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  • 99
    Thermo Fisher high capacity reverse transcription kit
    High Capacity Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5378 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 5378 article reviews
    Price from $9.99 to $1999.99
    high capacity reverse transcription kit - by Bioz Stars, 2020-09
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    99
    Thermo Fisher high capacity cdna reverse transcription kit
    α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. <t>RNA</t> was retrotranscribed, and 50 ng of <t>cDNA</t> was used to perform α 2 M and GAPDH PCR. n = 3.
    High Capacity Cdna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 122229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 122229 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher reverse transcription kit
    α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. <t>RNA</t> was retrotranscribed, and 50 ng of <t>cDNA</t> was used to perform α 2 M and GAPDH PCR. n = 3.
    Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 8397 article reviews
    Price from $9.99 to $1999.99
    reverse transcription kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Bio-Rad high capacity cdna reverse transcription kit
    Summary of cDNAs recovered from <t>DENV-resistant</t> Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. mRNA were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    High Capacity Cdna Reverse Transcription Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Bio-Rad
    Average 93 stars, based on 206 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit - by Bioz Stars, 2020-09
    93/100 stars
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    92
    Promega high capacity cdna reverse transcription kit
    Summary of cDNAs recovered from <t>DENV-resistant</t> Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. mRNA were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and <t>cDNA</t> inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.
    High Capacity Cdna Reverse Transcription Kit, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Promega
    Average 92 stars, based on 147 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit - by Bioz Stars, 2020-09
    92/100 stars
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    Image Search Results


    α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. RNA was retrotranscribed, and 50 ng of cDNA was used to perform α 2 M and GAPDH PCR. n = 3.

    Journal: Scientific Reports

    Article Title: Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion

    doi: 10.1038/s41598-020-66554-0

    Figure Lengend Snippet: α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. RNA was retrotranscribed, and 50 ng of cDNA was used to perform α 2 M and GAPDH PCR. n = 3.

    Article Snippet: Reverse transcription was performed with 1 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies).

    Techniques: Expressing, Purification, Polymerase Chain Reaction

    α 2 M* induced BeWo cell fusion through p-CREB, p-ERK1/2 and p-JNK activation, without affecting syncytin expression. ( A,B ) BeWo cells were seeded for 24 h prior to 24 h of starvation. Subsequently, cells were treated with 5 μM KT5720, 10 μM SP600125 or 10 μM UO126 for 1 h, and 100 pM of α 2 M* was added or not for 30 min. ( A ) Western blotting was performed. p-CREB, CREB, p-ERK1/2, ERK1/2, p-JNK and JNK levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. ( B ) Nuclei and syncytia were counted, and a fusion index was calculated. n = 3. Data represented as mean±SEM. ns (not significant), *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.005; ANOVA comparison test. ( C , D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M*. ( C ) RNA was retrotranscribed, and 10 ng of cDNA was used to perform qPCR using syncytin-1 and syncytin-2 primers. n = 3. Data represented as mean±SEM. **P ≤ 0.01; t-test comparison test. ( D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M* for 48 h. Western blotting was performed. Syncytin-1 and GAPDH levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation.

    Journal: Scientific Reports

    Article Title: Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion

    doi: 10.1038/s41598-020-66554-0

    Figure Lengend Snippet: α 2 M* induced BeWo cell fusion through p-CREB, p-ERK1/2 and p-JNK activation, without affecting syncytin expression. ( A,B ) BeWo cells were seeded for 24 h prior to 24 h of starvation. Subsequently, cells were treated with 5 μM KT5720, 10 μM SP600125 or 10 μM UO126 for 1 h, and 100 pM of α 2 M* was added or not for 30 min. ( A ) Western blotting was performed. p-CREB, CREB, p-ERK1/2, ERK1/2, p-JNK and JNK levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. ( B ) Nuclei and syncytia were counted, and a fusion index was calculated. n = 3. Data represented as mean±SEM. ns (not significant), *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.005; ANOVA comparison test. ( C , D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M*. ( C ) RNA was retrotranscribed, and 10 ng of cDNA was used to perform qPCR using syncytin-1 and syncytin-2 primers. n = 3. Data represented as mean±SEM. **P ≤ 0.01; t-test comparison test. ( D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M* for 48 h. Western blotting was performed. Syncytin-1 and GAPDH levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation.

    Article Snippet: Reverse transcription was performed with 1 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies).

