superscript iii system Thermo Fisher Search Results


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    Thermo Fisher thermo fisher superscript iii
    Nova2 expression levels and AS of its target are regulated in ECs. ( a ) RT–qPCR analysis of Nova2 and Nova1 mRNA expression levels in mouse ECs grown as confluent or sparse (left), in E9.5 and E15.5 mouse whole brain (centre), and RT–qPCR analysis of Muscleblind family members ( Mbnl1 , Mbnl2 and Mbnl3 ) in mouse ECs grown at different densities (right). ( b ) Immunoblotting analysis of Nova2 levels in mouse confluent and sparse ECs (left panel; Tubulin as the loading control) and in confluent ECs and the mouse brain cortex (right panel; GAPDH as loading control). ( c ) Nova2 mRNA and protein expression levels in HUVECs grown at different densities. ( d ) RT–qPCR analysis of Nova2 during endothelial differentiation of mouse ES cells at the indicated times. ( e ) Immunoblotting analysis of Nova2 in mouse EC lines derived from whole embryo, fetal heart and adult lung; Actin as the loading control. In all histograms, error bars indicate ±s.d. calculated from <t>three</t> independent experiments ( n =3). ( f ) <t>RT–PCR</t> analysis of AS of a known Nova2 target ( Ankyrin3 / Ank3 ) in mouse ECs (confluent and sparse; left), during endothelial differentiation of mouse ES cells (centre) and in mouse ECs of different origins (right). The schematic representation of the mouse gene structure (AS exon in grey; constitutive exons in black), the YCAY cluster predicted to function as Nova2 silencer (blue dot) and the Nova2-regulated exon-skipping event (blue bars) are also shown. NISS, Nova intronic splicing silencer. The percentage of exon inclusion is shown. Asterisk indicates a nonspecific PCR product.
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    Thermo Fisher superscript iii ssiii
    Effect of enzyme and priming on 16S rRNA transcript community composition. NMDS clustering of 16S rRNA cDNA community composition of the same sample derived from different enzyme and primer strategies: Sensiscript and Omniscript (top) and Superscript <t>III</t> and Superscript IV (Bottom). Distances were calculated using Bray-Curtis (left), Unifrac (middle) and WUnifrac (right) distances. Corresponding groups are indicated in the legend.
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    Thermo Fisher superscript iii ssiii kit
    Effect of enzyme and priming on 16S rRNA transcript community composition. NMDS clustering of 16S rRNA cDNA community composition of the same sample derived from different enzyme and primer strategies: Sensiscript and Omniscript (top) and Superscript <t>III</t> and Superscript IV (Bottom). Distances were calculated using Bray-Curtis (left), Unifrac (middle) and WUnifrac (right) distances. Corresponding groups are indicated in the legend.
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    Thermo Fisher superscript iii first strand synthesis ssiii supermix
    Effect of enzyme and priming on 16S rRNA transcript community composition. NMDS clustering of 16S rRNA cDNA community composition of the same sample derived from different enzyme and primer strategies: Sensiscript and Omniscript (top) and Superscript <t>III</t> and Superscript IV (Bottom). Distances were calculated using Bray-Curtis (left), Unifrac (middle) and WUnifrac (right) distances. Corresponding groups are indicated in the legend.
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    Thermo Fisher superscript iii first strand synthesis system thermo fisher scientific
    Effect of enzyme and priming on 16S rRNA transcript community composition. NMDS clustering of 16S rRNA cDNA community composition of the same sample derived from different enzyme and primer strategies: Sensiscript and Omniscript (top) and Superscript <t>III</t> and Superscript IV (Bottom). Distances were calculated using Bray-Curtis (left), Unifrac (middle) and WUnifrac (right) distances. Corresponding groups are indicated in the legend.
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    Thermo Fisher superscript iii ssiii fs reaction buffer
    Effect of enzyme and priming on 16S rRNA transcript community composition. NMDS clustering of 16S rRNA cDNA community composition of the same sample derived from different enzyme and primer strategies: Sensiscript and Omniscript (top) and Superscript <t>III</t> and Superscript IV (Bottom). Distances were calculated using Bray-Curtis (left), Unifrac (middle) and WUnifrac (right) distances. Corresponding groups are indicated in the legend.
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    Thermo Fisher superscript iii rt system
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Thermo Fisher ãŽâ¼l superscript iii ssiii rt
    Analyses of the <t>YF/NIEV</t> reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to <t>three</t> freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.
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    Image Search Results


