positive control). Mock inoculation (M) was a negative control. (A) RNA blot assay indicates U178G/U179G replication in rub-inoculated leaves. (B) RNA blot assay indicates U178G/U179G is unable to traffic to upper leaves following rub inoculation. (C) RT-PCR indicates U178G/U179G is not present in petioles and fails to exit inoculated leaves. In A and B, the region of the blot corresponding to circular progeny genomes is shown. Loading controls were ribosomal RNA (rRNA) (A and B) and RT-PCR of actin mRNA (C), detected by ethidium bromide staining. Images are representative of 10 (A and B) and three (C) independent experiments.

    Techniques Used: Infection, Positive Control, Negative Control, Northern blot, Reverse Transcription Polymerase Chain Reaction, Staining

    Stability of U178G/U179G is similar to wild type PSTVd. (A) RNA blot of in vitro degradation assays performed at 28°C in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM phenylmethylsulfonyl fluoride), or uninfected N . benthamiana leaf extract prepared with the same buffer. (B) Percentage of remaining PSTVd wild type (WT) and U178G/U179G RNA over time was determined using Quantity One software. Data are representative of three independent experiments.
    Figure Legend Snippet: Stability of U178G/U179G is similar to wild type PSTVd. (A) RNA blot of in vitro degradation assays performed at 28°C in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM phenylmethylsulfonyl fluoride), or uninfected N . benthamiana leaf extract prepared with the same buffer. (B) Percentage of remaining PSTVd wild type (WT) and U178G/U179G RNA over time was determined using Quantity One software. Data are representative of three independent experiments.

    Techniques Used: Northern blot, In Vitro, Software

    5) Product Images from "Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)"

    Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20205043

    Expression profiles of 24 selected NtMADS-box genes in tobacco. The relative transcript abundances of 24 selected NtMADS-box genes were examined via qPCR and visualized as a histogram. The tobacco flowers (120 day old plants) and 6–7 week old seedlings grown in the soil were collected. Three independent biological experiments with four individual plants were collected for RNA extraction and qPCR analysis. 26S was used as an internal control. Error bars represent the SD ( n = 3). Different letters a,b,c above the bars indicate a significant difference ( p
    Figure Legend Snippet: Expression profiles of 24 selected NtMADS-box genes in tobacco. The relative transcript abundances of 24 selected NtMADS-box genes were examined via qPCR and visualized as a histogram. The tobacco flowers (120 day old plants) and 6–7 week old seedlings grown in the soil were collected. Three independent biological experiments with four individual plants were collected for RNA extraction and qPCR analysis. 26S was used as an internal control. Error bars represent the SD ( n = 3). Different letters a,b,c above the bars indicate a significant difference ( p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, RNA Extraction

    Expression profiles of 168 NtMADS-box genes in tissues at different developmental stages. The relative transcript abundances of 168 NtMADS-box were examined via microarray and visualized as a heatmap. The expression profiles of NtMADS-box genes in the 23 different samples, including dry seeds, germination seeds, cotyledons, leaves from two-true leaf stage (labeled as two true leaf_leaf), roots from two-true leaf stage (two true leaf_root), leaves from four-true leaf stage (four true leaf_leaf), roots from four-true leaf stage (four true leaf_root), leaves from six-true leaf stage (six true leaf_leaf), roots from six-true leaf stage (six true leaf_root), leaves from ten-true leaf stage (ten ture leaf_leaf), roots from ten-true leaf stage (ten ture leaf_root), and squaring stage (sepal, fibrous root, and flower), vein, ovary, filament, style, corolla, calyx, stigma, and anther. The X axis is the samples in tissues at different developmental stages. The color scale represents Log2 expression values. The symbol of the star in the MIKC C subfamily represents selected genes for confirming the gene expression by qPCR. Three independent biological experiments with four individual plants were collected for RNA extraction.
    Figure Legend Snippet: Expression profiles of 168 NtMADS-box genes in tissues at different developmental stages. The relative transcript abundances of 168 NtMADS-box were examined via microarray and visualized as a heatmap. The expression profiles of NtMADS-box genes in the 23 different samples, including dry seeds, germination seeds, cotyledons, leaves from two-true leaf stage (labeled as two true leaf_leaf), roots from two-true leaf stage (two true leaf_root), leaves from four-true leaf stage (four true leaf_leaf), roots from four-true leaf stage (four true leaf_root), leaves from six-true leaf stage (six true leaf_leaf), roots from six-true leaf stage (six true leaf_root), leaves from ten-true leaf stage (ten ture leaf_leaf), roots from ten-true leaf stage (ten ture leaf_root), and squaring stage (sepal, fibrous root, and flower), vein, ovary, filament, style, corolla, calyx, stigma, and anther. The X axis is the samples in tissues at different developmental stages. The color scale represents Log2 expression values. The symbol of the star in the MIKC C subfamily represents selected genes for confirming the gene expression by qPCR. Three independent biological experiments with four individual plants were collected for RNA extraction.

    Techniques Used: Expressing, Microarray, Labeling, Real-time Polymerase Chain Reaction, RNA Extraction

    6) Product Images from "Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control"

    Article Title: Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-019-10113-9

    Identification of diap1* -dsRNA produced in C. glutamicum . Structural analysis of the produced RNA with RNase A ( a ) and RNase III ( b ). Total RNA from C. glutamicum strain 2256LΔ rnc harboring pVC7H2 or pVH2-HvIap-1 was prepared, and then, each RNA sample treated with RNase (plus sign) or not treated (minus sign) was subjected to 6% PAGE. Prominent RNA bands corresponding to diap1* -dsRNA are indicated with black arrows. Size marker of dsRNAs is also shown
    Figure Legend Snippet: Identification of diap1* -dsRNA produced in C. glutamicum . Structural analysis of the produced RNA with RNase A ( a ) and RNase III ( b ). Total RNA from C. glutamicum strain 2256LΔ rnc harboring pVC7H2 or pVH2-HvIap-1 was prepared, and then, each RNA sample treated with RNase (plus sign) or not treated (minus sign) was subjected to 6% PAGE. Prominent RNA bands corresponding to diap1* -dsRNA are indicated with black arrows. Size marker of dsRNAs is also shown

    Techniques Used: Produced, Polyacrylamide Gel Electrophoresis, Marker

    Production of diap1* -dsRNA by C. glutamicum in batch fermentation. a Growth of C. glutamicum 2256LΔ rnc harboring pVC7H2 as a control or pVH2-HvIap-1. Average values and standard deviations (SDs) for three independent experiments are shown, although the variation range is too small to distinguish the SDs on the graph. b PAGE analysis of total RNA prepared from each C. glutamicum strain during the fermentation. Lane M shows dsRNA marker, and each culture time (h) is indicated at the top of the gel. The arrow and asterisks indicate the positions of diap1* -dsRNA and its possible degradation products, respectively. The result presents one representative diap1* -dsRNA production experiment in a jar fermentor
    Figure Legend Snippet: Production of diap1* -dsRNA by C. glutamicum in batch fermentation. a Growth of C. glutamicum 2256LΔ rnc harboring pVC7H2 as a control or pVH2-HvIap-1. Average values and standard deviations (SDs) for three independent experiments are shown, although the variation range is too small to distinguish the SDs on the graph. b PAGE analysis of total RNA prepared from each C. glutamicum strain during the fermentation. Lane M shows dsRNA marker, and each culture time (h) is indicated at the top of the gel. The arrow and asterisks indicate the positions of diap1* -dsRNA and its possible degradation products, respectively. The result presents one representative diap1* -dsRNA production experiment in a jar fermentor

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker

    7) Product Images from "TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer"

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aau7523

    TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P
    Figure Legend Snippet: TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P

    Techniques Used: In Vivo, Flow Cytometry, Mouse Assay, In Vitro, Labeling, Cytometry, Quantitative RT-PCR, Two Tailed Test

    TET2/3 facilitate CSR by regulating expression of the cytidine deaminase AID. ( A ) qRT-PCR analysis of Aicda mRNA expression in WT and Tet2/3 DKO B cells activated 4 days with LPS and IL-4. Aicda expression was normalized to Gapdh and then to the level in WT. Data shown are representative of two independent experiments with three technical replicates. * P
    Figure Legend Snippet: TET2/3 facilitate CSR by regulating expression of the cytidine deaminase AID. ( A ) qRT-PCR analysis of Aicda mRNA expression in WT and Tet2/3 DKO B cells activated 4 days with LPS and IL-4. Aicda expression was normalized to Gapdh and then to the level in WT. Data shown are representative of two independent experiments with three technical replicates. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    8) Product Images from "Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations"

