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    Structured Review

    Thermo Fisher rnase free
    Rnase Free, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase free/product/Thermo Fisher
    Average 90 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    rnase free - by Bioz Stars, 2020-02
    90/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: .. RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. The isolated RNA was dissolved in RNA storage buffer (Ambion, Grand Island, NY, USA) and stored at −80 o C until used.

    Amplification:

    Article Title: Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties
    Article Snippet: In a final volume of 20 μL, 1 μg of RNase-free and DNase-treated total RNA was mixed with 5 × First-Strand buffer, 500 μM dNTPs, 500 nM OdT-T71 (5'-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG GGA GAT24 ), 2 mM DTT, 40 U RNaseOut (Invitrogen) and SuperScriptIII (200 U/μL). .. Quantitative amplification was performed in a Rotor-Gene (Rotor-Gene 3000, Corbett) using RealMasterMix SYBR Green kit (Eppendorf).

    Article Title: Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose
    Article Snippet: qPCR DNase treated (DNase I, RNase free; Fermentas) total RNA (5 μg) was reversely transcribed with the RevertAid™ First Strand cDNA Kit (Fermentas) according to the manufacturer’s protocol with a combination (1:1) of the provided oligo-dT and random hexamer primers. .. Primers, amplification efficiency and R-square values are given in Additional file : Table S8.

    Article Title: Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury
    Article Snippet: Quantitative RT-PCR Knees from WT mice (n = 8) and Mig-6 −/− mice (n = 7) were dissected under RNase-free conditions and homogenized in 1 ml of TRIzol reagent (Life Technologies, Carlsbad, CA, USA) with a Fast Prep-24 tissue and cell homogenizer (MP Biomedicals, Solon, OH, USA). .. RNA concentration was calculated and integrity was analyzed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively, and potential DNA contamination was removed by RNase-free DNase treatment (amplification grade DNaseI; Sigma-Aldrich). cDNA was made with a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA) on 0.5 μg of RNA.

    Article Title: Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum
    Article Snippet: The pipette contents were expelled into RNase-free 0.25 ml PCR tubes containing 5 μ l of DEPC-treated H2 0, 1 μ l of Superase RNase inhibitor (Ambion), 1 μ l of 0.1 M dithiothreitol, and 1 μ l of oligo-dT12–18 primer (Invitrogen), briefly centrifuged, and stored at −20°C. .. Multiplex PCR was performed with two rounds of amplification as described by with the modification of including the first-round PCR pairs for VMAT1 and VMAT2 in the first PCR round and amplifying these two signals separately in the second round with the corresponding nested primer sets.

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses. .. A threshold cycle (CT) value was determined from each amplification plot.

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. RNA integrity was tested by PCR amplification of the β -actin housekeeping gene.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses. .. A threshold cycle (CT ) value was determined from each amplification plot.

    DNA Synthesis:

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: .. Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses. .. Real-Time Quantitative Polymerase Chain Reaction (qPCR) was conducted using the StepOne™ detection system, EXPRESS SYBR® GreenER™ , and ROX™ qPCR SuperMix Universal, and sequence detector software v2.3 (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: .. The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses. .. Real-time quantitative polymerase chain reaction (qPCR) was conducted using the StepOne detection system, EXPRESS SYBR® GreenER™ qPCR SuperMix Universal, and sequence detector software (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Synthesized:

    Article Title: Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury
    Article Snippet: Total RNA was extracted under RNase-free conditions using an RNA isolation kit (RNAqueous Micro Kit; Ambion) according to the manufacturer's instructions. .. DNase was treated for 15 min to minimize genomic DNA contamination and quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). cDNA was synthesized from 200 ng of RNA using random hexamers of the Verso cDNA kit (Thermo Scientific).

    Quantitative RT-PCR:

    Article Title: Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury
    Article Snippet: Paragraph title: Real-time RT-PCR ... Total RNA was extracted under RNase-free conditions using an RNA isolation kit (RNAqueous Micro Kit; Ambion) according to the manufacturer's instructions.