    Nova2 expression levels and AS of its target are regulated in ECs. ( a ) RT–qPCR analysis of Nova2 and Nova1 mRNA expression levels in mouse ECs grown as confluent or sparse (left), in E9.5 and E15.5 mouse whole brain (centre), and RT–qPCR analysis of Muscleblind family members ( Mbnl1 , Mbnl2 and Mbnl3 ) in mouse ECs grown at different densities (right). ( b ) Immunoblotting analysis of Nova2 levels in mouse confluent and sparse ECs (left panel; Tubulin as the loading control) and in confluent ECs and the mouse brain cortex (right panel; GAPDH as loading control). ( c ) Nova2 mRNA and protein expression levels in HUVECs grown at different densities. ( d ) RT–qPCR analysis of Nova2 during endothelial differentiation of mouse ES cells at the indicated times. ( e ) Immunoblotting analysis of Nova2 in mouse EC lines derived from whole embryo, fetal heart and adult lung; Actin as the loading control. In all histograms, error bars indicate ±s.d. calculated from three independent experiments ( n =3). ( f ) RT–PCR analysis of AS of a known Nova2 target ( Ankyrin3 / Ank3 ) in mouse ECs (confluent and sparse; left), during endothelial differentiation of mouse ES cells (centre) and in mouse ECs of different origins (right). The schematic representation of the mouse gene structure (AS exon in grey; constitutive exons in black), the YCAY cluster predicted to function as Nova2 silencer (blue dot) and the Nova2-regulated exon-skipping event (blue bars) are also shown. NISS, Nova intronic splicing silencer. The percentage of exon inclusion is shown. Asterisk indicates a nonspecific PCR product.

    Journal: Nature Communications

    Article Title: The alternative splicing factor Nova2 regulates vascular development and lumen formation

    doi: 10.1038/ncomms9479

    Figure Lengend Snippet: Nova2 expression levels and AS of its target are regulated in ECs. ( a ) RT–qPCR analysis of Nova2 and Nova1 mRNA expression levels in mouse ECs grown as confluent or sparse (left), in E9.5 and E15.5 mouse whole brain (centre), and RT–qPCR analysis of Muscleblind family members ( Mbnl1 , Mbnl2 and Mbnl3 ) in mouse ECs grown at different densities (right). ( b ) Immunoblotting analysis of Nova2 levels in mouse confluent and sparse ECs (left panel; Tubulin as the loading control) and in confluent ECs and the mouse brain cortex (right panel; GAPDH as loading control). ( c ) Nova2 mRNA and protein expression levels in HUVECs grown at different densities. ( d ) RT–qPCR analysis of Nova2 during endothelial differentiation of mouse ES cells at the indicated times. ( e ) Immunoblotting analysis of Nova2 in mouse EC lines derived from whole embryo, fetal heart and adult lung; Actin as the loading control. In all histograms, error bars indicate ±s.d. calculated from three independent experiments ( n =3). ( f ) RT–PCR analysis of AS of a known Nova2 target ( Ankyrin3 / Ank3 ) in mouse ECs (confluent and sparse; left), during endothelial differentiation of mouse ES cells (centre) and in mouse ECs of different origins (right). The schematic representation of the mouse gene structure (AS exon in grey; constitutive exons in black), the YCAY cluster predicted to function as Nova2 silencer (blue dot) and the Nova2-regulated exon-skipping event (blue bars) are also shown. NISS, Nova intronic splicing silencer. The percentage of exon inclusion is shown. Asterisk indicates a nonspecific PCR product.

    Article Snippet: Total RNA was extracted from 24-hpf pooled embryos with TRIzol reagent (Invitrogen), purified with the RNeasy Mini Kit (QIAGEN) and retro-transcribed with d(T)18 oligo or gene-specific primer and Superscript III RT (Invitrogen) and then analysed in PCR for AS modification of nova2 targets.

    Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Effect of enzyme and priming on 16S rRNA transcript community composition. NMDS clustering of 16S rRNA cDNA community composition of the same sample derived from different enzyme and primer strategies: Sensiscript and Omniscript (top) and Superscript III and Superscript IV (Bottom). Distances were calculated using Bray-Curtis (left), Unifrac (middle) and WUnifrac (right) distances. Corresponding groups are indicated in the legend.

    Journal: bioRxiv

    Article Title: Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

    doi: 10.1101/2020.03.18.996603

    Figure Lengend Snippet: Effect of enzyme and priming on 16S rRNA transcript community composition. NMDS clustering of 16S rRNA cDNA community composition of the same sample derived from different enzyme and primer strategies: Sensiscript and Omniscript (top) and Superscript III and Superscript IV (Bottom). Distances were calculated using Bray-Curtis (left), Unifrac (middle) and WUnifrac (right) distances. Corresponding groups are indicated in the legend.

    Article Snippet: amoA and 16S rRNA Q-PCR standard curves amoA and 16S rRNA RNA dilutions were prepared and each individually reverse transcribed (RT) using four different enzymes: Superscript IV (SSIV), Superscript III (SSIII) (Invitrogen), Sensiscript (Sensi) and Omniscript (Omni) (Qiagen) and two priming strategies - gene specific (GS) and random hexamer (RH).

    Techniques: Derivative Assay

    rarefaction curves obtained from the sequencing of amoA transcripts from environmental samples using different RT protocols. The RT protocol used is indicated on the plots (SSIII= Superscript III; SSIV= Superscript IV/ RH= random hexamer; GS= gene specific). The rarefaction curves were drawn for each replicate as indicated on the legend.

    Journal: bioRxiv

    Article Title: Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

    doi: 10.1101/2020.03.18.996603

    Figure Lengend Snippet: rarefaction curves obtained from the sequencing of amoA transcripts from environmental samples using different RT protocols. The RT protocol used is indicated on the plots (SSIII= Superscript III; SSIV= Superscript IV/ RH= random hexamer; GS= gene specific). The rarefaction curves were drawn for each replicate as indicated on the legend.

    Article Snippet: amoA and 16S rRNA Q-PCR standard curves amoA and 16S rRNA RNA dilutions were prepared and each individually reverse transcribed (RT) using four different enzymes: Superscript IV (SSIV), Superscript III (SSIII) (Invitrogen), Sensiscript (Sensi) and Omniscript (Omni) (Qiagen) and two priming strategies - gene specific (GS) and random hexamer (RH).

    Techniques: Sequencing, Environmental Sampling, Random Hexamer Labeling

    The effect of SNHG20 knockdown on cell growth, apoptosis, and colony formation ( A ) The mRNA level of SNHG20 in ovarian cancer cells, which were transfected with siSNHG20, was detected, siNC acts as normal control. MTT assays showed SNHG20 knockdown inhibited cell proliferation of SKOV3 ( B ), OVCA429 ( C ), and OVCA433 ( D ) cells. ( E ) Flow cytometry analysis was performed for testing the effect of SNHG20 knockdown on the apoptosis of ovarian cancer cells. ( F ) Colony formation assay measuring colony formation of SNHG20 knockdown ovarian cancer cells. SNHG20 knockdown ovarian cancer cells were inoculated into nude mice and the tumor volume was detected every week. The growth curve of SKOV3 ( G ), OVCA429 ( H ), and OVCA433 ( I ) tumors were determined every week. Colony number was normalized to that obtained with cells transfected with siNC, which was set to 100%. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Journal: Bioscience Reports

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    doi: 10.1042/BSR20170681