    Article Title: Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations

    Journal: Frontiers in Neurology

    doi: 10.3389/fneur.2019.01219

    (A) Pedigree of family 1. The index case (III/4) is marked with an arrow. Black filled symbols represent patients with CCMs, gray striped symbols indicate relatives with neurological symptoms that are suggestive for CCM. Deceased family members are crossed out. Red stars indicate heterozygous carriers of the pathogenic CCM2 splice variant c.204+1G > A. (B,C) Representative axial T2-weighted (B) and sagittal T1-weighted (C) MR images of the index case at the age of 32. (D) Schematic exon-intron structure of CCM2 and selected protein-coding CCM2 transcripts that are listed in the ENSEMBL database. ENST00000258781.10 and ENST00000541586.5 are both expressed in blood lymphocytes but ENST00000541586.5 in which exon 3 is skipped is less abundant in brain or blood vessels. (E) Skipping of exon 3 on the c.204+1G > A CCM2 allele was confirmed by RT-PCR and cDNA sequencing. Lane 1: size marker, lane 2: control sample, lane 3: patient III/4. E, exon.
    Figure Legend Snippet: (A) Pedigree of family 1. The index case (III/4) is marked with an arrow. Black filled symbols represent patients with CCMs, gray striped symbols indicate relatives with neurological symptoms that are suggestive for CCM. Deceased family members are crossed out. Red stars indicate heterozygous carriers of the pathogenic CCM2 splice variant c.204+1G > A. (B,C) Representative axial T2-weighted (B) and sagittal T1-weighted (C) MR images of the index case at the age of 32. (D) Schematic exon-intron structure of CCM2 and selected protein-coding CCM2 transcripts that are listed in the ENSEMBL database. ENST00000258781.10 and ENST00000541586.5 are both expressed in blood lymphocytes but ENST00000541586.5 in which exon 3 is skipped is less abundant in brain or blood vessels. (E) Skipping of exon 3 on the c.204+1G > A CCM2 allele was confirmed by RT-PCR and cDNA sequencing. Lane 1: size marker, lane 2: control sample, lane 3: patient III/4. E, exon.

    Techniques Used: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Marker

    9) Product Images from "RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis"

    Article Title: RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2019/2841814

    Characterization of melanocytes and H 2 O 2 -induced oxidative death: (a) PIG1 and primary HEM were incubated with L-DOPA or PBS (control) for 5 h and images were obtained using phase contrast microscopy. Note that the melanocytes are branched and stained black by L-DOPA, confirming the presence of DOPA oxidase (tyrosinase) activity. (b) Indicated cells were cultured, and cell lysates were probed with MITF and TYRP-1 antibodies using western blotting. Equal loading was confirmed using β -actin antibodies. (c) PIG1 melanocytes were either left untreated or incubated with indicated concentration of H 2 O 2 for 2 h at 37°C. The oxidative stress was detected by staining cells with CellROX Orange dye for 30 min, and images were obtained by the Evos fluorescent microscope. (d) To observe the morphological changes, phase contrast microscopic images were obtained 3 h after exposure to H 2 O 2 . (e) PIG1 melanocytes were cultured with indicated concentration of H 2 O 2 for 6 h, 24 h, and 48 h. Cell viability was checked by trypan blue dye exclusion assay. Mean (three independent observations) values of viability percentages were plotted at different time intervals. Greater reduction in cell viability was found with increasing concentration of H 2 O 2 . Grey box includes the time points chosen for RNA-seq experiments. (f) Flow chart showing the different time points and concentrations of H 2 O 2 used in RNA-seq experiments.
    Figure Legend Snippet: Characterization of melanocytes and H 2 O 2 -induced oxidative death: (a) PIG1 and primary HEM were incubated with L-DOPA or PBS (control) for 5 h and images were obtained using phase contrast microscopy. Note that the melanocytes are branched and stained black by L-DOPA, confirming the presence of DOPA oxidase (tyrosinase) activity. (b) Indicated cells were cultured, and cell lysates were probed with MITF and TYRP-1 antibodies using western blotting. Equal loading was confirmed using β -actin antibodies. (c) PIG1 melanocytes were either left untreated or incubated with indicated concentration of H 2 O 2 for 2 h at 37°C. The oxidative stress was detected by staining cells with CellROX Orange dye for 30 min, and images were obtained by the Evos fluorescent microscope. (d) To observe the morphological changes, phase contrast microscopic images were obtained 3 h after exposure to H 2 O 2 . (e) PIG1 melanocytes were cultured with indicated concentration of H 2 O 2 for 6 h, 24 h, and 48 h. Cell viability was checked by trypan blue dye exclusion assay. Mean (three independent observations) values of viability percentages were plotted at different time intervals. Greater reduction in cell viability was found with increasing concentration of H 2 O 2 . Grey box includes the time points chosen for RNA-seq experiments. (f) Flow chart showing the different time points and concentrations of H 2 O 2 used in RNA-seq experiments.

    Techniques Used: Incubation, Microscopy, Staining, Activity Assay, Cell Culture, Western Blot, Concentration Assay, Exclusion Assay, RNA Sequencing Assay, Flow Cytometry

    10) Product Images from "A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress"

    Article Title: A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress

    Journal: Journal of Molecular Cell Biology

    doi: 10.1093/jmcb/mjz094

    CircRNA profiles in human cancer cells. ( A ) Comparison of high-confidence circRNAs across A549, HeLa, and MCF-7 (supported by a minimum of 5 back-splice reads in any 2 samples). Most circRNAs are expressed in at least two cell types. A similar partition occurs when a further filter on minimum expression of the host gene is applied ( Supplementary Figure S3H ). ( B ) Scatter plot comparing the expression of circRNAs in back-splice RPM to the expression of the host gene in TPM. circRNA expression does not generally reflect the abundance of the host gene. Mean expression across replicates is shown for each cell line under hypoxic (blue) and normoxic (red) conditions. Linear regression lines and Pearson correlation coefficients with associated P -values are reported. ( C ) Scheme showing how ‘percent circularized’ metric is computed from back-splice reads and reads spanning the corresponding linear splice junctions. ( D ) In total, 210 circRNAs are more abundant than their linear counterparts, as exemplified by circATXN7 (hsa_circ_0007761; labeled in orange; Huang et al., 2019 ) in MCF-7 cells. Violin plot shows distribution of ‘percent circularized’ values for circRNAs from the three cell lines (mean per cell line across all replicates and conditions). Orange lines indicate circRNAs with > 20% and 50% relative abundance. ( E ) Genome browser view of ATXN7 gene showing RNA-Seq data from MCF-7 cells under normoxic conditions. Coverage of chimeric alignments (back-splice reads, red, bottom) documents back-splicing of exon 4 to exon 3 to produce circATXN7. The high abundance of circATXN7 is reflected in a peak in the coverage of linearly mapped reads (blue, top) corresponding to internal regions of the circRNA, while the remainder of the linear transcript shows less coverage.
    Figure Legend Snippet: CircRNA profiles in human cancer cells. ( A ) Comparison of high-confidence circRNAs across A549, HeLa, and MCF-7 (supported by a minimum of 5 back-splice reads in any 2 samples). Most circRNAs are expressed in at least two cell types. A similar partition occurs when a further filter on minimum expression of the host gene is applied ( Supplementary Figure S3H ). ( B ) Scatter plot comparing the expression of circRNAs in back-splice RPM to the expression of the host gene in TPM. circRNA expression does not generally reflect the abundance of the host gene. Mean expression across replicates is shown for each cell line under hypoxic (blue) and normoxic (red) conditions. Linear regression lines and Pearson correlation coefficients with associated P -values are reported. ( C ) Scheme showing how ‘percent circularized’ metric is computed from back-splice reads and reads spanning the corresponding linear splice junctions. ( D ) In total, 210 circRNAs are more abundant than their linear counterparts, as exemplified by circATXN7 (hsa_circ_0007761; labeled in orange; Huang et al., 2019 ) in MCF-7 cells. Violin plot shows distribution of ‘percent circularized’ values for circRNAs from the three cell lines (mean per cell line across all replicates and conditions). Orange lines indicate circRNAs with > 20% and 50% relative abundance. ( E ) Genome browser view of ATXN7 gene showing RNA-Seq data from MCF-7 cells under normoxic conditions. Coverage of chimeric alignments (back-splice reads, red, bottom) documents back-splicing of exon 4 to exon 3 to produce circATXN7. The high abundance of circATXN7 is reflected in a peak in the coverage of linearly mapped reads (blue, top) corresponding to internal regions of the circRNA, while the remainder of the linear transcript shows less coverage.