    Article Title: Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury
    Article Snippet: .. Quantitative RT-PCR Knees from WT mice (n = 8) and Mig-6 −/− mice (n = 7) were dissected under RNase-free conditions and homogenized in 1 ml of TRIzol reagent (Life Technologies, Carlsbad, CA, USA) with a Fast Prep-24 tissue and cell homogenizer (MP Biomedicals, Solon, OH, USA). .. RNA was chloroform-precipitated and extracted with an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions.

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: .. RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. The isolated RNA was dissolved in RNA storage buffer (Ambion, Grand Island, NY, USA) and stored at −80 o C until used.

    Article Title: ABA Alleviates Uptake and Accumulation of Zinc in Grapevine (Vitis vinifera L.) by Inducing Expression of ZIP and Detoxification-Related Genes
    Article Snippet: RNA Extraction and Analysis of Transcriptional Level by Real-Time PCR Total RNA extraction and quantitative real-time PCR (qRT-PCR) were performed as described by . .. All RNA samples were digested to remove genomic DNA and reverse-transcribed in a 20 mL reaction mixture for cDNA synthesis using a DNase I, RNase-free, and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States), respectively, following the manufacturer’s manuals.

    SYBR Green Assay:

    Article Title: Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties
    Article Snippet: In a final volume of 20 μL, 1 μg of RNase-free and DNase-treated total RNA was mixed with 5 × First-Strand buffer, 500 μM dNTPs, 500 nM OdT-T71 (5'-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG GGA GAT24 ), 2 mM DTT, 40 U RNaseOut (Invitrogen) and SuperScriptIII (200 U/μL). .. Quantitative amplification was performed in a Rotor-Gene (Rotor-Gene 3000, Corbett) using RealMasterMix SYBR Green kit (Eppendorf).

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses. .. In brief, CMKLR1 mRNA expression was performed in a final reaction volume of 20 μ L (10 μ M forward and reverse primer, 500 nM ROX, 1X SYBR Green qPCR master mix, and cDNA 1000 ng).

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses. .. In brief, CD36 mRNA expression was performed in a final reaction volume of 20 mL (40 μ M forward and reverse primer, 50 nM ROX, 2x SYBR Green qPCR master mix and cDNA 100 ng).

    Article Title: ABA Alleviates Uptake and Accumulation of Zinc in Grapevine (Vitis vinifera L.) by Inducing Expression of ZIP and Detoxification-Related Genes
    Article Snippet: All RNA samples were digested to remove genomic DNA and reverse-transcribed in a 20 mL reaction mixture for cDNA synthesis using a DNase I, RNase-free, and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States), respectively, following the manufacturer’s manuals. .. The expression levels of 10 Zn-related genes, whose functions will be introduced in discussion, VviZIP2, VviZIP6, VviZIP7, VviZIP13, VviHMA2, VviNAS, VviNRAMP3, VviYSL1, VviPCR2 , and VvibZIP23 , were determined in root and leaf tissues by using an Applied BiosystemsTM 7500 Fast Dx Real-Time PCR Instrument (Thermo Fisher Scientific, MA, United States) and a PowerUP SYBR Green Master Mix (Thermo Fisher Scientific, TX, United States).

    IA:

    Article Title: Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury
    Article Snippet: Quantitative RT-PCR Knees from WT mice (n = 8) and Mig-6 −/− mice (n = 7) were dissected under RNase-free conditions and homogenized in 1 ml of TRIzol reagent (Life Technologies, Carlsbad, CA, USA) with a Fast Prep-24 tissue and cell homogenizer (MP Biomedicals, Solon, OH, USA). .. Primers used for markers of osteogenesis and chondrogenesis (Integrated DNA Technologies, Coralville, IA, USA) were as follows: sox9 : 5′- GCAGACCAGTACCCGCATCT-3′ and 5′-CTCGTTCAGCAGCCTCCAG-3′; aggrecan : 5′- AGGACCTGGTAGTGCGAGTG-3′ and 5′-GCGTGTGGCGAAGAA-3′; col2a1 : 5′- AATGGGCAGAGGTATAAAGATAAGGA-3′ and 5′-CATTCCCAGTGTCACACACACA-3′; col10a1 : 5′-TTCATCCCATACGCCATAAAG-3′ and 5′-AGCTGGGCCAATATCTCCTT-3′; col1a1 : 5′-CCTGAGTCAGCAGATTGAGAACA-3′ and 5′-CCAGTACTCTCCGCTCTTCCA-3′, runx2 : 5′- CTGCAAGAAGGCTCTGGCGT-3′ and 5′-CGGTTGGTCTCGGTGGCTGG-3′; opn : 5′- GGCATTGCCTCCTCCCTC-3′ and 5′-GCAGGCTGTAAAGCTTCTCC-3′; osx : 5′-GCAACTGGCTAGGTGGTGGTC-3′ and 5′-GCAAAGTCAGATGGGTAAGTAGGC-3′; ocn-1 : 5′-CTCTGCTGACCCTGGCTGCG-3′ and 5′-TGAGGCTCCAAGGTAGCGCC-3′; ocn-2 : 5′- TGACCTCACAGATGCCAAGCCCA-3′ and 5′-AGGGCTCTGGCCACTTACCCA-3′; and 18 s : 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′.