    Figure Lengend Snippet: The effect of SNHG20 knockdown on cell growth, apoptosis, and colony formation ( A ) The mRNA level of SNHG20 in ovarian cancer cells, which were transfected with siSNHG20, was detected, siNC acts as normal control. MTT assays showed SNHG20 knockdown inhibited cell proliferation of SKOV3 ( B ), OVCA429 ( C ), and OVCA433 ( D ) cells. ( E ) Flow cytometry analysis was performed for testing the effect of SNHG20 knockdown on the apoptosis of ovarian cancer cells. ( F ) Colony formation assay measuring colony formation of SNHG20 knockdown ovarian cancer cells. SNHG20 knockdown ovarian cancer cells were inoculated into nude mice and the tumor volume was detected every week. The growth curve of SKOV3 ( G ), OVCA429 ( H ), and OVCA433 ( I ) tumors were determined every week. Colony number was normalized to that obtained with cells transfected with siNC, which was set to 100%. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Article Snippet: The total RNA was reverse transcribed into first-strand cDNA using the SuperScript III® (Invitrogen) according to the manufacturer’s guide. q-PCR was performed on the SYBR Premix ExTaq kit (TaKaRa) on ABIPRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, FosterCity, CA, U.S.A.) according to the instructions.

    Techniques: Transfection, MTT Assay, Flow Cytometry, Cytometry, Colony Assay, Mouse Assay, Quantitative RT-PCR

    LncRNA SNHG20 expression was increased in ovarian cancer ( A ) The expression of SNHG20 in 17 ovarian tumor samples and paired adjacent non-tumorous normal tissues was examined by q-PCR. ( B ) The expression in 13 metastatic ovarian tumor samples and paired primary tumor samples was also examined by q-PCR. ( C ) The expression of SNHG20 in HOSE and ovarian cancer cells was determined by q-PCR. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Journal: Bioscience Reports

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    doi: 10.1042/BSR20170681

    Figure Lengend Snippet: LncRNA SNHG20 expression was increased in ovarian cancer ( A ) The expression of SNHG20 in 17 ovarian tumor samples and paired adjacent non-tumorous normal tissues was examined by q-PCR. ( B ) The expression in 13 metastatic ovarian tumor samples and paired primary tumor samples was also examined by q-PCR. ( C ) The expression of SNHG20 in HOSE and ovarian cancer cells was determined by q-PCR. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Article Snippet: The total RNA was reverse transcribed into first-strand cDNA using the SuperScript III® (Invitrogen) according to the manufacturer’s guide. q-PCR was performed on the SYBR Premix ExTaq kit (TaKaRa) on ABIPRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, FosterCity, CA, U.S.A.) according to the instructions.

    Techniques: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

    The effect of SNHG20 overexpression on normal ovarian epithelial cell growth, apoptosis, and colony formation ( A ) HOSE cells were infected with lentivirus carrying SNHG20 and then the mRNA level was examined by q-PCR. MTT assays showed the effects of SNHG20 overexpression on HOSE cell growth ( B ), cell apoptosis ( C ), and colony formation ( D ). Colony number was normalized to that obtained from cells transfected with siNC, which was set to 100%. ( E ) SNHG20 overexpressing HOSE cells were inoculated into nude mice and the tumor volumes were evaluated every week. ( F ) The tumor weight was detected at the end point after we killed all groups of mice. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Journal: Bioscience Reports

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    doi: 10.1042/BSR20170681

    Figure Lengend Snippet: The effect of SNHG20 overexpression on normal ovarian epithelial cell growth, apoptosis, and colony formation ( A ) HOSE cells were infected with lentivirus carrying SNHG20 and then the mRNA level was examined by q-PCR. MTT assays showed the effects of SNHG20 overexpression on HOSE cell growth ( B ), cell apoptosis ( C ), and colony formation ( D ). Colony number was normalized to that obtained from cells transfected with siNC, which was set to 100%. ( E ) SNHG20 overexpressing HOSE cells were inoculated into nude mice and the tumor volumes were evaluated every week. ( F ) The tumor weight was detected at the end point after we killed all groups of mice. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Article Snippet: The total RNA was reverse transcribed into first-strand cDNA using the SuperScript III® (Invitrogen) according to the manufacturer’s guide. q-PCR was performed on the SYBR Premix ExTaq kit (TaKaRa) on ABIPRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, FosterCity, CA, U.S.A.) according to the instructions.