    Techniques Used: Expressing, Labeling, RNA Sequencing Assay

    11) Product Images from "The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature"

    Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12897-w

    ERG regulates thrombomodulin expression and activity in vitro. a qPCR analysis of thrombomodulin ( TM ) mRNA expression in control (siCtrl) and ERG-deficient (siERG) HUVEC after 12, 24 and 48 h treatment ( n = 3 independent experiments). Data were normalized to GAPDH . b Representative immunoblot and quantification of TM following siCtrl or siERG treatment on HUVEC for 12, 24 and 48 h ( n = 4 independent experiments). Data were normalized to GAPDH. c Representative image and quantification of TM expression (green) in HUVEC transfected with siCtrl or siERG siRNA for 48 h by immunofluorescence; nuclei are identified by DAPI (blue) and cells are co- stained for ERG (magenta). Scale bar 40 µm. Quantification represents the mean pixel intensity for TM signal (arbitrary unit, A.U.) per cell. d , e Representative immunoblot and quantification of ERG and TM expression in control (siCtrl) and ERG-deficient (siERG) d HDMEC (microvascular EC) or e HDBEC (microvascular EC) after 48 h siRNA treatment ( n = 3). f qPCR analysis of TM mRNA expression in HUVEC transfected with control pcDNA or ERG cDNA expression plasmid (ERG) ( n = 3). Data were normalised to GAPDH . g siCtrl or siERG-treated HUVEC for 48 h were incubated with protein C (300 nM), CaCl 2 (5 mM), thrombin (10 nM). After 10, 20, 30 and 60 min of incubation at 37 °C, anti-thrombin III (100 nM) and heparin (15 U per ml) were added to neutralise thrombin, and protein C activity was measured using chromogenic substrate S-2366 (0.5 mM) ( n = 3). Data are expressed as relative activated protein C (APC) concentration normalised to siCtrl-treated condition following 60 min of incubation. All graphical data are mean ± s.e.m., * P
    Figure Legend Snippet: ERG regulates thrombomodulin expression and activity in vitro. a qPCR analysis of thrombomodulin ( TM ) mRNA expression in control (siCtrl) and ERG-deficient (siERG) HUVEC after 12, 24 and 48 h treatment ( n = 3 independent experiments). Data were normalized to GAPDH . b Representative immunoblot and quantification of TM following siCtrl or siERG treatment on HUVEC for 12, 24 and 48 h ( n = 4 independent experiments). Data were normalized to GAPDH. c Representative image and quantification of TM expression (green) in HUVEC transfected with siCtrl or siERG siRNA for 48 h by immunofluorescence; nuclei are identified by DAPI (blue) and cells are co- stained for ERG (magenta). Scale bar 40 µm. Quantification represents the mean pixel intensity for TM signal (arbitrary unit, A.U.) per cell. d , e Representative immunoblot and quantification of ERG and TM expression in control (siCtrl) and ERG-deficient (siERG) d HDMEC (microvascular EC) or e HDBEC (microvascular EC) after 48 h siRNA treatment ( n = 3). f qPCR analysis of TM mRNA expression in HUVEC transfected with control pcDNA or ERG cDNA expression plasmid (ERG) ( n = 3). Data were normalised to GAPDH . g siCtrl or siERG-treated HUVEC for 48 h were incubated with protein C (300 nM), CaCl 2 (5 mM), thrombin (10 nM). After 10, 20, 30 and 60 min of incubation at 37 °C, anti-thrombin III (100 nM) and heparin (15 U per ml) were added to neutralise thrombin, and protein C activity was measured using chromogenic substrate S-2366 (0.5 mM) ( n = 3). Data are expressed as relative activated protein C (APC) concentration normalised to siCtrl-treated condition following 60 min of incubation. All graphical data are mean ± s.e.m., * P

    Techniques Used: Expressing, Activity Assay, In Vitro, Real-time Polymerase Chain Reaction, Transfection, Immunofluorescence, Staining, Plasmid Preparation, Incubation, Concentration Assay

    12) Product Images from "Strigolactones enhance root‐knot nematode (Meloidogyne graminicola) infection in rice by antagonizing the jasmonate pathway"

    Article Title: Strigolactones enhance root‐knot nematode (Meloidogyne graminicola) infection in rice by antagonizing the jasmonate pathway

    Journal: The New Phytologist

    doi: 10.1111/nph.15953

    Meloidogyne graminicola infection in rice roots influences the strigolactone ( SL ) and jasmonic acid ( JA ) pathways in opposite ways. (a) Relative expression of SL biosynthesis and signaling genes with and without M. graminicola infection 1 d post‐inoculation (dpi). The messenger RNA levels were measured by quantitative real‐time PCR in three technical replicates per sample. Transcripts of each gene were normalized to OsExp transcripts. Data are presented as the mean + SE ( n = 4), and each biological replicate was a pool of four to six plants. (b) Biosynthesis and signaling genes of SL pathway in rice. (c) Concentrations of jasmonates in rice roots with and without M. graminicola infection 1 dpi. Hormones were analyzed by LC – MS / MS . Data are presented as the mean + SE ( n = 5), and each replicate was a pool of four to six plants. Data were analyzed using a two‐tailed Student's t ‐test: *, P
    Figure Legend Snippet: Meloidogyne graminicola infection in rice roots influences the strigolactone ( SL ) and jasmonic acid ( JA ) pathways in opposite ways. (a) Relative expression of SL biosynthesis and signaling genes with and without M. graminicola infection 1 d post‐inoculation (dpi). The messenger RNA levels were measured by quantitative real‐time PCR in three technical replicates per sample. Transcripts of each gene were normalized to OsExp transcripts. Data are presented as the mean + SE ( n = 4), and each biological replicate was a pool of four to six plants. (b) Biosynthesis and signaling genes of SL pathway in rice. (c) Concentrations of jasmonates in rice roots with and without M. graminicola infection 1 dpi. Hormones were analyzed by LC – MS / MS . Data are presented as the mean + SE ( n = 5), and each replicate was a pool of four to six plants. Data were analyzed using a two‐tailed Student's t ‐test: *, P

    Techniques Used: Infection, Expressing, Real-time Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Two Tailed Test

    13) Product Images from "Functional and structural characterization of the chikungunya virus translational recoding signals"

    Article Title: Functional and structural characterization of the chikungunya virus translational recoding signals

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.005606

    Structural analyses of the CHIKV recoding signals. A–C , stimulatory elements for Af/As and Carib CHIKV TCR and −1 PRF signals resolved through SHAPE. RNA templates containing the translational recoding sequences were transcribed from corresponding Dual-Luciferase reporter vectors and treated with NMIA. Untreated RNAs were used as negative controls. γ- 32 P-radiolabeled cDNA products were separated by 8% urea-PAGE and visualized via a Fujifilm phosphorimaging system. Autoradiograms are annotated to indicate the respective sequencing lanes (G, A, U, and C), an untreated control lane (−), and the NMIA-labeled experimental lane (+). Circles denote the relative reactivity of bases where white is the most unreactive and black is the most reactive. For added visual clarity of the CHIKV TCR gels, a longer run of the samples has been provided to further separate the 5′ sequence information. D and F , structures of CHIKV recoding signals derived from the above SHAPE data. Circles correspond to the previously described nucleotide reactivity scale. Polymorphisms between the Af/As and Carib consensus sequences are indicated in red. E and G , three-dimensional models of the CHIKV TCR and −1 PRF signals were generated by molecular dynamics simulations and visualized in PyMOL.
    Figure Legend Snippet: Structural analyses of the CHIKV recoding signals. A–C , stimulatory elements for Af/As and Carib CHIKV TCR and −1 PRF signals resolved through SHAPE. RNA templates containing the translational recoding sequences were transcribed from corresponding Dual-Luciferase reporter vectors and treated with NMIA. Untreated RNAs were used as negative controls. γ- 32 P-radiolabeled cDNA products were separated by 8% urea-PAGE and visualized via a Fujifilm phosphorimaging system. Autoradiograms are annotated to indicate the respective sequencing lanes (G, A, U, and C), an untreated control lane (−), and the NMIA-labeled experimental lane (+). Circles denote the relative reactivity of bases where white is the most unreactive and black is the most reactive. For added visual clarity of the CHIKV TCR gels, a longer run of the samples has been provided to further separate the 5′ sequence information. D and F , structures of CHIKV recoding signals derived from the above SHAPE data. Circles correspond to the previously described nucleotide reactivity scale. Polymorphisms between the Af/As and Carib consensus sequences are indicated in red. E and G , three-dimensional models of the CHIKV TCR and −1 PRF signals were generated by molecular dynamics simulations and visualized in PyMOL.