    Article Title: ABA Alleviates Uptake and Accumulation of Zinc in Grapevine (Vitis vinifera L.) by Inducing Expression of ZIP and Detoxification-Related Genes
    Article Snippet: All RNA samples were digested to remove genomic DNA and reverse-transcribed in a 20 mL reaction mixture for cDNA synthesis using a DNase I, RNase-free, and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States), respectively, following the manufacturer’s manuals. .. The primers were designed with PrimerQuest Tool (Integrated DNA Technologies, IA, United States) , and the annealing temperature was 60°C for all primer pairs except VviUbiquitin , which annealed at 56°C.

    Random Hexamer Labeling:

    Article Title: Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose
    Article Snippet: .. qPCR DNase treated (DNase I, RNase free; Fermentas) total RNA (5 μg) was reversely transcribed with the RevertAid™ First Strand cDNA Kit (Fermentas) according to the manufacturer’s protocol with a combination (1:1) of the provided oligo-dT and random hexamer primers. ..

    Expressing:

    Article Title: Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer
    Article Snippet: .. All RNA preparation and handling steps took place in a dedicated area in a laminar flow hood, under RNase-free conditions. mRNA was isolated from the total RNA by use of the Dynabeads mRNA purification Kit (Invitrogen, USA) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, USA) and was used for CK-19 expression studies in CTCs as previously described [ ]. .. Isolation of ctDNA from plasma Peripheral blood in EDTA was collected and processed immediately for plasma isolation.

    Article Title: Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury
    Article Snippet: Total RNA was extracted under RNase-free conditions using an RNA isolation kit (RNAqueous Micro Kit; Ambion) according to the manufacturer's instructions. .. Real-time RT-PCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) using TaqMan gene expression assays for murine cGKI (Mm00440954_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1), purchased from Applied Biosystems.

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: .. Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses. .. Real-Time Quantitative Polymerase Chain Reaction (qPCR) was conducted using the StepOne™ detection system, EXPRESS SYBR® GreenER™ , and ROX™ qPCR SuperMix Universal, and sequence detector software v2.3 (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: .. The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses. .. Real-time quantitative polymerase chain reaction (qPCR) was conducted using the StepOne detection system, EXPRESS SYBR® GreenER™ qPCR SuperMix Universal, and sequence detector software (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Article Title: ABA Alleviates Uptake and Accumulation of Zinc in Grapevine (Vitis vinifera L.) by Inducing Expression of ZIP and Detoxification-Related Genes
    Article Snippet: All RNA samples were digested to remove genomic DNA and reverse-transcribed in a 20 mL reaction mixture for cDNA synthesis using a DNase I, RNase-free, and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States), respectively, following the manufacturer’s manuals. .. The expression levels of 10 Zn-related genes, whose functions will be introduced in discussion, VviZIP2, VviZIP6, VviZIP7, VviZIP13, VviHMA2, VviNAS, VviNRAMP3, VviYSL1, VviPCR2 , and VvibZIP23 , were determined in root and leaf tissues by using an Applied BiosystemsTM 7500 Fast Dx Real-Time PCR Instrument (Thermo Fisher Scientific, MA, United States) and a PowerUP SYBR Green Master Mix (Thermo Fisher Scientific, TX, United States).