    Techniques: Activation Assay, Expressing, Western Blot, Software, Real-time Polymerase Chain Reaction

    Plk1 mRNA and protein expression in BT-474 cells after incubating with nanoparticles. (A) An RT-PCR analysis was performed 48 hours after incubating with nanoparticle formulations using primers specific for Plk1 and for GAPDH for standardization. Representative gels show Plk1 cDNA (top) and GAPDH cDNA for standardization (bottom). Percentage of Plk1 cDNA expression is given as percentage of GAPDH-standardized Plk1 cDNA expression in control cells ( n = 8, mean ± SD). A quantitative real-time PCR analysis was performed (B) 24 and (C) 48 hours after incubating with the nanoparticle formulations using Plk1- and GAPDH-specific primers. Graphical summary of gene expression values of treated cells standardized to control cells are shown ( n = 3–5, mean ± SD). (D–F) Western blot analyses were performed using anti-Plk1 antibodies 24, 48, and 72 hours after incubating with the nanoparticle formulations. To control for variability of loading, membranes were reprobed with antibodies against p38 (24 and 48 hours) or β-actin (72 hours). Representative Western blots show Plk1 (top) and β-actin or p38 protein (bottom) for standardization. Percentage of Plk1 protein expression is given as percentage of β-actin- or p38-standardized Plk1 levels in control cells ( n = 3, mean ± SD).

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Downregulation of Plk1 Expression By Receptor-Mediated Uptake of Antisense Oligonucleotide-Loaded Nanoparticles 1

    doi:

    Figure Lengend Snippet: Plk1 mRNA and protein expression in BT-474 cells after incubating with nanoparticles. (A) An RT-PCR analysis was performed 48 hours after incubating with nanoparticle formulations using primers specific for Plk1 and for GAPDH for standardization. Representative gels show Plk1 cDNA (top) and GAPDH cDNA for standardization (bottom). Percentage of Plk1 cDNA expression is given as percentage of GAPDH-standardized Plk1 cDNA expression in control cells ( n = 8, mean ± SD). A quantitative real-time PCR analysis was performed (B) 24 and (C) 48 hours after incubating with the nanoparticle formulations using Plk1- and GAPDH-specific primers. Graphical summary of gene expression values of treated cells standardized to control cells are shown ( n = 3–5, mean ± SD). (D–F) Western blot analyses were performed using anti-Plk1 antibodies 24, 48, and 72 hours after incubating with the nanoparticle formulations. To control for variability of loading, membranes were reprobed with antibodies against p38 (24 and 48 hours) or β-actin (72 hours). Representative Western blots show Plk1 (top) and β-actin or p38 protein (bottom) for standardization. Percentage of Plk1 protein expression is given as percentage of β-actin- or p38-standardized Plk1 levels in control cells ( n = 3, mean ± SD).

    Article Snippet: After isolation of total RNA using RNeasy Mini Kits (Qiagen) according to the manufacturer's protocol 24 and 48 hours after incubating with the nanoparticle formulations, the mRNA was transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Darmstadt, Germany).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

    Volcano plot of the AI data from 105 heterozygous cSNPs in 13 cell lines. AI for each SNP was determined by calculating the fluorescence signal ratio between the two alleles ( S Allele1 / S Allele2 ) in RNA (cDNA) and genomic DNA for each heterozygous SNP. The level of AI obtained by dividing the signal ratio in RNA by the corresponding ratio in DNA is plotted on the horizontal axis. The P -value for the difference between allelic ratios in RNA and DNA based on five replicate assays is plotted on the vertical axis. Spots above the horizontal dashed line represent the SNPs showing AI at a P -value

    Journal: Nucleic Acids Research

    Article Title: Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells

    doi: 10.1093/nar/gkl1152

    Figure Lengend Snippet: Volcano plot of the AI data from 105 heterozygous cSNPs in 13 cell lines. AI for each SNP was determined by calculating the fluorescence signal ratio between the two alleles ( S Allele1 / S Allele2 ) in RNA (cDNA) and genomic DNA for each heterozygous SNP. The level of AI obtained by dividing the signal ratio in RNA by the corresponding ratio in DNA is plotted on the horizontal axis. The P -value for the difference between allelic ratios in RNA and DNA based on five replicate assays is plotted on the vertical axis. Spots above the horizontal dashed line represent the SNPs showing AI at a P -value

    Article Snippet: Five micrograms of purified RNA was reverse transcribed to cDNA using the High-Capacity cDNA archive kit (Applied Biosystems, 4322171).