    Techniques Used: Luciferase, Polyacrylamide Gel Electrophoresis, Sequencing, Labeling, Derivative Assay, Generated

    14) Product Images from "Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1"

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083114

    SIRT1 overexpression partially restores the transcriptional repression within specific loci in sir2Δ mutant. RT-PCR transcriptional analysis in WT and sir2Δ strains transformed with empty plasmid (-), SIRT1 construct (+) or SIRT1 - H363Y mutant construct (+*) both in repression (glucose) and induction (galactose) conditions. (A) rDNA locus: NTS1r and NTS2 ; (B) TEL VI locus: YFR057W and IRC7 ; (C) HM loci: HMLalpha1 . Histograms indicate averages and Std. Dev. bars from at least three independent biological replicates. Two−tailed t−test was applied for statistical analysis. Asterisks indicate statistically significant differences between sir2Δ - and sir2Δ + or between sir2Δ +* and sir2Δ + in galactose medium; α = 0.05. (Percentages of p−value: *p
    Figure Legend Snippet: SIRT1 overexpression partially restores the transcriptional repression within specific loci in sir2Δ mutant. RT-PCR transcriptional analysis in WT and sir2Δ strains transformed with empty plasmid (-), SIRT1 construct (+) or SIRT1 - H363Y mutant construct (+*) both in repression (glucose) and induction (galactose) conditions. (A) rDNA locus: NTS1r and NTS2 ; (B) TEL VI locus: YFR057W and IRC7 ; (C) HM loci: HMLalpha1 . Histograms indicate averages and Std. Dev. bars from at least three independent biological replicates. Two−tailed t−test was applied for statistical analysis. Asterisks indicate statistically significant differences between sir2Δ - and sir2Δ + or between sir2Δ +* and sir2Δ + in galactose medium; α = 0.05. (Percentages of p−value: *p

    Techniques Used: Over Expression, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Plasmid Preparation, Construct, Two Tailed Test

    15) Product Images from "KIS, a Kinase Associated with Microtubule Regulators, Enhances Translation of AMPA Receptors and Stimulates Dendritic Spine Remodeling"

    Article Title: KIS, a Kinase Associated with Microtubule Regulators, Enhances Translation of AMPA Receptors and Stimulates Dendritic Spine Remodeling

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1573-14.2014

    KIS promotes CPEB3 dissociation from GluR2 3′UTR and regulates its polyadenylation. A , Luciferase reporter analysis of the GluR2 3′UTR. HEK293T cells were transfected with the Gaussia luciferase ORF fused to the 3′UTR of GluR2, in combination with plasmid vectors expressing KIS, CPEB3, or CPEB3 + KIS. Values were made relative to those obtained from an empty vector (Ctrl). Mean values ( n = 6) and confidence limits (α = 0.05) for the mean are represented. Obtained p values for relevant pairwise t tests are shown. B , Effect of RNase treatment on the association of KIS to CPEB3. Total extracts (input) from HEK293T cells expressing CPEB3 and FLAG or FLAG-KIS were incubated with (+) or without (−) RNase prior to immunoprecipitation (IP), and the corresponding αFLAG immunoprecipitates (FLAG IP), were analyzed by immunoblotting to detect CPEB3 and FLAG-KIS proteins. C , RNA immunoprecipitation and quantitative PCR. HEK293T cells were transfected with the firefly luciferase ORF fused to the 3′UTR of GluR2, in combination with vectors expressing FLAG, FLAG-CPEB3, or FLAG-CPEB3 + KIS. Cell lysates were immunoprecipitated with FLAG IgG, and precipitated mRNAs were reverse transcribed and quantified by RT-qPCR. Luc-GluR2 3′UTR mRNA levels were made relative to GADPH mRNA. Mean values from three independent experiments and confidence limits (α = 0.05) for the mean are plotted. Obtained p values for relevant pairwise t tests are shown. D , Polyadenylation assays of the GluR2 3′UTR. Cell extracts from HEK293T cells as in C were used to amplify poly(A) (top) and total (bottom) Luc-GluR2 3′UTR mRNAs, and the resulting products were separated by agarose electrophoresis. E , Cell extracts as in D were analyzed by immunoblotting to detect CPEB3 and KIS proteins. F , Polyadenylation profiles obtained by densitometric analysis of samples as in D . G , Quantification of relative polyadenylation levels from profiles in F as poly(A)/nonpoly(A) ratios. Mean values ( n = 3) and confidence limits (α = 0.05) for the mean are represented. Obtained p values for relevant pairwise t tests are shown.
    Figure Legend Snippet: KIS promotes CPEB3 dissociation from GluR2 3′UTR and regulates its polyadenylation. A , Luciferase reporter analysis of the GluR2 3′UTR. HEK293T cells were transfected with the Gaussia luciferase ORF fused to the 3′UTR of GluR2, in combination with plasmid vectors expressing KIS, CPEB3, or CPEB3 + KIS. Values were made relative to those obtained from an empty vector (Ctrl). Mean values ( n = 6) and confidence limits (α = 0.05) for the mean are represented. Obtained p values for relevant pairwise t tests are shown. B , Effect of RNase treatment on the association of KIS to CPEB3. Total extracts (input) from HEK293T cells expressing CPEB3 and FLAG or FLAG-KIS were incubated with (+) or without (−) RNase prior to immunoprecipitation (IP), and the corresponding αFLAG immunoprecipitates (FLAG IP), were analyzed by immunoblotting to detect CPEB3 and FLAG-KIS proteins. C , RNA immunoprecipitation and quantitative PCR. HEK293T cells were transfected with the firefly luciferase ORF fused to the 3′UTR of GluR2, in combination with vectors expressing FLAG, FLAG-CPEB3, or FLAG-CPEB3 + KIS. Cell lysates were immunoprecipitated with FLAG IgG, and precipitated mRNAs were reverse transcribed and quantified by RT-qPCR. Luc-GluR2 3′UTR mRNA levels were made relative to GADPH mRNA. Mean values from three independent experiments and confidence limits (α = 0.05) for the mean are plotted. Obtained p values for relevant pairwise t tests are shown. D , Polyadenylation assays of the GluR2 3′UTR. Cell extracts from HEK293T cells as in C were used to amplify poly(A) (top) and total (bottom) Luc-GluR2 3′UTR mRNAs, and the resulting products were separated by agarose electrophoresis. E , Cell extracts as in D were analyzed by immunoblotting to detect CPEB3 and KIS proteins. F , Polyadenylation profiles obtained by densitometric analysis of samples as in D . G , Quantification of relative polyadenylation levels from profiles in F as poly(A)/nonpoly(A) ratios. Mean values ( n = 3) and confidence limits (α = 0.05) for the mean are represented. Obtained p values for relevant pairwise t tests are shown.

    Techniques Used: Luciferase, Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Electrophoresis

    16) Product Images from "Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia"

    Article Title: Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-8-123

    ROS drive cytokine and chemokine mRNA expression in virus-infected microglia . Microglial cell cultures were pre-treated with the NADPH oxidase inhibitors DPI or APO for 1 h prior to a 5 h exposure to HSV. Following viral infection, RNA was extracted and cDNA synthesized to assess mRNA expression through quantitative real-time PCR for A) TNF-α; B) IL-1β; C) CCL2; and D) CXCL10. mRNA levels were normalized to the housekeeping gene HPRT and are presented as fold induction over uninfected controls. Data shown are representative of three individual experiments using microglial cells obtained from different animals.
    Figure Legend Snippet: ROS drive cytokine and chemokine mRNA expression in virus-infected microglia . Microglial cell cultures were pre-treated with the NADPH oxidase inhibitors DPI or APO for 1 h prior to a 5 h exposure to HSV. Following viral infection, RNA was extracted and cDNA synthesized to assess mRNA expression through quantitative real-time PCR for A) TNF-α; B) IL-1β; C) CCL2; and D) CXCL10. mRNA levels were normalized to the housekeeping gene HPRT and are presented as fold induction over uninfected controls. Data shown are representative of three individual experiments using microglial cells obtained from different animals.