    Modification:

    Article Title: Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum
    Article Snippet: The pipette contents were expelled into RNase-free 0.25 ml PCR tubes containing 5 μ l of DEPC-treated H2 0, 1 μ l of Superase RNase inhibitor (Ambion), 1 μ l of 0.1 M dithiothreitol, and 1 μ l of oligo-dT12–18 primer (Invitrogen), briefly centrifuged, and stored at −20°C. .. Multiplex PCR was performed with two rounds of amplification as described by with the modification of including the first-round PCR pairs for VMAT1 and VMAT2 in the first PCR round and amplifying these two signals separately in the second round with the corresponding nested primer sets.

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: Total RNA was isolated from purified mononuclear cells, using TRIzol® LS Reagent (Ambion RNA Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008) based on the single-step RNA isolation modified method reported by Chomczynski [ ]. .. Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: Purified mononuclear cells were used directly for total RNA isolation; it was performed according to the manufacturer's procedure using TRIzol® LS Reagent (Ambion, Invitrogen™ , by Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008) based on the single-step RNA isolation modified method reported by Chomczynski and Sacchi [ , ]. .. The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses.

    Flow Cytometry:

    Article Title: Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer
    Article Snippet: .. All RNA preparation and handling steps took place in a dedicated area in a laminar flow hood, under RNase-free conditions. mRNA was isolated from the total RNA by use of the Dynabeads mRNA purification Kit (Invitrogen, USA) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, USA) and was used for CK-19 expression studies in CTCs as previously described [ ]. .. Isolation of ctDNA from plasma Peripheral blood in EDTA was collected and processed immediately for plasma isolation.

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: .. RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. The isolated RNA was dissolved in RNA storage buffer (Ambion, Grand Island, NY, USA) and stored at −80 o C until used.

    Article Title: HuR Function and Translational State Analysis Following Global Brain Ischemia and Reperfusion
    Article Snippet: Consumables were RNase-free and containers and surfaces were treated with RNaseZap (Invitrogen, Carlsbad, CA) and rinsed x5 with sterile water. .. Procedures were performed under a laminar flow hood whenever possible, and all solutions were prepared with DEPC-treated water.

    Dissection:

    Article Title: piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues
    Article Snippet: Paragraph title: 2.3. Mosquito Tissue Dissection and RNA Isolation ... Midguts were placed into RNase-free 1.5 mL microcentrifuge tubes containing 1 mL of RNAlater (Life Technologies, Thermo Fisher Scientific, Walthman, MA, USA) and were then flash frozen in a dry ice/ethanol bath and stored at −80 °C until subsequent RNA extraction.

    Article Title: HuR Function and Translational State Analysis Following Global Brain Ischemia and Reperfusion
    Article Snippet: For all dissection procedures and all procedures involving mRNA, RNAse contamination was minimized by taking the following steps. .. Consumables were RNase-free and containers and surfaces were treated with RNaseZap (Invitrogen, Carlsbad, CA) and rinsed x5 with sterile water.

    Infection:

    Article Title: piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues
    Article Snippet: Pools of three groups of infected/uninfected midguts (n = 100 per group) were collected at 1 dpi by dissecting anesthetized DENV-2-positive mosquitoes onto a drop of pre-chilled physiological saline, and the slide was then placed on a dissecting microscope. .. Midguts were placed into RNase-free 1.5 mL microcentrifuge tubes containing 1 mL of RNAlater (Life Technologies, Thermo Fisher Scientific, Walthman, MA, USA) and were then flash frozen in a dry ice/ethanol bath and stored at −80 °C until subsequent RNA extraction.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues
    Article Snippet: RT-PCR was employed to identify infected mosquitos with DENV-2-specific primers [ ] ( ). .. Midguts were placed into RNase-free 1.5 mL microcentrifuge tubes containing 1 mL of RNAlater (Life Technologies, Thermo Fisher Scientific, Walthman, MA, USA) and were then flash frozen in a dry ice/ethanol bath and stored at −80 °C until subsequent RNA extraction.