    Techniques: Over Expression, Infection, Polymerase Chain Reaction, MTT Assay, Transfection, Mouse Assay, Quantitative RT-PCR

    The effect of SNHG20 knockdown on Wnt/β-catenin signaling pathway in ovarian cancer cells Ovarian cancer cells were transfected with siSNHG20 or siNC, then these cells were subjected to q-PCR ( A ) and Western blot ( B ) for testing the mRNA and protein level of E-cadherin. ( C–E ) The mRNA level and protein level of β-catenin, GSK-3β, p-GSK-3β and several targets of Wnt/β-catenin signaling were examined by q-PCR and western blot in SKOV3 and OVCA429 cells. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Journal: Bioscience Reports

    Article Title: Up-regulation of long non-coding RNA SNHG20 promotes ovarian cancer progression via Wnt/β-catenin signaling

    doi: 10.1042/BSR20170681

    Figure Lengend Snippet: The effect of SNHG20 knockdown on Wnt/β-catenin signaling pathway in ovarian cancer cells Ovarian cancer cells were transfected with siSNHG20 or siNC, then these cells were subjected to q-PCR ( A ) and Western blot ( B ) for testing the mRNA and protein level of E-cadherin. ( C–E ) The mRNA level and protein level of β-catenin, GSK-3β, p-GSK-3β and several targets of Wnt/β-catenin signaling were examined by q-PCR and western blot in SKOV3 and OVCA429 cells. qRT-PCR results were normalized by β-actin. Data represent the mean ± S.E.M. from three independent experiments; *** P

    Article Snippet: The total RNA was reverse transcribed into first-strand cDNA using the SuperScript III® (Invitrogen) according to the manufacturer’s guide. q-PCR was performed on the SYBR Premix ExTaq kit (TaKaRa) on ABIPRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, FosterCity, CA, U.S.A.) according to the instructions.

    Techniques: Transfection, Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR

    Detection of processed transcripts of E. coli CRISPR I cassette A. The E. coli CRISPR I locus is schematically shown. Genes are indicated by arrows and identified. In CRISPR I cassette, repeats are indicated by black rhombuses, spacers – by white rectangles. Numbers below the scheme indicate spacer numbers. The last repeat of the cassette is colored grey, to indicate that its sequence differs from other repeats. The leader sequence is located between the CRISPR I cassette and the cas2 gene and is indicated by a thicker black line. B. A Northern blot showing changes in abundance of spacer 4 transcript in E. coli strains with indicated cas genes disruptions. The probe was designed to reveal RNA produced by rightward (see Fig. 1A) transcription. C. Northern blots with total RNA purified from E. coli cells with disrupted casA gene were performed with probes specific for spacers indicated (direction of transcription the same as in Fig. 1B). In each panel, the first three lanes contained increasing amounts of DNA oligonucleotides complementary to probes used for blotting. Approximate calculated numbers of processed CRISPR transcript per cell are shown below.

    Journal: Molecular microbiology

    Article Title: Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli

    doi: 10.1111/j.1365-2958.2010.07265.x

    Figure Lengend Snippet: Detection of processed transcripts of E. coli CRISPR I cassette A. The E. coli CRISPR I locus is schematically shown. Genes are indicated by arrows and identified. In CRISPR I cassette, repeats are indicated by black rhombuses, spacers – by white rectangles. Numbers below the scheme indicate spacer numbers. The last repeat of the cassette is colored grey, to indicate that its sequence differs from other repeats. The leader sequence is located between the CRISPR I cassette and the cas2 gene and is indicated by a thicker black line. B. A Northern blot showing changes in abundance of spacer 4 transcript in E. coli strains with indicated cas genes disruptions. The probe was designed to reveal RNA produced by rightward (see Fig. 1A) transcription. C. Northern blots with total RNA purified from E. coli cells with disrupted casA gene were performed with probes specific for spacers indicated (direction of transcription the same as in Fig. 1B). In each panel, the first three lanes contained increasing amounts of DNA oligonucleotides complementary to probes used for blotting. Approximate calculated numbers of processed CRISPR transcript per cell are shown below.

    Article Snippet: For a single extension reaction up to 0.5 µg of in vitro transcribed RNA was reverse-transcribed with 100 U of SuperScript III enzyme of First-Strand Synthesis Kit for RT-PCR (Invitrogen) according to the manufacturer’s protocol in the presence of 1 pmoles of [32 P] 5’-end-labelled primers.