    Techniques Used: Expressing, Infection, Synthesized, Real-time Polymerase Chain Reaction

    17) Product Images from "Unique features of the grapevine VvK5.1 channel support novel functions for outward K+ channels in plants"

    Article Title: Unique features of the grapevine VvK5.1 channel support novel functions for outward K+ channels in plants

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erz341

    VvK5.1 transcript levels in grapevine organs and during berry development. RT-qPCR was performed on first-strand cDNAs synthesized from total RNAs of different organs. (A) VvK5.1 transcript levels in roots from rooted canes, or in vegetative organs (stems, leaves, tendrils, and stalks) collected at fruit set (15 d after flowering), flowers, or in berries at three different developmental stages (fruit set, veraison, and ripeness). Vegetative organs, flowers, and berries were collected from grapevines grown in open field conditions under standard irrigation. (B) VvK5.1 transcript levels of berries collected at different developmental stages in field conditions. The fruit set, veraison, and ripening phases are indicated. Note that VvK5.1 expression suddenly and strongly increased in grape berries at veraison. The mean values and SE of two biological replicates are presented.
    Figure Legend Snippet: VvK5.1 transcript levels in grapevine organs and during berry development. RT-qPCR was performed on first-strand cDNAs synthesized from total RNAs of different organs. (A) VvK5.1 transcript levels in roots from rooted canes, or in vegetative organs (stems, leaves, tendrils, and stalks) collected at fruit set (15 d after flowering), flowers, or in berries at three different developmental stages (fruit set, veraison, and ripeness). Vegetative organs, flowers, and berries were collected from grapevines grown in open field conditions under standard irrigation. (B) VvK5.1 transcript levels of berries collected at different developmental stages in field conditions. The fruit set, veraison, and ripening phases are indicated. Note that VvK5.1 expression suddenly and strongly increased in grape berries at veraison. The mean values and SE of two biological replicates are presented.

    Techniques Used: Quantitative RT-PCR, Synthesized, Expressing

    18) Product Images from "The lrp Gene and Its Role in Type I Fimbriation in Citrobacter rodentium"

    Article Title: The lrp Gene and Its Role in Type I Fimbriation in Citrobacter rodentium

    Journal:

    doi: 10.1128/JB.187.20.7009-7017.2005

    Yeast agglutination experiments. We mixed 10 μl of 3% (wt/vol) Saccharomyces cerevisiae yeast cells on a glass slide with the same volume of three bacterial cultures of Citrobacter rodentium EM2 (A), wild-type (wt) (B), and EM3 (C) strains at
    Figure Legend Snippet: Yeast agglutination experiments. We mixed 10 μl of 3% (wt/vol) Saccharomyces cerevisiae yeast cells on a glass slide with the same volume of three bacterial cultures of Citrobacter rodentium EM2 (A), wild-type (wt) (B), and EM3 (C) strains at

    Techniques Used: Agglutination

    Related Articles

    Clone Assay:

    Article Title: RNA-binding protein FXR1 is presented in rat brain in amyloid form
    Article Snippet: .. Cloning of the FXR1 gene fragments Total rat RNA was extracted from brain homogenates using TRIzol reagent (Invitrogen) according to the manufacturer protocol. cDNA synthesis was performed with SuperScript III Reverse Transcriptase (Invitrogen). cDNA was further used for FXR1 fragments synthesis. .. The fragment of FXR1 gene coding for 1-379 aa for expression in E. coli was amplified with fxr1EcoRIforward and fxr1(379)BamHIreverse primers and inserted into pET302 vector to obtain the pET302-FXR1(1-379) plasmid.

    Amplification:

    Article Title: Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations
    Article Snippet: Three hundred nanogram of total RNA were reverse described into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA). .. The exons 1, 3, 4, and 5 (exon numbering according to LRG_664) of the CCM2 transcript LRG_664t2 were amplified using specific forward (5′-GCGGCGATATGGAAGAGG-3′) and reverse (5'-GCACCCTGAGGATGATATC-3′) primers ( , ).

    Article Title: RNA-binding protein FXR1 is presented in rat brain in amyloid form
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    Article Title: Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi
    Article Snippet: Contaminating DNA in the RNA samples was removed by DNase I (New England BioLabs, Ipswich, MA) digestions and confirmed by PCR amplification of the flaB gene of B. burgdorferi . .. The cDNA was prepared from 1 μg RNA using Superscript III reverse transcriptase with random primers (Invitrogen, Carlsbad, CA). qRT-PCR was performed using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) on an ABI 7000 sequence detection system.

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
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    Article Title: Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein
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    DNA Synthesis:

    Article Title: Strigolactones enhance root‐knot nematode (Meloidogyne graminicola) infection in rice by antagonizing the jasmonate pathway
    Article Snippet: Paragraph title: RNA isolation and complementary DNA synthesis ... 500 ng total RNA into complementary DNA (cDNA) was achieved by using SuperScript III reverse transcriptase (Invitrogen) and 50 pmol Oligo (dT)12‐18 Primer (Invitrogen) in a reaction volume of 20 μl.

    Synthesized:

    Article Title: Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control
    Article Snippet: .. First-strand cDNA was synthesized from 1 μg of the total RNA using SuperScript III Reverse Transcriptase (Life Technologies, Tokyo, Japan) and primer P19. qPCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) according to the manufacturer’s protocol in a LightCycler 96 instrument (Roche, Basel, Switzerland). .. The expression level of diap1 relative to the internal control, ribosomal protein 49 gene (rp49 ; accession number: AB480201), was calculated by the 2−ΔΔC t method (Livak and Schmittgen ); primer pairs P17 and P18, and P15 and P16, were used respectively (nucleotides 1649–1783 of diap1 -cDNA [Supplemental Fig. ]; Chikami et al. ).

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: .. Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific). .. Gene expression was normalized to Gapdh .

    Quantitative RT-PCR:

    Article Title: RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis
    Article Snippet: RT-PCR and Western Blotting About 2 μ g of RNA was reverse transcribed to generate cDNA using random primers and Superscript III reverse transcriptase (Invitrogen). .. Quantitative RT-PCR reactions were carried out in triplicates on a QuantStudio 12K Flex Real Time PCR machine.

    Article Title: A calmodulin‐like protein regulates plasmodesmal closure during bacterial immune responses
    Article Snippet: .. Quantitative RT‐PCR analysis RNA was extracted from leaves using TRIzol (Invitrogen) and treated with Turbo DNA‐free kit (Ambion, Thermo Fisher Scientific) before cDNA synthesis using SuperScript® III Reverse Transcriptase (Invitrogen). .. Quantitative reverse transcription polymerase chain reaction (RT‐PCR) was performed on the cDNA samples with primers listed in Table using the fluorescence output from a QuantStudio™ 12K Flex Real‐Time PCR System.

    Article Title: Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi
    Article Snippet: .. The cDNA was prepared from 1 μg RNA using Superscript III reverse transcriptase with random primers (Invitrogen, Carlsbad, CA). qRT-PCR was performed using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) on an ABI 7000 sequence detection system. ..

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: .. Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific). .. Gene expression was normalized to Gapdh .

    SYBR Green Assay:

    Article Title: Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi
    Article Snippet: .. The cDNA was prepared from 1 μg RNA using Superscript III reverse transcriptase with random primers (Invitrogen, Carlsbad, CA). qRT-PCR was performed using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) on an ABI 7000 sequence detection system. ..

    Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
    Article Snippet: Quantitative Real-Time PCR of Selected NtMADS-Box Genes A total of 2 μg of total RNA in a 20 μL reaction was converted to cDNA with a SuperScript III Reverse Transcriptase (Invitrogen, Waltham, Massachusetts, USA) by the manufacturer’s instructions on an Eppendorf Mastercycler thermocycler (Eppendorf AG, Germany) with the following conditions: 25 °C for 5 min, 50 °C for 60 min, 70 °C for 15 min, followed by a hold at 4 °C until use in a qPCR reaction. .. A total of 60 μL of deionized water was added into 20 μL cDNA, and 1 μL of diluted cDNA mixture was used as the input for the qPCR reaction. qPCR reactions were made with a SuperReal PreMix Plus SYBR Green Kit (TIANGEN Biotech, BeiJing, China) following manufacturer’s instructions in a 20 μL volume. qPCR was done on an Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System (ThemoFisher Scientific, Waltham, Massachusetts, USA) with the following cycling conditions: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 32 s. The melt curve conditions were 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 sec. All samples had only one melt temperature peak.

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: .. Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific). .. Gene expression was normalized to Gapdh .

    Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature
    Article Snippet: Real-time polymerase chain reaction Total RNA from tissues and HUVEC was isolated by using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using Superscript III Reverse Transcriptase (Invitrogen). .. Quantitative real-time PCR was performed using PerfeCTa SYBR Green Fastmix (Quanta Biosciences) on a Bio-Rad CFX96 system.

    Incubation:

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: .. RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. ..

    Article Title: A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress
    Article Snippet: For the RNase R treatment, 10 μg total RNA were incubated at 37°C for 40 min with or without 10 units of RNase R (Epicenter), followed by 3 min incubation at 95°C for RNase R inactivation. .. After treatment and precipitation, RNA was recovered and cDNAs were synthetized by RT-PCR using SuperScript III Reverse Transcriptase (Life Technologies), dNTPs, and random hexamers (dNTP Mix and Hexanucleotide Mix, Sigma-Aldrich) following the SuperScript III protocol recommended by the manufacturer.