    Techniques: Fluorescence

    Analysis of mRNA expression levels of galectins in normal and infected corneas by qRT-PCR. Complementary DNA was synthesized from 100 ng each of total RNA preparations of normal and infected corneas using the High-Capacity cDNA Reverse Transcriptase Kit, and PCR amplification was performed in triplicate using gene-specific primers for β-actin, Gal-1, -3, -7, -8, and -9 and a Taqman master mix according to the manufacturer's instructions. A threshold cycle value (C t ) was calculated from each amplification plot. Quantification data of each gene were normalized to the expression of β-actin, a value of 1.0 was given to the expression of each gene in the control cornea and the expression values for galectins in infected corneas were calculated as a change in expression level with respect to the control cornea. At least four corneas were pooled and considered one biological replica. N = 4 for all galectins. Data are plotted as mean ± SEM and analyzed using one-way ANOVA. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Fingerprinting of Galectins in Normal, P. aeruginosa–Infected, and Chemically Burned Mouse Corneas

    doi: 10.1167/iovs.14-15338

    Figure Lengend Snippet: Analysis of mRNA expression levels of galectins in normal and infected corneas by qRT-PCR. Complementary DNA was synthesized from 100 ng each of total RNA preparations of normal and infected corneas using the High-Capacity cDNA Reverse Transcriptase Kit, and PCR amplification was performed in triplicate using gene-specific primers for β-actin, Gal-1, -3, -7, -8, and -9 and a Taqman master mix according to the manufacturer's instructions. A threshold cycle value (C t ) was calculated from each amplification plot. Quantification data of each gene were normalized to the expression of β-actin, a value of 1.0 was given to the expression of each gene in the control cornea and the expression values for galectins in infected corneas were calculated as a change in expression level with respect to the control cornea. At least four corneas were pooled and considered one biological replica. N = 4 for all galectins. Data are plotted as mean ± SEM and analyzed using one-way ANOVA. * P

    Article Snippet: Complementary DNA was synthesized from 100 ng total RNA using the High-Capacity cDNA Reverse Transcriptase Kit (Invitrogen).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Synthesized, Polymerase Chain Reaction, Amplification

    Male Balb/c mice were injected with either control buffer (Con) or tunicamycin (Tun). After 24 hrs, the epididymal fat pads were dissected and ( A ) total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated genes were quantified using real-time PCR and normalized to 18S rRNA. The value obtained for the control for each gene was assigned a value of 1. The value obtained following injection with tunicamycin was expressed relative to the control value. Results represent the mean ± SD of n = 3–8 experiments. ▪ Control, □ Tunicamycin. ( B ) Equal amounts of protein were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing the indicated proteins. For IRE1α, GRP78 and GAPDH intervening lanes have been removed. ( C – E ) The intensities of the bands in ( B ) were quantified from n = 3–6 experiments. The control values were set to 1. The values obtained from tunicamycin treated mice were expressed relative to the control. * Indicates P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Endoplasmic reticulum stress in adipose tissue augments lipolysis

    doi: 10.1111/jcmm.12384

    Figure Lengend Snippet: Male Balb/c mice were injected with either control buffer (Con) or tunicamycin (Tun). After 24 hrs, the epididymal fat pads were dissected and ( A ) total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated genes were quantified using real-time PCR and normalized to 18S rRNA. The value obtained for the control for each gene was assigned a value of 1. The value obtained following injection with tunicamycin was expressed relative to the control value. Results represent the mean ± SD of n = 3–8 experiments. ▪ Control, □ Tunicamycin. ( B ) Equal amounts of protein were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing the indicated proteins. For IRE1α, GRP78 and GAPDH intervening lanes have been removed. ( C – E ) The intensities of the bands in ( B ) were quantified from n = 3–6 experiments. The control values were set to 1. The values obtained from tunicamycin treated mice were expressed relative to the control. * Indicates P

    Article Snippet: RNA (2 μg) was transcribed to cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Burlington, ON, Canada).

    Techniques: Mouse Assay, Injection, Real-time Polymerase Chain Reaction, SDS Page

    Subcutaneous white adipose tissue was obtained during surgery from burned patients (burn) or non-burned patients undergoing elective surgery (non-burn). ( A ) Total RNA was extracted from adipose tissue obtained from non-burned ( n = 3–4) and burned patients ( n = 5–8) and transcribed to cDNA. Real-time quantitative PCR was performed to evaluate the relative mRNA levels of the indicated genes that were normalized to IDH1 mRNA. The value obtained from one non-burn sample was set to 1. The values obtained from all other samples were expressed relative to the non-burn sample. ▪ Non-burn, □ Burn. ( B , Top and Bottom Panels ) Adipose tissue was homogenized and equal amounts of total protein in the tissue homogenates were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing GRP78 and alpha/beta tubulin. ( C ) The intensities of the bands corresponding to GRP78 in ( B ) were quantified. ( D ) Human adipocytes (non-burn) were isolated and cultured either in the presence or absence of tunicamycin (5 μg/ml) at 37°C for the indicated times. The culture media was collected and the concentration of free glycerol determined. To account for the variability between patients in basal lipolysis rates, the amount of glycerol released from control cells from each patient and at each time-point was given a value of 100%. The amount of glycerol released from adipocytes in the presence of tunicamycin is expressed as a percentage of the value obtained from the control cells. Results represent the mean ± SD from n = 3 female patients ages 48 and 61 years, ▪ Control, □ Tunicamycin. * indicates P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Endoplasmic reticulum stress in adipose tissue augments lipolysis

