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    Thermo Fisher high capacity cdna reverse transcription kit
    Schematic workflow of optimized CLIP. Cells are irradiated with 254 nm UV light on ice to form crosslinked <t>RNA-RBP</t> complexes, followed by partial RNase digestion and immunoprecipitation with RBP-specific antibodies. RNA crosslinked with RBPs is dephosphorylated for linker ligation, phosphorylated with [γ- 32 P]- ATP if necessary, separated by NuPAGE, and then transferred to a membrane. Isolated RNA is ligated with a 5'-App linker at the 3'-end and is subjected to BrdU-labeled <t>cDNA</t> synthesis by reverse transcription using a primer with a barcode and cleavage site. The cDNA is purified with the anti-BrdU antibody-coupled magnetic beads, circularized, digested with APE I, and amplified by PCR. Lastly, the CLIP cDNA library is subjected to size purification by PAGE. Steps modified from the original BrdU-CLIP protocol are indicated in red.
    High Capacity Cdna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 119037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher high capacity cdna reverse transcription kit thermo fisher scientific
    Schematic workflow of optimized CLIP. Cells are irradiated with 254 nm UV light on ice to form crosslinked <t>RNA-RBP</t> complexes, followed by partial RNase digestion and immunoprecipitation with RBP-specific antibodies. RNA crosslinked with RBPs is dephosphorylated for linker ligation, phosphorylated with [γ- 32 P]- ATP if necessary, separated by NuPAGE, and then transferred to a membrane. Isolated RNA is ligated with a 5'-App linker at the 3'-end and is subjected to BrdU-labeled <t>cDNA</t> synthesis by reverse transcription using a primer with a barcode and cleavage site. The cDNA is purified with the anti-BrdU antibody-coupled magnetic beads, circularized, digested with APE I, and amplified by PCR. Lastly, the CLIP cDNA library is subjected to size purification by PAGE. Steps modified from the original BrdU-CLIP protocol are indicated in red.
    High Capacity Cdna Reverse Transcription Kit Thermo Fisher Scientific, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity cdna reverse transcription kit thermo fisher scientific/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit thermo fisher scientific - by Bioz Stars, 2020-07
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    Thermo Fisher high capacity cdna reverse transcription kit thermo fisher scientific 4368813 insulin
    Schematic workflow of optimized CLIP. Cells are irradiated with 254 nm UV light on ice to form crosslinked <t>RNA-RBP</t> complexes, followed by partial RNase digestion and immunoprecipitation with RBP-specific antibodies. RNA crosslinked with RBPs is dephosphorylated for linker ligation, phosphorylated with [γ- 32 P]- ATP if necessary, separated by NuPAGE, and then transferred to a membrane. Isolated RNA is ligated with a 5'-App linker at the 3'-end and is subjected to BrdU-labeled <t>cDNA</t> synthesis by reverse transcription using a primer with a barcode and cleavage site. The cDNA is purified with the anti-BrdU antibody-coupled magnetic beads, circularized, digested with APE I, and amplified by PCR. Lastly, the CLIP cDNA library is subjected to size purification by PAGE. Steps modified from the original BrdU-CLIP protocol are indicated in red.
    High Capacity Cdna Reverse Transcription Kit Thermo Fisher Scientific 4368813 Insulin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity cdna reverse transcription kit thermo fisher scientific 4368813 insulin/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit thermo fisher scientific 4368813 insulin - by Bioz Stars, 2020-07
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    Schematic workflow of optimized CLIP. Cells are irradiated with 254 nm UV light on ice to form crosslinked RNA-RBP complexes, followed by partial RNase digestion and immunoprecipitation with RBP-specific antibodies. RNA crosslinked with RBPs is dephosphorylated for linker ligation, phosphorylated with [γ- 32 P]- ATP if necessary, separated by NuPAGE, and then transferred to a membrane. Isolated RNA is ligated with a 5'-App linker at the 3'-end and is subjected to BrdU-labeled cDNA synthesis by reverse transcription using a primer with a barcode and cleavage site. The cDNA is purified with the anti-BrdU antibody-coupled magnetic beads, circularized, digested with APE I, and amplified by PCR. Lastly, the CLIP cDNA library is subjected to size purification by PAGE. Steps modified from the original BrdU-CLIP protocol are indicated in red.