    Expressing:

    Article Title: RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis
    Article Snippet: RT-PCR and Western Blotting About 2 μ g of RNA was reverse transcribed to generate cDNA using random primers and Superscript III reverse transcriptase (Invitrogen). .. The relative expression of each gene was calculated using the 2-DDCT method with GAPDH as reference.

    Article Title: A calmodulin‐like protein regulates plasmodesmal closure during bacterial immune responses
    Article Snippet: Quantitative RT‐PCR analysis RNA was extracted from leaves using TRIzol (Invitrogen) and treated with Turbo DNA‐free kit (Ambion, Thermo Fisher Scientific) before cDNA synthesis using SuperScript® III Reverse Transcriptase (Invitrogen). .. Quantitative RT‐PCR analysis via the 2−ΔC т method to calculate the gene expression level relative to either GAPDH‐A (At3g26650) or UBI10 (At4g05320) as an internal control (Schmittgen & Livak, ).

    Article Title: RNA-binding protein FXR1 is presented in rat brain in amyloid form
    Article Snippet: Cloning of the FXR1 gene fragments Total rat RNA was extracted from brain homogenates using TRIzol reagent (Invitrogen) according to the manufacturer protocol. cDNA synthesis was performed with SuperScript III Reverse Transcriptase (Invitrogen). cDNA was further used for FXR1 fragments synthesis. .. The fragment of FXR1 gene coding for 1-379 aa for expression in E. coli was amplified with fxr1EcoRIforward and fxr1(379)BamHIreverse primers and inserted into pET302 vector to obtain the pET302-FXR1(1-379) plasmid.

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: .. RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. ..

    Article Title: Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control
    Article Snippet: First-strand cDNA was synthesized from 1 μg of the total RNA using SuperScript III Reverse Transcriptase (Life Technologies, Tokyo, Japan) and primer P19. qPCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) according to the manufacturer’s protocol in a LightCycler 96 instrument (Roche, Basel, Switzerland). .. The expression level of diap1 relative to the internal control, ribosomal protein 49 gene (rp49 ; accession number: AB480201), was calculated by the 2−ΔΔC t method (Livak and Schmittgen ); primer pairs P17 and P18, and P15 and P16, were used respectively (nucleotides 1649–1783 of diap1 -cDNA [Supplemental Fig. ]; Chikami et al. ).

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific). .. Gene expression was normalized to Gapdh .

    Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature
    Article Snippet: Real-time polymerase chain reaction Total RNA from tissues and HUVEC was isolated by using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using Superscript III Reverse Transcriptase (Invitrogen). .. Gene expression values were normalized to GAPDH expression (human) or 18s (mouse).

    Modification:

    Article Title: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana
    Article Snippet: Chemical probing of loop 27 structure DMS and SHAPE modification with benzoyl cyanide BzCN, as well as data analysis, were carried out essentially as described by Tijerina et al . (2007) [ ] and Giguère et al . (2014) [ ], respectively. .. Templates for in vitro transcription of these strands were prepared by performing reverse transcription using SuperScript III Reverse Transcriptase (ThermoFisher Scientific, Waltham, MA) with RNA extracts obtained from infected plants to generate unit-length cDNAs from circular genomic PSTVd RNA.

    Article Title: Functional and structural characterization of the chikungunya virus translational recoding signals
    Article Snippet: .. The primer was annealed to modified RNA, and subsequent reverse transcription (using Superscript III reverse transcriptase, Thermo Scientific, catalogue number 18080044) was carried out as reported previously ( , ). .. Radioactivity of cDNA samples was standardized with a liquid scintillator prior to electrophoresis though 8% urea-PAGE (SequaGel UreaGel system, National Diagnostics, catalogue number EC-833). cDNA products were visualized on a Fujifilm phosphorimaging system.

    Western Blot:

    Article Title: RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis
    Article Snippet: .. RT-PCR and Western Blotting About 2 μ g of RNA was reverse transcribed to generate cDNA using random primers and Superscript III reverse transcriptase (Invitrogen). .. Quantitative RT-PCR reactions were carried out in triplicates on a QuantStudio 12K Flex Real Time PCR machine.

    Infection:

    Article Title: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana
    Article Snippet: .. Templates for in vitro transcription of these strands were prepared by performing reverse transcription using SuperScript III Reverse Transcriptase (ThermoFisher Scientific, Waltham, MA) with RNA extracts obtained from infected plants to generate unit-length cDNAs from circular genomic PSTVd RNA. ..

    Article Title: Strigolactones enhance root‐knot nematode (Meloidogyne graminicola) infection in rice by antagonizing the jasmonate pathway
    Article Snippet: The whole root systems from both the infected and uninfected control plants were collected 1 d after inoculation. .. 500 ng total RNA into complementary DNA (cDNA) was achieved by using SuperScript III reverse transcriptase (Invitrogen) and 50 pmol Oligo (dT)12‐18 Primer (Invitrogen) in a reaction volume of 20 μl.

    Polymerase Chain Reaction:

    Article Title: Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations
    Article Snippet: Three hundred nanogram of total RNA were reverse described into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA). .. PCR products were size-separated by agarose gel electrophoresis and visualized on a Gel Doc™ EZ Imager (Bio-Rad, Hercules, USA).

    Article Title: Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi
    Article Snippet: .. The cDNA was prepared from 1 μg RNA using Superscript III reverse transcriptase with random primers (Invitrogen, Carlsbad, CA). qRT-PCR was performed using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) on an ABI 7000 sequence detection system. ..

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. .. The resulting cDNAs were amplified by PCR co-amplification using the following primer pairs: SIRT1-F/SIRT1-R, NTS2-F/NTS2-R each with ACT1-450-F/ACT1-450-R; NTS1r-F/NTS1r-R, YFR057W-F/ YFR057W-R, IRC7-F/IRC7-R, HML1α-F/ HML1α-R each co-amplified with ACT1-182-F/ACT1-182-R. PCR was performed under the following conditions: 95°C for 30 s, 60°C for 30 s, and 68°C for 1 min, with 18 cycles for ACT1 , 24 cycles for SIRT1 , NTS1r and NTS2 , 27 cycles for YFR057W , IRC7 and HMLα1 .

    Article Title: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana
    Article Snippet: Templates for in vitro transcription of these strands were prepared by performing reverse transcription using SuperScript III Reverse Transcriptase (ThermoFisher Scientific, Waltham, MA) with RNA extracts obtained from infected plants to generate unit-length cDNAs from circular genomic PSTVd RNA. .. This was followed by PCR using a reverse primer and a forward primer with an appended T3 RNA polymerase promoter sequence: PSTVd-175F-5'-GGGGACAAGTTTGTACAAAAAAGCAGAATTAACCCTCACTAAAGGTTTTCACCCTTCCTTT-3' (forward) and PSTVd-175R-5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCGAGCTCTGTTTCGGCGGGAATTAC-3' (reverse) for strand 175; PSTVd-321F-5'-GGGGACAAGTTTGTACAAAAAAGCAGAATTAACCCTCACTAAAGGCGAGGGTGTTTAGCC-3' (forward) and PSTVd-321R-5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCGAGCTCCGAAGCAAGTAAGATAGAGA-3' (reverse) for strand 321.

    Article Title: A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress
    Article Snippet: After treatment and precipitation, RNA was recovered and cDNAs were synthetized by RT-PCR using SuperScript III Reverse Transcriptase (Life Technologies), dNTPs, and random hexamers (dNTP Mix and Hexanucleotide Mix, Sigma-Aldrich) following the SuperScript III protocol recommended by the manufacturer. .. The presence of the circRNAs specifically in the polyA(−) fraction and the RNase R-treated samples was confirmed using divergent primers flanking the back-splice junctions (primers were designed using SnapGene and ordered at Sigma-Aldrich) by semi-quantitative PCR.

    Sequencing:

    Article Title: Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations
    Article Snippet: Three hundred nanogram of total RNA were reverse described into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA). .. The excised fragments were purified with the Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Freiburg, Germany) and analyzed by Sanger sequencing.

    Article Title: Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi
    Article Snippet: .. The cDNA was prepared from 1 μg RNA using Superscript III reverse transcriptase with random primers (Invitrogen, Carlsbad, CA). qRT-PCR was performed using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) on an ABI 7000 sequence detection system. ..