    doi: 10.1111/jcmm.12384

    Figure Lengend Snippet: Subcutaneous white adipose tissue was obtained during surgery from burned patients (burn) or non-burned patients undergoing elective surgery (non-burn). ( A ) Total RNA was extracted from adipose tissue obtained from non-burned ( n = 3–4) and burned patients ( n = 5–8) and transcribed to cDNA. Real-time quantitative PCR was performed to evaluate the relative mRNA levels of the indicated genes that were normalized to IDH1 mRNA. The value obtained from one non-burn sample was set to 1. The values obtained from all other samples were expressed relative to the non-burn sample. ▪ Non-burn, □ Burn. ( B , Top and Bottom Panels ) Adipose tissue was homogenized and equal amounts of total protein in the tissue homogenates were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing GRP78 and alpha/beta tubulin. ( C ) The intensities of the bands corresponding to GRP78 in ( B ) were quantified. ( D ) Human adipocytes (non-burn) were isolated and cultured either in the presence or absence of tunicamycin (5 μg/ml) at 37°C for the indicated times. The culture media was collected and the concentration of free glycerol determined. To account for the variability between patients in basal lipolysis rates, the amount of glycerol released from control cells from each patient and at each time-point was given a value of 100%. The amount of glycerol released from adipocytes in the presence of tunicamycin is expressed as a percentage of the value obtained from the control cells. Results represent the mean ± SD from n = 3 female patients ages 48 and 61 years, ▪ Control, □ Tunicamycin. * indicates P

    Article Snippet: RNA (2 μg) was transcribed to cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Burlington, ON, Canada).

    Techniques: Real-time Polymerase Chain Reaction, SDS Page, Isolation, Cell Culture, Concentration Assay

    HAC-driven expression of dystrophin sequences originally deleted in DMD patients. ( a ) The genomic organization of the dystrophin gene: the gray vertical bars represent the exons; the green arrows indicate the promoters driving the expression of the different dystrophin isoforms within the gene 1 , 2 ; primer pairs used to amplify exon–exon junctions inside the deleted region are indicated by red arrowheads, and those used to amplify specific isoforms are indicated by blue arrowheads. Exon number here reported refers to the muscle dystrophin isoform Dp427m. ( b ) RT-PCR of specific dystrophin sequences localized inside the patient with deletion of exons (Ex) 4–43. Primers were constructed to spam five different exon–exon junctions (Ex. J.). cDNA from human heart and skeletal muscle was used as positive control. Analyses were performed at three different stages of the differentiation process: undifferentiated colonies, differentiated EBs cultured in suspension (day 20), and adhered cells (day 24), for each hiPS cell lines (DMD, healthy, and DYS-HAC hiPS cells). cDNA from human tissues was used as positive control. cDNA, complementary DNA; DMD, Duchenne muscular dystrophy; DYS-HAC, human artificial chromosome carrying the whole dystrophin genomic sequence; EBs, embryoid bodies; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAC, human artificial chromosome; hiPS cells, human induced pluripotent stem cells; RT-PCR, reverse transcriptase–polymerase chain reaction.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

    doi: 10.1038/mtm.2013.1

    Figure Lengend Snippet: HAC-driven expression of dystrophin sequences originally deleted in DMD patients. ( a ) The genomic organization of the dystrophin gene: the gray vertical bars represent the exons; the green arrows indicate the promoters driving the expression of the different dystrophin isoforms within the gene 1 , 2 ; primer pairs used to amplify exon–exon junctions inside the deleted region are indicated by red arrowheads, and those used to amplify specific isoforms are indicated by blue arrowheads. Exon number here reported refers to the muscle dystrophin isoform Dp427m. ( b ) RT-PCR of specific dystrophin sequences localized inside the patient with deletion of exons (Ex) 4–43. Primers were constructed to spam five different exon–exon junctions (Ex. J.). cDNA from human heart and skeletal muscle was used as positive control. Analyses were performed at three different stages of the differentiation process: undifferentiated colonies, differentiated EBs cultured in suspension (day 20), and adhered cells (day 24), for each hiPS cell lines (DMD, healthy, and DYS-HAC hiPS cells). cDNA from human tissues was used as positive control. cDNA, complementary DNA; DMD, Duchenne muscular dystrophy; DYS-HAC, human artificial chromosome carrying the whole dystrophin genomic sequence; EBs, embryoid bodies; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAC, human artificial chromosome; hiPS cells, human induced pluripotent stem cells; RT-PCR, reverse transcriptase–polymerase chain reaction.