    Article Title: Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum
    Article Snippet: In 10 cases at the completion of the recording experiment, the cytoplasm of the recorded neuron was aspirated into the recording micropipette, and the resulting material was used for multiplex reverse transcription (RT)-PCR analysis as described previously ( ; ) with modifications, including the addition of GTP and ATP to the recording solution as described in detail by . .. The pipette contents were expelled into RNase-free 0.25 ml PCR tubes containing 5 μ l of DEPC-treated H2 0, 1 μ l of Superase RNase inhibitor (Ambion), 1 μ l of 0.1 M dithiothreitol, and 1 μ l of oligo-dT12–18 primer (Invitrogen), briefly centrifuged, and stored at −20°C.

    Sequencing:

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses. .. Real-Time Quantitative Polymerase Chain Reaction (qPCR) was conducted using the StepOne™ detection system, EXPRESS SYBR® GreenER™ , and ROX™ qPCR SuperMix Universal, and sequence detector software v2.3 (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses. .. Real-time quantitative polymerase chain reaction (qPCR) was conducted using the StepOne detection system, EXPRESS SYBR® GreenER™ qPCR SuperMix Universal, and sequence detector software (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    DNA Extraction:

    Article Title: Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer
    Article Snippet: All DNA isolation and handling steps took place in a dedicated area and in a laminal flow hood. gDNA was extracted from the EpCAM-positive CTC-fraction as previously described [ , ]. .. All RNA preparation and handling steps took place in a dedicated area in a laminar flow hood, under RNase-free conditions. mRNA was isolated from the total RNA by use of the Dynabeads mRNA purification Kit (Invitrogen, USA) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, USA) and was used for CK-19 expression studies in CTCs as previously described [ ].

    RNA Sequencing Assay:

    Article Title: A novel environment-evoked transcriptional signature predicts reactivity in single dentate granule neurons
    Article Snippet: .. For RNA-seq experiments, tools and solutions were made RNAse-free and RNAse inhibitors were used (Ambion #AM2684 at 1:1000 in both isolation and storage buffers). .. Slice immunohistochemistry Mice were deeply anesthetized with a cocktail of ketamine/xylazine/acepromazine and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde.

    Article Title: ABA Alleviates Uptake and Accumulation of Zinc in Grapevine (Vitis vinifera L.) by Inducing Expression of ZIP and Detoxification-Related Genes
    Article Snippet: All RNA samples were digested to remove genomic DNA and reverse-transcribed in a 20 mL reaction mixture for cDNA synthesis using a DNase I, RNase-free, and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States), respectively, following the manufacturer’s manuals. .. The genes were selected according to whole-genome array data organized from publicly available RNA-sequencing experiments in our previous publication ( ).

    Isolation:

    Article Title: piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues
    Article Snippet: Paragraph title: 2.3. Mosquito Tissue Dissection and RNA Isolation ... Midguts were placed into RNase-free 1.5 mL microcentrifuge tubes containing 1 mL of RNAlater (Life Technologies, Thermo Fisher Scientific, Walthman, MA, USA) and were then flash frozen in a dry ice/ethanol bath and stored at −80 °C until subsequent RNA extraction.

    Article Title: Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties
    Article Snippet: For isolation of total RNA trophozoites were treated with TRIZOL reagent (Invitrogen) following the manufacturer's instructions. .. In a final volume of 20 μL, 1 μg of RNase-free and DNase-treated total RNA was mixed with 5 × First-Strand buffer, 500 μM dNTPs, 500 nM OdT-T71 (5'-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG GGA GAT24 ), 2 mM DTT, 40 U RNaseOut (Invitrogen) and SuperScriptIII (200 U/μL).

    Article Title: Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer
    Article Snippet: .. All RNA preparation and handling steps took place in a dedicated area in a laminar flow hood, under RNase-free conditions. mRNA was isolated from the total RNA by use of the Dynabeads mRNA purification Kit (Invitrogen, USA) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, USA) and was used for CK-19 expression studies in CTCs as previously described [ ]. .. Isolation of ctDNA from plasma Peripheral blood in EDTA was collected and processed immediately for plasma isolation.