    Article Title: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana
    Article Snippet: Templates for in vitro transcription of these strands were prepared by performing reverse transcription using SuperScript III Reverse Transcriptase (ThermoFisher Scientific, Waltham, MA) with RNA extracts obtained from infected plants to generate unit-length cDNAs from circular genomic PSTVd RNA. .. This was followed by PCR using a reverse primer and a forward primer with an appended T3 RNA polymerase promoter sequence: PSTVd-175F-5'-GGGGACAAGTTTGTACAAAAAAGCAGAATTAACCCTCACTAAAGGTTTTCACCCTTCCTTT-3' (forward) and PSTVd-175R-5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCGAGCTCTGTTTCGGCGGGAATTAC-3' (reverse) for strand 175; PSTVd-321F-5'-GGGGACAAGTTTGTACAAAAAAGCAGAATTAACCCTCACTAAAGGCGAGGGTGTTTAGCC-3' (forward) and PSTVd-321R-5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCCGAGCTCCGAAGCAAGTAAGATAGAGA-3' (reverse) for strand 321.

    Article Title: Functional and structural characterization of the chikungunya virus translational recoding signals
    Article Snippet: Complementary primers for the Renilla and firefly regions were used to amplify the inserted CHIKV sequence and attach a T7 promoter sequence to the 5′ end of the amplicons. .. The primer was annealed to modified RNA, and subsequent reverse transcription (using Superscript III reverse transcriptase, Thermo Scientific, catalogue number 18080044) was carried out as reported previously ( , ).

    Radioactivity:

    Article Title: Functional and structural characterization of the chikungunya virus translational recoding signals
    Article Snippet: The primer was annealed to modified RNA, and subsequent reverse transcription (using Superscript III reverse transcriptase, Thermo Scientific, catalogue number 18080044) was carried out as reported previously ( , ). .. Radioactivity of cDNA samples was standardized with a liquid scintillator prior to electrophoresis though 8% urea-PAGE (SequaGel UreaGel system, National Diagnostics, catalogue number EC-833). cDNA products were visualized on a Fujifilm phosphorimaging system.

    Fluorescence:

    Article Title: A calmodulin‐like protein regulates plasmodesmal closure during bacterial immune responses
    Article Snippet: Quantitative RT‐PCR analysis RNA was extracted from leaves using TRIzol (Invitrogen) and treated with Turbo DNA‐free kit (Ambion, Thermo Fisher Scientific) before cDNA synthesis using SuperScript® III Reverse Transcriptase (Invitrogen). .. Quantitative reverse transcription polymerase chain reaction (RT‐PCR) was performed on the cDNA samples with primers listed in Table using the fluorescence output from a QuantStudio™ 12K Flex Real‐Time PCR System.

    Isolation:

    Article Title: Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations
    Article Snippet: CCM2 Transcript Analyses RNA was isolated from peripheral blood lymphocytes using the PAXgene Blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland). .. Three hundred nanogram of total RNA were reverse described into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA).

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: .. RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. ..

    Article Title: Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein
    Article Snippet: .. Isolation of novel exons, open reading frame, isoforms, and conserved domains in CCM2 gene Multi-tissue reference RNAs pool from four different suppliers (Super Array, BiotaQ, Biochain, NTomics) were used to amplify CCM2 cDNA fragments with SuperScript® III reverse transcriptase (Invitrogen). .. Rapid amplification of cDNA end (RACE) was applied for using either human brain Marathon-Ready cDNA or SMART RACE kit reagents (BD Clontech) to define full-length cDNA.

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: .. Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific). .. Gene expression was normalized to Gapdh .

    Article Title: Strigolactones enhance root‐knot nematode (Meloidogyne graminicola) infection in rice by antagonizing the jasmonate pathway
    Article Snippet: Paragraph title: RNA isolation and complementary DNA synthesis ... 500 ng total RNA into complementary DNA (cDNA) was achieved by using SuperScript III reverse transcriptase (Invitrogen) and 50 pmol Oligo (dT)12‐18 Primer (Invitrogen) in a reaction volume of 20 μl.

    Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature
    Article Snippet: .. Real-time polymerase chain reaction Total RNA from tissues and HUVEC was isolated by using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using Superscript III Reverse Transcriptase (Invitrogen). .. Quantitative real-time PCR was performed using PerfeCTa SYBR Green Fastmix (Quanta Biosciences) on a Bio-Rad CFX96 system.

    Size-exclusion Chromatography:

    Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
    Article Snippet: Quantitative Real-Time PCR of Selected NtMADS-Box Genes A total of 2 μg of total RNA in a 20 μL reaction was converted to cDNA with a SuperScript III Reverse Transcriptase (Invitrogen, Waltham, Massachusetts, USA) by the manufacturer’s instructions on an Eppendorf Mastercycler thermocycler (Eppendorf AG, Germany) with the following conditions: 25 °C for 5 min, 50 °C for 60 min, 70 °C for 15 min, followed by a hold at 4 °C until use in a qPCR reaction. .. A total of 60 μL of deionized water was added into 20 μL cDNA, and 1 μL of diluted cDNA mixture was used as the input for the qPCR reaction. qPCR reactions were made with a SuperReal PreMix Plus SYBR Green Kit (TIANGEN Biotech, BeiJing, China) following manufacturer’s instructions in a 20 μL volume. qPCR was done on an Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System (ThemoFisher Scientific, Waltham, Massachusetts, USA) with the following cycling conditions: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 32 s. The melt curve conditions were 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 sec. All samples had only one melt temperature peak.

    Purification:

    Article Title: Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations
    Article Snippet: Three hundred nanogram of total RNA were reverse described into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA). .. The excised fragments were purified with the Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Freiburg, Germany) and analyzed by Sanger sequencing.

    Article Title: Functional and structural characterization of the chikungunya virus translational recoding signals
    Article Snippet: Transcribed RNA was purified with a MEGAclear cleanup kit (Life Technologies, catalogue number 1908), and the quality of the RNA samples was assessed through urea-PAGE. .. The primer was annealed to modified RNA, and subsequent reverse transcription (using Superscript III reverse transcriptase, Thermo Scientific, catalogue number 18080044) was carried out as reported previously ( , ).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis
    Article Snippet: .. RT-PCR and Western Blotting About 2 μ g of RNA was reverse transcribed to generate cDNA using random primers and Superscript III reverse transcriptase (Invitrogen). .. Quantitative RT-PCR reactions were carried out in triplicates on a QuantStudio 12K Flex Real Time PCR machine.

    Article Title: A calmodulin‐like protein regulates plasmodesmal closure during bacterial immune responses
    Article Snippet: Quantitative RT‐PCR analysis RNA was extracted from leaves using TRIzol (Invitrogen) and treated with Turbo DNA‐free kit (Ambion, Thermo Fisher Scientific) before cDNA synthesis using SuperScript® III Reverse Transcriptase (Invitrogen). .. Quantitative reverse transcription polymerase chain reaction (RT‐PCR) was performed on the cDNA samples with primers listed in Table using the fluorescence output from a QuantStudio™ 12K Flex Real‐Time PCR System.

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: .. RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. ..

    Article Title: Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein
    Article Snippet: Isolation of novel exons, open reading frame, isoforms, and conserved domains in CCM2 gene Multi-tissue reference RNAs pool from four different suppliers (Super Array, BiotaQ, Biochain, NTomics) were used to amplify CCM2 cDNA fragments with SuperScript® III reverse transcriptase (Invitrogen). .. The RACE and RT-PCR resulting products were sequenced and further analyzed with Vector NTI (Invitrogen) and CLC genomic workbench (Qiagen) to define the potential novel fragments and open reading frames (ORFs) as described before .

    Article Title: A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress
    Article Snippet: .. After treatment and precipitation, RNA was recovered and cDNAs were synthetized by RT-PCR using SuperScript III Reverse Transcriptase (Life Technologies), dNTPs, and random hexamers (dNTP Mix and Hexanucleotide Mix, Sigma-Aldrich) following the SuperScript III protocol recommended by the manufacturer. .. The presence of the circRNAs specifically in the polyA(−) fraction and the RNase R-treated samples was confirmed using divergent primers flanking the back-splice junctions (primers were designed using SnapGene and ordered at Sigma-Aldrich) by semi-quantitative PCR.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Functional Complementation of sir2Δ Yeast Mutation by the Human Orthologous Gene SIRT1
    Article Snippet: .. RT-PCR RNA from logarithmically growing cultures was isolated as previously described [ ].A 1.5-μg amount of DNase I–treated RNA was subjected to cDNA synthesis, starting from 2.5 μM oligo(dT) for evaluation of SIRT1, NTS1r, YFR057W, IRC7 and HMLα1 mRNA expression levels (50ng/μl Random hexamers at 25°C 10min for NTS2 ), by incubation with 30 U of SuperScript III Reverse Transcriptase (Invitrogen, Cat.No. ..