    Article Snippet: For the cardiac marker analyses, first-strand complementary DNA (cDNA) synthesis was performed using an oligo-(dT)20 primer and the cDNA Reverse Transcription Kit (Life Technologies).

    Techniques: HAC Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Construct, Positive Control, Cell Culture, Sequencing, Polymerase Chain Reaction

    HAC-driven expression of multiple dystrophin isoforms. ( a ) RT-PCR of dystrophin isoforms Dp427m, Dp260, Dp140, and Dp71 at three different stages of the differentiation process: undifferentiated colonies, differentiated EBs cultured in suspension (day 20), and adhered cells (day 24), for each hiPS cell lines (DMD, healthy, and DYS-HAC hiPS cells). cDNA from human tissues was used as positive control. ( b ) Real-time PCR for dystrophin isoforms present in cardiac tissue. Muscle-specific Dp427m is double checked with Dp427m-specific primer set spanning exons 1–3 and an all Dp427 isoform-specific primer set spanning exons 25–26. Data are presented as mean ± SD. cDNA, complementary DNA; DYS-HAC, human artificial chromosome carrying the whole dystrophin genomic sequence; EBs, embryoid bodies; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAC, human artificial chromosome; hiPS cells, human induced pluripotent stem cells; RT-PCR, reverse transcriptase–polymerase chain reaction; DMD, Duchenne muscular dystrophy.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

    doi: 10.1038/mtm.2013.1

    Figure Lengend Snippet: HAC-driven expression of multiple dystrophin isoforms. ( a ) RT-PCR of dystrophin isoforms Dp427m, Dp260, Dp140, and Dp71 at three different stages of the differentiation process: undifferentiated colonies, differentiated EBs cultured in suspension (day 20), and adhered cells (day 24), for each hiPS cell lines (DMD, healthy, and DYS-HAC hiPS cells). cDNA from human tissues was used as positive control. ( b ) Real-time PCR for dystrophin isoforms present in cardiac tissue. Muscle-specific Dp427m is double checked with Dp427m-specific primer set spanning exons 1–3 and an all Dp427 isoform-specific primer set spanning exons 25–26. Data are presented as mean ± SD. cDNA, complementary DNA; DYS-HAC, human artificial chromosome carrying the whole dystrophin genomic sequence; EBs, embryoid bodies; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HAC, human artificial chromosome; hiPS cells, human induced pluripotent stem cells; RT-PCR, reverse transcriptase–polymerase chain reaction; DMD, Duchenne muscular dystrophy.

    Article Snippet: For the cardiac marker analyses, first-strand complementary DNA (cDNA) synthesis was performed using an oligo-(dT)20 primer and the cDNA Reverse Transcription Kit (Life Technologies).

    Techniques: HAC Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Positive Control, Real-time Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction

    Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g RNA was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into cDNA. mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p

    Journal: Journal of Cell Communication and Signaling

    Article Title: K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

    doi: 10.1007/s12079-017-0412-8

    Figure Lengend Snippet: Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g RNA was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into cDNA. mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p

    Article Snippet: The resulting RNA was reverse transcribed using high capacity cDNA synthesis kit and oligodT primers at 37°C for 120 min. Real-time PCR was performed using Power SyBr Green reagents in an ABI 7500 system (Thermo Fisher Scientific).

    Techniques: Modification, Expressing, Flow Cytometry, Cytometry, Marker, Cell Culture, Derivative Assay, Staining, Real-time Polymerase Chain Reaction

    The evolution of miR-351 and miR-298.mr. ( A ) Evolutionary conservation of miR-351 and miR-298. Images are reproduced from miRviewer ( http://people.csail.mit.edu/artzi/miRviewer ). miR-351 and miR-298.mr are unique to mice (mmu – Mus musculus ) and rats (rno – Rattus norvegicus ). Bolded nucleotides represent the mature miRNA. Nucleotide variations are italicized and underlined. ( B ) Alignment of mature miRNA sequences from the miR-125 family (cel – Caenorhabditis elegans ). Bolded nucleotides represent the regulatory ‘seed’ region which is identical to all family members. ( C ) An on-scale representative of the genomic locus of the mouse miR-351 gene. Arrows indicate downregulation of the miRNA expression level 48 h following activation to the level indicated (mean ± SEM, N = 3). miR-450a-1 and 2 could not be distinguished. ND – not detected. All miRNAs genes in this cluster, except for miR-351 gene are evolutionarily highly conserved as can be seen in the conservation plot below (reproduced from UCSC Genome Browser). ( D ) Reverse transcription PCR of the RNA transcript precursor of miR-351. Using primers complementary to the pre-miRNAs sequences of the mice miR-351, miR-503 and miR-322 that were designed to amplify the regions indicated in (C), we observed that miR-351 gene is transcribed in a single transcriptional unit together with miR-503 (a) which is transcribed together with miR-322 (b). Bands can be observed in cDNA and genomic DNA (gDNA) amplification but not in RNA (to exclude gDNA contamination).