    Journal: PLoS ONE

    Article Title: Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method

    doi: 10.1371/journal.pone.0231450

    Figure Lengend Snippet: Schematic workflow of optimized CLIP. Cells are irradiated with 254 nm UV light on ice to form crosslinked RNA-RBP complexes, followed by partial RNase digestion and immunoprecipitation with RBP-specific antibodies. RNA crosslinked with RBPs is dephosphorylated for linker ligation, phosphorylated with [γ- 32 P]- ATP if necessary, separated by NuPAGE, and then transferred to a membrane. Isolated RNA is ligated with a 5'-App linker at the 3'-end and is subjected to BrdU-labeled cDNA synthesis by reverse transcription using a primer with a barcode and cleavage site. The cDNA is purified with the anti-BrdU antibody-coupled magnetic beads, circularized, digested with APE I, and amplified by PCR. Lastly, the CLIP cDNA library is subjected to size purification by PAGE. Steps modified from the original BrdU-CLIP protocol are indicated in red.

    Article Snippet: The cDNA from 100 ng of total RNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions.

    Techniques: Cross-linking Immunoprecipitation, Irradiation, Immunoprecipitation, Ligation, Isolation, Labeling, Purification, Magnetic Beads, Amplification, Polymerase Chain Reaction, cDNA Library Assay, Polyacrylamide Gel Electrophoresis, Modification

    Optimization of dephosphorylation, linker ligation and circularization. (A–B) Quantification of reverse transcribed cDNA to evaluate the yield of linker-ligated RNA. Template RNA was isolated from HNRNPU-RNA, dephosphorylated with PNK or CIAP, and subsequently ligated using linkers with phosphorylated or adenylated 5’-ends. Linkers were phosphorylated or adenylated prior to polyacrylmide gel electrophoresis (PAGE) or after isolation from the membrane. cDNA that was reverse transcribed in the presence of [α- 32 P]-dCTP was purified with anti-BrdU beads and separated in a 10% TBE-urea gel (A). Signal density was quantified from the autoradiograph (B). (C) Schematics for optimization of elution during anti-BrdU purification and during circularization using RNA ladders. (D–E) Reverse transcribed cDNAs from linker-ligated RNA ladders were eluted from anti-BrdU beads as indicated in (C), and were subsequently circularized and re-linearized in tubes (EL2-1, -2, -3) or on beads (EL2-4).

    Journal: PLoS ONE

    Article Title: Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method

    doi: 10.1371/journal.pone.0231450

    Figure Lengend Snippet: Optimization of dephosphorylation, linker ligation and circularization. (A–B) Quantification of reverse transcribed cDNA to evaluate the yield of linker-ligated RNA. Template RNA was isolated from HNRNPU-RNA, dephosphorylated with PNK or CIAP, and subsequently ligated using linkers with phosphorylated or adenylated 5’-ends. Linkers were phosphorylated or adenylated prior to polyacrylmide gel electrophoresis (PAGE) or after isolation from the membrane. cDNA that was reverse transcribed in the presence of [α- 32 P]-dCTP was purified with anti-BrdU beads and separated in a 10% TBE-urea gel (A). Signal density was quantified from the autoradiograph (B). (C) Schematics for optimization of elution during anti-BrdU purification and during circularization using RNA ladders. (D–E) Reverse transcribed cDNAs from linker-ligated RNA ladders were eluted from anti-BrdU beads as indicated in (C), and were subsequently circularized and re-linearized in tubes (EL2-1, -2, -3) or on beads (EL2-4).

    Article Snippet: The cDNA from 100 ng of total RNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions.

    Techniques: De-Phosphorylation Assay, Ligation, Isolation, Nucleic Acid Electrophoresis, Polyacrylamide Gel Electrophoresis, Purification, Autoradiography

    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

    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

    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

    RNA and protein content of cellular exosomes from HCC cells (A) RNA was extracted from Hep3B derived exosomes (lane 1) or their corresponding donor cells (lane 2) and analyzed by capillary electrophoresis (Bioanalyzer, Agilent). The RNA content is strikingly different, with the majority of RNA in Hep3B derived exosomes below 2 kb in size and with a very low fraction of 18S ribosomal RNA (rRNA) and 28S rRNA compared to RNA from donor cells. (B) An equivalent amount (600 ng) of RNA from Hep3B or PLC/PRF/5 donor cells, or exosomes obtained from these cells was transcribed to cDNA and the expression of 18S ribosomal RNA (18S rRNA) and small nucleolar RNA U43 (snoRNA U43) examined by real-time PCR. Amplification curves and the mean value ± SEM from four independent samples of threshold cycles for 18S rRNA and snoRNA U43 are shown. Both 18S rRNA and snoRNA U43 show higher CT values in exosomes than in their corresponding donor cells. (C) Protein was isolated from Hep3B derived exosomes or their donor cells and 15 μg of protein was separated on a Bis-Tris gel and stained with SYPRO Ruby (lane 1, donor cells; lane 2, exosomes). The protein content in exosomes was different from that in their corresponding donor cells.