    Techniques: CRISPR, Sequencing, Northern Blot, Produced, Purification

    DNA methylation analysis of FBN1 CpG island shores of WT and KO alleles in FBN1 het KO fibroblast cells. A diagram of the porcine FBN1 upstream region is illustrated in the upper panel. The SNP between the WT and KO alleles identified in this study is located in the FBN1 CpG island shore. Sequenced sodium bisulfite PCR fragments including the FBN1 CpG island shore were divided into three sub-regions based on methylation status (O, outside the FBN1 CpG island shore. S, shore region. I, CpG island.). The DNA methylation status of each allele in FBN1 het KO fibroblast cells is represented in the lower panel. Depending on the SNP (G or A) present in the sodium bisulfite PCR fragments, sequenced reads were defined as WT (G) or KO (A) alleles for each cell line collected at P6 (early passage) and P20 (late passage). Horizontal and vertical axes indicate the position of the CpG sites (O, 2 CpGs; S, 9 CpGs; I, 13 CpGs) and the number of sequenced reads (29 to 45 reads), respectively. White and black bars indicate unmethylated and methylated CpGs, respectively, consistent with our previous study 15 . CpGs within the two dotted lines are in the shore region (S) and whose RM allele ratio was calculated in the present study.

    Journal: Scientific Reports

    Article Title: DNA methylation ambiguity in the Fibrillin-1 (FBN1) CpG island shore possibly involved in Marfan syndrome

    doi: 10.1038/s41598-020-62127-3

    Figure Lengend Snippet: DNA methylation analysis of FBN1 CpG island shores of WT and KO alleles in FBN1 het KO fibroblast cells. A diagram of the porcine FBN1 upstream region is illustrated in the upper panel. The SNP between the WT and KO alleles identified in this study is located in the FBN1 CpG island shore. Sequenced sodium bisulfite PCR fragments including the FBN1 CpG island shore were divided into three sub-regions based on methylation status (O, outside the FBN1 CpG island shore. S, shore region. I, CpG island.). The DNA methylation status of each allele in FBN1 het KO fibroblast cells is represented in the lower panel. Depending on the SNP (G or A) present in the sodium bisulfite PCR fragments, sequenced reads were defined as WT (G) or KO (A) alleles for each cell line collected at P6 (early passage) and P20 (late passage). Horizontal and vertical axes indicate the position of the CpG sites (O, 2 CpGs; S, 9 CpGs; I, 13 CpGs) and the number of sequenced reads (29 to 45 reads), respectively. White and black bars indicate unmethylated and methylated CpGs, respectively, consistent with our previous study 15 . CpGs within the two dotted lines are in the shore region (S) and whose RM allele ratio was calculated in the present study.

    Article Snippet: First-strand cDNA synthesis was performed using a Superscript III First-strand Synthesis System (Thermo Fisher Scientific) with transcript-specific primers: FBN1 _del1 (for FBN1 mRNA): 5′-GCACTCTCCACGGAGGTCC-3′, FBN1 -del3 (for FBN1 preRNA): 5′-GCAATAGGCAAATTGAGAAACTTC-3′, and GAPDH : 5′-TCTGGGATGGAAACTGGAAG-3′.

    Techniques: DNA Methylation Assay, Polymerase Chain Reaction, Methylation

    DNA methylation status of FBN1 CpG island shore and FBN1 expression in human cells. The DNA methylation status of five human somatic cell lines (MRC5, AM936EP, Edom22, PAE551, and UtE1104) ( a ) and twelve human iPS cell lines ( b ) were analyzed with the Infinium Human Methylation450K BeadChip. In the human FBN1 upstream region, three probes (probe I, II, and III) that are located in the outside region (probe I), the CpG island shore (probe II), and inside the CpG island (probe III) of the human FBN1 locus. DNA methylation levels are presented as a pie chart; black and white areas indicate the percentages of methylated and unmethylated CpGs, respectively. ( c ) The correlation between FBN1 mRNA level and DNA methylation level of FBN1 CpG island shore (probe II) was calculated for human iPS cell lines (n = 12). FBN1 expression and DNA methylation data were based on the gene expression array and the Infinium Human Methylation450K BeadChip, respectively.