    Journal: Nucleic Acids Research

    Article Title: Species-specific microRNA roles elucidated following astrocyte activation

    doi: 10.1093/nar/gkq1325

    Figure Lengend Snippet: The evolution of miR-351 and miR-298.mr. ( A ) Evolutionary conservation of miR-351 and miR-298. Images are reproduced from miRviewer ( http://people.csail.mit.edu/artzi/miRviewer ). miR-351 and miR-298.mr are unique to mice (mmu – Mus musculus ) and rats (rno – Rattus norvegicus ). Bolded nucleotides represent the mature miRNA. Nucleotide variations are italicized and underlined. ( B ) Alignment of mature miRNA sequences from the miR-125 family (cel – Caenorhabditis elegans ). Bolded nucleotides represent the regulatory ‘seed’ region which is identical to all family members. ( C ) An on-scale representative of the genomic locus of the mouse miR-351 gene. Arrows indicate downregulation of the miRNA expression level 48 h following activation to the level indicated (mean ± SEM, N = 3). miR-450a-1 and 2 could not be distinguished. ND – not detected. All miRNAs genes in this cluster, except for miR-351 gene are evolutionarily highly conserved as can be seen in the conservation plot below (reproduced from UCSC Genome Browser). ( D ) Reverse transcription PCR of the RNA transcript precursor of miR-351. Using primers complementary to the pre-miRNAs sequences of the mice miR-351, miR-503 and miR-322 that were designed to amplify the regions indicated in (C), we observed that miR-351 gene is transcribed in a single transcriptional unit together with miR-503 (a) which is transcribed together with miR-322 (b). Bands can be observed in cDNA and genomic DNA (gDNA) amplification but not in RNA (to exclude gDNA contamination).

    Article Snippet: miRNA profiling First-strand cDNA was synthesized from total RNA using Megaplex reverse transcriptase reaction with the High Capacity cDNA kit (Applied Biosystems, USA).

    Techniques: Mouse Assay, Expressing, Activation Assay, Polymerase Chain Reaction, Amplification

    Increased expression of GFAT in lung cancer cell lines and tissues. ( A ) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total RNA was extracted from cell lines; cDNA was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and β-actin as loading control. Products were run in agarose gel with EB. ( B ) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. β-Actin was probed as a loading control. ( C ) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective β-actin, and then cancer over normal expression was calculated. * P

    Journal: Molecular carcinogenesis

    Article Title: Inhibition of the hexosamine biosynthesis pathway potentiates cisplatin cytotoxicity by decreasing BiP expression in non-small cell lung cancer cells

    doi: 10.1002/mc.22992

    Figure Lengend Snippet: Increased expression of GFAT in lung cancer cell lines and tissues. ( A ) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total RNA was extracted from cell lines; cDNA was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and β-actin as loading control. Products were run in agarose gel with EB. ( B ) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. β-Actin was probed as a loading control. ( C ) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective β-actin, and then cancer over normal expression was calculated. * P

    Article Snippet: Briefly, total RNA extracted with Trizol (Invitrogen) was used for reverse transcription with High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Synthesized, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, TaqMan Assay

    Knockdown of PABPN1 in human cells. HeLa and/or HEK293 cells were grown on 35 mm plates to approximately 30% confluence and transiently transfected with PABPN1-Si or PABPN1-UTR RNAi for indicated length of time. Non transfected (NT) control cells were also maintained in culture for the same duration as transfected cells. (A, B) Following transfection, cells were harvested at 45 h, 66 h and 80 h in Laemmli buffer. Whole cell extracts from Si, UTR, and NT HeLa (A) and HEK293 (B) cells were analyzed for PABPN1 protein by western blotting; GAPDH was used as the loading control. (C) Forty five hours after transfection, HeLa cells grown on coverslip were fixed with Para-formaldehyde and processed for immunostaining with PABPN1 specific antibody and counterstained with Texas-red-conjugated secondary antibody. Processed specimens were then examined with a confocal microscope. Images from two different sections of the slide are shown here. (D) Forty five hours and sixty six hours after transfection, RNA was extracted from HeLa cells using Trizol and reverse transcribed; cDNA thus made were used for PCR with primers specific to β-actin, PABP and PABPN1. Samples from PABPN1-UTR Si and non transfected (NT) cells were collected at 66 h time point. The bands on the gel representing the PCR products were scanned and quantified as described in Materials and Methods . The band intensities are shown in arbitrary unit.