    Plasmid Preparation:

    Article Title: RNA-binding protein FXR1 is presented in rat brain in amyloid form
    Article Snippet: Cloning of the FXR1 gene fragments Total rat RNA was extracted from brain homogenates using TRIzol reagent (Invitrogen) according to the manufacturer protocol. cDNA synthesis was performed with SuperScript III Reverse Transcriptase (Invitrogen). cDNA was further used for FXR1 fragments synthesis. .. The fragment of FXR1 gene coding for 1-379 aa for expression in E. coli was amplified with fxr1EcoRIforward and fxr1(379)BamHIreverse primers and inserted into pET302 vector to obtain the pET302-FXR1(1-379) plasmid.

    Article Title: Alternatively spliced isoforms reveal a novel type of PTB domain in CCM2 protein
    Article Snippet: Isolation of novel exons, open reading frame, isoforms, and conserved domains in CCM2 gene Multi-tissue reference RNAs pool from four different suppliers (Super Array, BiotaQ, Biochain, NTomics) were used to amplify CCM2 cDNA fragments with SuperScript® III reverse transcriptase (Invitrogen). .. The RACE and RT-PCR resulting products were sequenced and further analyzed with Vector NTI (Invitrogen) and CLC genomic workbench (Qiagen) to define the potential novel fragments and open reading frames (ORFs) as described before .

    Real-time Polymerase Chain Reaction:

    Article Title: RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis
    Article Snippet: RT-PCR and Western Blotting About 2 μ g of RNA was reverse transcribed to generate cDNA using random primers and Superscript III reverse transcriptase (Invitrogen). .. Quantitative RT-PCR reactions were carried out in triplicates on a QuantStudio 12K Flex Real Time PCR machine.

    Article Title: A calmodulin‐like protein regulates plasmodesmal closure during bacterial immune responses
    Article Snippet: Quantitative RT‐PCR analysis RNA was extracted from leaves using TRIzol (Invitrogen) and treated with Turbo DNA‐free kit (Ambion, Thermo Fisher Scientific) before cDNA synthesis using SuperScript® III Reverse Transcriptase (Invitrogen). .. Quantitative reverse transcription polymerase chain reaction (RT‐PCR) was performed on the cDNA samples with primers listed in Table using the fluorescence output from a QuantStudio™ 12K Flex Real‐Time PCR System.

    Article Title: Genome-Wide Identification, Gene Structure and Expression Analysis of the MADS-Box Gene Family Indicate Their Function in the Development of Tobacco (Nicotiana tabacum L.)
    Article Snippet: .. Quantitative Real-Time PCR of Selected NtMADS-Box Genes A total of 2 μg of total RNA in a 20 μL reaction was converted to cDNA with a SuperScript III Reverse Transcriptase (Invitrogen, Waltham, Massachusetts, USA) by the manufacturer’s instructions on an Eppendorf Mastercycler thermocycler (Eppendorf AG, Germany) with the following conditions: 25 °C for 5 min, 50 °C for 60 min, 70 °C for 15 min, followed by a hold at 4 °C until use in a qPCR reaction. .. A total of 60 μL of deionized water was added into 20 μL cDNA, and 1 μL of diluted cDNA mixture was used as the input for the qPCR reaction. qPCR reactions were made with a SuperReal PreMix Plus SYBR Green Kit (TIANGEN Biotech, BeiJing, China) following manufacturer’s instructions in a 20 μL volume. qPCR was done on an Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System (ThemoFisher Scientific, Waltham, Massachusetts, USA) with the following cycling conditions: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 32 s. The melt curve conditions were 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 sec. All samples had only one melt temperature peak.

    Article Title: Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control
    Article Snippet: .. First-strand cDNA was synthesized from 1 μg of the total RNA using SuperScript III Reverse Transcriptase (Life Technologies, Tokyo, Japan) and primer P19. qPCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) according to the manufacturer’s protocol in a LightCycler 96 instrument (Roche, Basel, Switzerland). .. The expression level of diap1 relative to the internal control, ribosomal protein 49 gene (rp49 ; accession number: AB480201), was calculated by the 2−ΔΔC t method (Livak and Schmittgen ); primer pairs P17 and P18, and P15 and P16, were used respectively (nucleotides 1649–1783 of diap1 -cDNA [Supplemental Fig. ]; Chikami et al. ).

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: .. Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific). .. Gene expression was normalized to Gapdh .

    Article Title: The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature
    Article Snippet: .. Real-time polymerase chain reaction Total RNA from tissues and HUVEC was isolated by using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using Superscript III Reverse Transcriptase (Invitrogen). .. Quantitative real-time PCR was performed using PerfeCTa SYBR Green Fastmix (Quanta Biosciences) on a Bio-Rad CFX96 system.

    RNA Extraction:

    Article Title: Positive and Negative Regulation of Glycerol Utilization by the c-di-GMP Binding Protein PlzA in Borrelia burgdorferi
    Article Snippet: Paragraph title: RNA extraction and qRT-PCR. ... The cDNA was prepared from 1 μg RNA using Superscript III reverse transcriptase with random primers (Invitrogen, Carlsbad, CA). qRT-PCR was performed using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) on an ABI 7000 sequence detection system.

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer
    Article Snippet: Paragraph title: RNA extraction, cDNA synthesis, and qRT-PCR ... Total RNA was isolated with RNeasy Plus Kit (Qiagen, Germany) or with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers’ instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific), and qRT-PCR was performed using FastStart Universal SYBR Green Master Mix (Roche, Germany) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific).

    Agarose Gel Electrophoresis:

    Article Title: Novel Pathogenic Variants in a Cassette Exon of CCM2 in Patients With Cerebral Cavernous Malformations
    Article Snippet: Three hundred nanogram of total RNA were reverse described into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, USA). .. PCR products were size-separated by agarose gel electrophoresis and visualized on a Gel Doc™ EZ Imager (Bio-Rad, Hercules, USA).

    In Vitro:

    Article Title: A three-dimensional RNA motif mediates directional trafficking of Potato spindle tuber viroid from epidermal to palisade mesophyll cells in Nicotiana benthamiana
    Article Snippet: .. Templates for in vitro transcription of these strands were prepared by performing reverse transcription using SuperScript III Reverse Transcriptase (ThermoFisher Scientific, Waltham, MA) with RNA extracts obtained from infected plants to generate unit-length cDNAs from circular genomic PSTVd RNA. ..

    Article Title: Functional and structural characterization of the chikungunya virus translational recoding signals
    Article Snippet: In vitro transcription was carried out with a T7 MEGAscript kit from Life Technologies (catalogue number AM1334). .. The primer was annealed to modified RNA, and subsequent reverse transcription (using Superscript III reverse transcriptase, Thermo Scientific, catalogue number 18080044) was carried out as reported previously ( , ).

    Electrophoresis:

    Article Title: Functional and structural characterization of the chikungunya virus translational recoding signals
    Article Snippet: The primer was annealed to modified RNA, and subsequent reverse transcription (using Superscript III reverse transcriptase, Thermo Scientific, catalogue number 18080044) was carried out as reported previously ( , ). .. Radioactivity of cDNA samples was standardized with a liquid scintillator prior to electrophoresis though 8% urea-PAGE (SequaGel UreaGel system, National Diagnostics, catalogue number EC-833). cDNA products were visualized on a Fujifilm phosphorimaging system.

    Spectrophotometry:

    Article Title: Strigolactones enhance root‐knot nematode (Meloidogyne graminicola) infection in rice by antagonizing the jasmonate pathway
    Article Snippet: The quantity and quality of the RNA was checked by spectrophotometry (NanoDrop 2000; Thermo Fisher Scientific, Wilmington, DE USA). .. 500 ng total RNA into complementary DNA (cDNA) was achieved by using SuperScript III reverse transcriptase (Invitrogen) and 50 pmol Oligo (dT)12‐18 Primer (Invitrogen) in a reaction volume of 20 μl.

    Concentration Assay:

    Article Title: A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress
    Article Snippet: After washing, eluted polyA(+) RNA and polyA(−) RNA suspension were precipitated overnight by adding ethanol to a final concentration (f.c.) of 70% and sodium acetate (0.3 M f.c.). .. After treatment and precipitation, RNA was recovered and cDNAs were synthetized by RT-PCR using SuperScript III Reverse Transcriptase (Life Technologies), dNTPs, and random hexamers (dNTP Mix and Hexanucleotide Mix, Sigma-Aldrich) following the SuperScript III protocol recommended by the manufacturer.

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    • superscript iii reverse transcriptase  (Thermo Fisher)
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    Thermo Fisher superscript iii reverse transcriptase
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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