    Journal: Scientific Reports

    Article Title: DNA methylation ambiguity in the Fibrillin-1 (FBN1) CpG island shore possibly involved in Marfan syndrome

    doi: 10.1038/s41598-020-62127-3

    Figure Lengend Snippet: DNA methylation status of FBN1 CpG island shore and FBN1 expression in human cells. The DNA methylation status of five human somatic cell lines (MRC5, AM936EP, Edom22, PAE551, and UtE1104) ( a ) and twelve human iPS cell lines ( b ) were analyzed with the Infinium Human Methylation450K BeadChip. In the human FBN1 upstream region, three probes (probe I, II, and III) that are located in the outside region (probe I), the CpG island shore (probe II), and inside the CpG island (probe III) of the human FBN1 locus. DNA methylation levels are presented as a pie chart; black and white areas indicate the percentages of methylated and unmethylated CpGs, respectively. ( c ) The correlation between FBN1 mRNA level and DNA methylation level of FBN1 CpG island shore (probe II) was calculated for human iPS cell lines (n = 12). FBN1 expression and DNA methylation data were based on the gene expression array and the Infinium Human Methylation450K BeadChip, respectively.

    Article Snippet: First-strand cDNA synthesis was performed using a Superscript III First-strand Synthesis System (Thermo Fisher Scientific) with transcript-specific primers: FBN1 _del1 (for FBN1 mRNA): 5′-GCACTCTCCACGGAGGTCC-3′, FBN1 -del3 (for FBN1 preRNA): 5′-GCAATAGGCAAATTGAGAAACTTC-3′, and GAPDH : 5′-TCTGGGATGGAAACTGGAAG-3′.

    Techniques: DNA Methylation Assay, Expressing, Methylation

    Antisense transcripts in DM2 and tetrapeptide RAN proteins expressed across CCUG and CAGG expansion RNAs. (A) Schematic diagram of CAGG antisense transcripts and relative location of primers for strand-specific RT-PCR. (B, C) qRT-PCR showing elevated antisense mRNA relative to β-actin in DM2 compared with controls. n=3 samples/group, experiments performed at least three times, error bars show standard deviation (SD) with at least three technical replicates (D) Diagram of putative proteins translated from sense and antisense DM2 transcripts. (E) Non-ATG CCTG and CAGG constructs with 6X stop-codon cassette, two stops in each frame, upstream of the CCTG expansion with C-terminal tags in all three reading frames. Immunoblots of transfected HEK293T cells show expanded LPAC proteins (F) and QAGR proteins (H) are expressed in all three frames. IF detection of (LPAC) EXP proteins (G) and (QAGR) EXP .

    Journal: Neuron

    Article Title: RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2

    doi: 10.1016/j.neuron.2017.08.039

    Figure Lengend Snippet: Antisense transcripts in DM2 and tetrapeptide RAN proteins expressed across CCUG and CAGG expansion RNAs. (A) Schematic diagram of CAGG antisense transcripts and relative location of primers for strand-specific RT-PCR. (B, C) qRT-PCR showing elevated antisense mRNA relative to β-actin in DM2 compared with controls. n=3 samples/group, experiments performed at least three times, error bars show standard deviation (SD) with at least three technical replicates (D) Diagram of putative proteins translated from sense and antisense DM2 transcripts. (E) Non-ATG CCTG and CAGG constructs with 6X stop-codon cassette, two stops in each frame, upstream of the CCTG expansion with C-terminal tags in all three reading frames. Immunoblots of transfected HEK293T cells show expanded LPAC proteins (F) and QAGR proteins (H) are expressed in all three frames. IF detection of (LPAC) EXP proteins (G) and (QAGR) EXP .

