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    Thermo Fisher highcapacity cdna reverse transcription kits
    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 <t>DNA</t> 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.
    Highcapacity Cdna Reverse Transcription Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    SOCE is diminished in invasive melanoma cells. (A and B) RNA was extracted from melanoma cell lines, and then cDNA was prepared by reverse transcription. Custom qRT-PCR primers were designed to amplify 50- to 60-bp products against human Wnt5A (A) or

    Journal: Molecular and Cellular Biology

    Article Title: Novel Protein Kinase C-Mediated Control of Orai1 Function in Invasive Melanoma

    doi: 10.1128/MCB.01500-14

    Figure Lengend Snippet: SOCE is diminished in invasive melanoma cells. (A and B) RNA was extracted from melanoma cell lines, and then cDNA was prepared by reverse transcription. Custom qRT-PCR primers were designed to amplify 50- to 60-bp products against human Wnt5A (A) or

    Article Snippet: Reverse transcription-PCR (RT-PCR) was completed as a two-step process using high-capacity cDNA reverse transcription (Applied Biosystems) followed by Sybr green PCR (Invitrogen) on a 7300 real-time PCR machine (Applied Biosystems).

    Techniques: Quantitative RT-PCR

    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

    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