    Journal: PLoS ONE

    Article Title: Depletion of Nuclear Poly(A) Binding Protein PABPN1 Produces a Compensatory Response by Cytoplasmic PABP4 and PABP5 in Cultured Human Cells

    doi: 10.1371/journal.pone.0053036

    Figure Lengend Snippet: Knockdown of PABPN1 in human cells. HeLa and/or HEK293 cells were grown on 35 mm plates to approximately 30% confluence and transiently transfected with PABPN1-Si or PABPN1-UTR RNAi for indicated length of time. Non transfected (NT) control cells were also maintained in culture for the same duration as transfected cells. (A, B) Following transfection, cells were harvested at 45 h, 66 h and 80 h in Laemmli buffer. Whole cell extracts from Si, UTR, and NT HeLa (A) and HEK293 (B) cells were analyzed for PABPN1 protein by western blotting; GAPDH was used as the loading control. (C) Forty five hours after transfection, HeLa cells grown on coverslip were fixed with Para-formaldehyde and processed for immunostaining with PABPN1 specific antibody and counterstained with Texas-red-conjugated secondary antibody. Processed specimens were then examined with a confocal microscope. Images from two different sections of the slide are shown here. (D) Forty five hours and sixty six hours after transfection, RNA was extracted from HeLa cells using Trizol and reverse transcribed; cDNA thus made were used for PCR with primers specific to β-actin, PABP and PABPN1. Samples from PABPN1-UTR Si and non transfected (NT) cells were collected at 66 h time point. The bands on the gel representing the PCR products were scanned and quantified as described in Materials and Methods . The band intensities are shown in arbitrary unit.

    Article Snippet: An aliquot of total RNA (100–500 ng) was reverse transcribed using High Capacity cDNA transcription kit (Applied Biosystems, Life Technologies).

    Techniques: Transfection, Western Blot, Immunostaining, Microscopy, Polymerase Chain Reaction

    Coagulation factor gene expression is elevated in IP mice and malaria-exposed murine trophoblasts. (A) RNA was isolated from conceptuses removed from ED 10 UP (n = 5) and IP (n = 6) mice. Primers specific for the genes indicated were utilized to measure cDNA expression levels in IP relative to UP mice. Data are normalized against murine 18S RNA. Data are expressed as the ratio of fold increase in IP mice to that of UP mice ± SEM. (B) SM9-1 trophoblasts were stimulated with P. chabaudi AS-iRBCs and RNA isolated over the time course indicated. QRT-PCR was conducted as in panel A. Data are expressed as the ratio of fold increase relative to time matched SM9-1 trophoblasts stimulated with uninfected RBC ± SEM and are representative of four separate experiments.

    Journal: PLoS ONE

    Article Title: Maternal Malaria Induces a Procoagulant and Antifibrinolytic State That Is Embryotoxic but Responsive to Anticoagulant Therapy

    doi: 10.1371/journal.pone.0031090

    Figure Lengend Snippet: Coagulation factor gene expression is elevated in IP mice and malaria-exposed murine trophoblasts. (A) RNA was isolated from conceptuses removed from ED 10 UP (n = 5) and IP (n = 6) mice. Primers specific for the genes indicated were utilized to measure cDNA expression levels in IP relative to UP mice. Data are normalized against murine 18S RNA. Data are expressed as the ratio of fold increase in IP mice to that of UP mice ± SEM. (B) SM9-1 trophoblasts were stimulated with P. chabaudi AS-iRBCs and RNA isolated over the time course indicated. QRT-PCR was conducted as in panel A. Data are expressed as the ratio of fold increase relative to time matched SM9-1 trophoblasts stimulated with uninfected RBC ± SEM and are representative of four separate experiments.

    Article Snippet: Briefly, RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription and DNA-free Kits (Applied Biosystems, Carlsbad, CA, USA) or RNeasy® Plus Mini Kit (Qiagen, Valencia, CA, USA) as described by the manufacturers' protocols.

    Techniques: Coagulation, Expressing, Mouse Assay, Isolation, Quantitative RT-PCR

    2.3. Total RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (qPCR)

    Journal: Food & function

    Article Title: Dietary resistant starch type 4-derived butyrate attenuates nuclear factor-kappa-B1 through modulation of lysine 27 trimethylation of histone H3

    doi: 10.1039/c6fo00856a

    Figure Lengend Snippet: 2.3. Total RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (qPCR)

    Article Snippet: The cDNAs were synthesized using 3 µg of RNA for each sample using the High-Capacity cDNA Reverse Transcription (RT) Kit (Invitrogen, Grand Island, NY), following the manufacturers’ protocol.