    Article Snippet: Total RNA from cells was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was generated from total RNA using random-hexamer primers and the SuperScript III system (Invitrogen). qRT-PCR was performed on a StepOnePlus Real-Time PCR Systems (Applied Biosystems) using Power SYBR Green PCR Master Mix and 3xTag primer sets.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Construct, Western Blot, Transfection

    Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total RNA was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Journal: PLoS ONE

    Article Title: Regulation of Microglia Activity by Glaucocalyxin-A: Attenuation of Lipopolysaccharide-Stimulated Neuroinflammation through NF-?B and p38 MAPK Signaling Pathways

    doi: 10.1371/journal.pone.0055792

    Figure Lengend Snippet: Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total RNA was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Article Snippet: RNA (2.5 µg) was reverse-transcribed using a Superscript™-III kit (Invitrogen), according to the manufacturer’s instruction.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Effect of glaucocalyxin-A (GLA) on inducible nitric oxide synthase (iNOS) expression and enzyme activity in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary microglia, B: BV-2 microglia. Lysates were analyzed by immunoblotting with an anti-iNOS antibody. Representative quantification data were shown in the lower panel. Results are expressed as a ratio of iNOS to β-actin. C: BV-2 cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 6 h. The total RNA was isolated and iNOS mRNA level was determined by RT-PCR. Quantification data are shown in the lower panel. Results are expressed as a ratio of iNOS to GAPDH. D: BV-2 microglial cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 18 h. iNOS activity was analyzed with enzyme immunoassay kit. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Journal: PLoS ONE

    Article Title: Regulation of Microglia Activity by Glaucocalyxin-A: Attenuation of Lipopolysaccharide-Stimulated Neuroinflammation through NF-?B and p38 MAPK Signaling Pathways

    doi: 10.1371/journal.pone.0055792

    Figure Lengend Snippet: Effect of glaucocalyxin-A (GLA) on inducible nitric oxide synthase (iNOS) expression and enzyme activity in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary microglia, B: BV-2 microglia. Lysates were analyzed by immunoblotting with an anti-iNOS antibody. Representative quantification data were shown in the lower panel. Results are expressed as a ratio of iNOS to β-actin. C: BV-2 cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 6 h. The total RNA was isolated and iNOS mRNA level was determined by RT-PCR. Quantification data are shown in the lower panel. Results are expressed as a ratio of iNOS to GAPDH. D: BV-2 microglial cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 18 h. iNOS activity was analyzed with enzyme immunoassay kit. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Article Snippet: RNA (2.5 µg) was reverse-transcribed using a Superscript™-III kit (Invitrogen), according to the manufacturer’s instruction.

    Techniques: Expressing, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Analyses of the YF/NIEV reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to three freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.

    Journal: mSphere

    Article Title: Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle

    doi: 10.1128/mSphere.00375-16

    Figure Lengend Snippet: Analyses of the YF/NIEV reporter virus construct in vertebrate cells. (A) RNA replication of YF/NIEV_Ren in BHK cells. BHK cells were electroporated with the indicated in vitro -transcribed reporter virus RNAs, and Renilla luciferase expression levels were measured at 24 h p.e. Data represent means and ranges of results of duplicate electroporation experiments. (B) Plaque formation of YFV_Ren and YF/NIEV_Ren in BHK cells. BHK cells were electroporated with in vitro RNA transcripts of the indicated constructs and processed for ICA analyses. Seeded cells were overlaid with agarose. At 3 days p.e., cells were fixed and subjected to crystal violet staining. (C) Infectivity assay. Supernatants from BHK cells electroporated with either YF_Ren or YF/NIEV_Ren in vitro -transcribed RNAs were used to infect C6/36 cells. To test for infectivity, Renilla luciferase levels were determined at the indicated time points p.i. Data represent means and ranges of results of duplicate infection experiments. (D) Intra- and extracellular infectivity titers. In vitro transcripts from the full-length clones specified at the bottom were electroporated in C6/36 or BHK cells as indicated. At 6 (C6/36) or 2 (BHK) days p.e., cell lysates and supernatants were subjected to three freeze and thaw cycles. Titers were determined by TCID 50 assay using C6/36 cells and Renilla luciferase as the readout. Dashed line, detection limit. Data represent means and ranges of results of duplicate electroporation experiments.

    Article Snippet: For NIEV RNAs, cDNA was synthesized using a SuperScript III RT system (Life Technologies, Inc.) and random hexamer primers.

    Techniques: Construct, In Vitro, Luciferase, Expressing, Electroporation, Staining, Infection, Clone Assay