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Inter-cellular nanovesicle mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth

    doi: 10.1002/hep.24504

    Figure Lengend Snippet: RNA and protein content of cellular exosomes from HCC cells (A) RNA was extracted from Hep3B derived exosomes (lane 1) or their corresponding donor cells (lane 2) and analyzed by capillary electrophoresis (Bioanalyzer, Agilent). The RNA content is strikingly different, with the majority of RNA in Hep3B derived exosomes below 2 kb in size and with a very low fraction of 18S ribosomal RNA (rRNA) and 28S rRNA compared to RNA from donor cells. (B) An equivalent amount (600 ng) of RNA from Hep3B or PLC/PRF/5 donor cells, or exosomes obtained from these cells was transcribed to cDNA and the expression of 18S ribosomal RNA (18S rRNA) and small nucleolar RNA U43 (snoRNA U43) examined by real-time PCR. Amplification curves and the mean value ± SEM from four independent samples of threshold cycles for 18S rRNA and snoRNA U43 are shown. Both 18S rRNA and snoRNA U43 show higher CT values in exosomes than in their corresponding donor cells. (C) Protein was isolated from Hep3B derived exosomes or their donor cells and 15 μg of protein was separated on a Bis-Tris gel and stained with SYPRO Ruby (lane 1, donor cells; lane 2, exosomes). The protein content in exosomes was different from that in their corresponding donor cells.

    Article Snippet: cDNA was transcribed from a total of 600ng of DNase I-treated RNA using the cDNA reverse transcription kit and random primers (Invitrogen, Carlsbad, CA).

    Techniques: Derivative Assay, Electrophoresis, Planar Chromatography, Expressing, Real-time Polymerase Chain Reaction, Amplification, Isolation, Staining

    Cell-to-cell transfer of firefly luciferase by exosomes (A) Real-tine PCR was performed on an cDNA transcribed from an equivalent amount (600 ng) of RNA from PLC-luc derived exosomes or their donor cells (n = 3, each in duplicate). PCR amplification curves for Fluc mRNA and 18S rRNA are shown. (B) PLC/PRF/5 cells were incubated with 15 μg/ml of PLC-luc derived exosomes for 16 hours. RNA was isolated and equivalent amount of RNA (300 ng) was transcribed to cDNA (n = 3). Amplification curves by quantitative real-time PCR for Fluc mRNA and 18S rRNA in PLC/PRF/5 (recipient cells) and PLC-luc (donor cells) are shown. (C) PLC/PRF/5 cells in a 96-well plate were incubated with various concentrations of PLC-luc derived exosomes, and luciferase activity was assessed in these cells after 16 hours. Bars express the mean value of luminescence ± SEM of four separate determinations. *, p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Inter-cellular nanovesicle mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth

    doi: 10.1002/hep.24504

    Figure Lengend Snippet: Cell-to-cell transfer of firefly luciferase by exosomes (A) Real-tine PCR was performed on an cDNA transcribed from an equivalent amount (600 ng) of RNA from PLC-luc derived exosomes or their donor cells (n = 3, each in duplicate). PCR amplification curves for Fluc mRNA and 18S rRNA are shown. (B) PLC/PRF/5 cells were incubated with 15 μg/ml of PLC-luc derived exosomes for 16 hours. RNA was isolated and equivalent amount of RNA (300 ng) was transcribed to cDNA (n = 3). Amplification curves by quantitative real-time PCR for Fluc mRNA and 18S rRNA in PLC/PRF/5 (recipient cells) and PLC-luc (donor cells) are shown. (C) PLC/PRF/5 cells in a 96-well plate were incubated with various concentrations of PLC-luc derived exosomes, and luciferase activity was assessed in these cells after 16 hours. Bars express the mean value of luminescence ± SEM of four separate determinations. *, p

    Article Snippet: cDNA was transcribed from a total of 600ng of DNase I-treated RNA using the cDNA reverse transcription kit and random primers (Invitrogen, Carlsbad, CA).

    Techniques: Luciferase, Polymerase Chain Reaction, Planar Chromatography, Derivative Assay, Amplification, Incubation, Isolation, Real-time Polymerase Chain Reaction, Activity Assay

    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