    Techniques: RNA Extraction, Real-time Polymerase Chain Reaction

    Variation in the relative abundance of the two forms of the HLA-A 3’UTR RNA was obtained from peripheral blood lymphocytes of healthy donors. Rapid amplification of cDNA ends (3’ RACE) of 3’UTRs of HLA-A alleles from 6 individuals with distinct HLA-A genotypes showed preferential use of either the proximal or distal PAS. HLA-A*01 and A*11 use only the proximal PAS, resulting in the short form of the 3’UTR, whereas others use various fractions of each. HLA-A genotypes of the individuals are indicated below each amplicon.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Post transcriptional regulation of HLA-A protein expression by alternative polyadenylation signals involving the RNA binding protein Syncrip

    doi: 10.4049/jimmunol.1700697

    Figure Lengend Snippet: Variation in the relative abundance of the two forms of the HLA-A 3’UTR RNA was obtained from peripheral blood lymphocytes of healthy donors. Rapid amplification of cDNA ends (3’ RACE) of 3’UTRs of HLA-A alleles from 6 individuals with distinct HLA-A genotypes showed preferential use of either the proximal or distal PAS. HLA-A*01 and A*11 use only the proximal PAS, resulting in the short form of the 3’UTR, whereas others use various fractions of each. HLA-A genotypes of the individuals are indicated below each amplicon.

    Article Snippet: Briefly, reverse transcription was performed with 900ng of total RNA using the high capacity RNA to cDNA kit (Applied Bioscience) in a volume of 10µl.

    Techniques: Rapid Amplification of cDNA Ends, Amplification

    Zika virus (ZIKV) transcriptional expression in glomerular cells. A, Expression of ZIKV messenger RNA as shown by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in human podocytes. B, C, Human glomerular endothelial cells ( B ) and human mesangial cells ( C ) exposed to wild-type ZIKV along with heat-killed (HK) ZIKV and mock-infected controls. All cells were exposed to ZIKV for 72 hours. Total RNA was extracted from infected cells, followed by complementary DNA amplification and qRT-PCR. Fold expression was normalized to glyceraldehyde 3-phosphate dehydrogenase. Error bars represent standard errors of the mean for triplicate experiments. The units are comparative mRNA transcriptional units.

    Journal: The Journal of Infectious Diseases

    Article Title: Zika Virus Infection of the Human Glomerular Cells: Implications for Viral Reservoirs and Renal Pathogenesis

    doi: 10.1093/infdis/jix171

    Figure Lengend Snippet: Zika virus (ZIKV) transcriptional expression in glomerular cells. A, Expression of ZIKV messenger RNA as shown by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in human podocytes. B, C, Human glomerular endothelial cells ( B ) and human mesangial cells ( C ) exposed to wild-type ZIKV along with heat-killed (HK) ZIKV and mock-infected controls. All cells were exposed to ZIKV for 72 hours. Total RNA was extracted from infected cells, followed by complementary DNA amplification and qRT-PCR. Fold expression was normalized to glyceraldehyde 3-phosphate dehydrogenase. Error bars represent standard errors of the mean for triplicate experiments. The units are comparative mRNA transcriptional units.

    Article Snippet: Messenger RNA (mRNA) in 0.5 μg of each sample was primed using random hexamers and reverse-transcribed with a high-capacity complementary DNA (cDNA) reverse-transcription kit (Applied Biosystems).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Amplification

    Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. mRNA were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and cDNA inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Summary of cDNAs recovered from DENV-resistant Huh7.5 cell clones. Fifty-two colonies of clonal Huh7.5 cells that survived a chellenge infection of DENV-2 were infected with DENV at an MOI of 1, and culture supernatants of infected cells were subjected to plaque assay. mRNA were isolated from cells whose supernatant formed 10 or less DENV plaques less (DENV-resistant clones, total 32 clones), and cDNA inserts expressing in the clones were analyzed by sequencing. More than 50 plaques were formed with a supernatant of control protein (BAP)-expressing Huh7.5 cells.

    Article Snippet: In some experiments for the detection of DENV-2 RNA, qRT-PCR was performed by High-Capacity cDNA Reverse Transcription Kit and SsoFast Probes Supermix (Bio-Rad) using previously described primers and fluorescent probe targeting 3’UTR of the DENV genome [ ].

    Techniques: Clone Assay, Infection, Plaque Assay, Isolation, Expressing, Sequencing

    Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.

    Journal: PLoS Pathogens

    Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

    doi: 10.1371/journal.ppat.1005357

    Figure Lengend Snippet: Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.

    Article Snippet: In some experiments for the detection of DENV-2 RNA, qRT-PCR was performed by High-Capacity cDNA Reverse Transcription Kit and SsoFast Probes Supermix (Bio-Rad) using previously described primers and fluorescent probe targeting 3’UTR of the DENV genome [ ].

    Techniques: cDNA Library Assay, Generated, Plasmid Preparation, Produced, Transduction, Expressing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Migration, Molecular Weight, Transformation Assay, Clone Assay, Infection, Staining, Plaque Assay, Isolation, Sequencing