    Article Title: Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury
    Article Snippet: .. Total RNA was extracted under RNase-free conditions using an RNA isolation kit (RNAqueous Micro Kit; Ambion) according to the manufacturer's instructions. .. DNase was treated for 15 min to minimize genomic DNA contamination and quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). cDNA was synthesized from 200 ng of RNA using random hexamers of the Verso cDNA kit (Thermo Scientific).

    Article Title: A novel environment-evoked transcriptional signature predicts reactivity in single dentate granule neurons
    Article Snippet: .. For RNA-seq experiments, tools and solutions were made RNAse-free and RNAse inhibitors were used (Ambion #AM2684 at 1:1000 in both isolation and storage buffers). .. Slice immunohistochemistry Mice were deeply anesthetized with a cocktail of ketamine/xylazine/acepromazine and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde.

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: Total RNA was isolated from purified mononuclear cells, using TRIzol® LS Reagent (Ambion RNA Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008) based on the single-step RNA isolation modified method reported by Chomczynski [ ]. .. Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses.

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: .. RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. The isolated RNA was dissolved in RNA storage buffer (Ambion, Grand Island, NY, USA) and stored at −80 o C until used.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: Purified mononuclear cells were used directly for total RNA isolation; it was performed according to the manufacturer's procedure using TRIzol® LS Reagent (Ambion, Invitrogen™ , by Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008) based on the single-step RNA isolation modified method reported by Chomczynski and Sacchi [ , ]. .. The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses.

    Transferring:

    Article Title: Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum
    Article Snippet: .. The pipette contents were expelled into RNase-free 0.25 ml PCR tubes containing 5 μ l of DEPC-treated H2 0, 1 μ l of Superase RNase inhibitor (Ambion), 1 μ l of 0.1 M dithiothreitol, and 1 μ l of oligo-dT12–18 primer (Invitrogen), briefly centrifuged, and stored at −20°C. .. Multiplex PCR was performed with two rounds of amplification as described by with the modification of including the first-round PCR pairs for VMAT1 and VMAT2 in the first PCR round and amplifying these two signals separately in the second round with the corresponding nested primer sets.

    Microscopy:

    Article Title: piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues
    Article Snippet: Pools of three groups of infected/uninfected midguts (n = 100 per group) were collected at 1 dpi by dissecting anesthetized DENV-2-positive mosquitoes onto a drop of pre-chilled physiological saline, and the slide was then placed on a dissecting microscope. .. Midguts were placed into RNase-free 1.5 mL microcentrifuge tubes containing 1 mL of RNAlater (Life Technologies, Thermo Fisher Scientific, Walthman, MA, USA) and were then flash frozen in a dry ice/ethanol bath and stored at −80 °C until subsequent RNA extraction.

    Purification:

    Article Title: Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties
    Article Snippet: Extracted RNA was purified using the RNeasy mini kit (Qiagen) without β-mercaptoethanol and DNA was digested with DNase (Qiagen). cDNA synthesis was accomplished with SuperScriptIII Reverse Transcriptase (Invitrogen). .. In a final volume of 20 μL, 1 μg of RNase-free and DNase-treated total RNA was mixed with 5 × First-Strand buffer, 500 μM dNTPs, 500 nM OdT-T71 (5'-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG GGA GAT24 ), 2 mM DTT, 40 U RNaseOut (Invitrogen) and SuperScriptIII (200 U/μL).

    Article Title: Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer
    Article Snippet: .. All RNA preparation and handling steps took place in a dedicated area in a laminar flow hood, under RNase-free conditions. mRNA was isolated from the total RNA by use of the Dynabeads mRNA purification Kit (Invitrogen, USA) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, USA) and was used for CK-19 expression studies in CTCs as previously described [ ]. .. Isolation of ctDNA from plasma Peripheral blood in EDTA was collected and processed immediately for plasma isolation.

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: Total RNA was isolated from purified mononuclear cells, using TRIzol® LS Reagent (Ambion RNA Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008) based on the single-step RNA isolation modified method reported by Chomczynski [ ]. .. Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: Purified mononuclear cells were used directly for total RNA isolation; it was performed according to the manufacturer's procedure using TRIzol® LS Reagent (Ambion, Invitrogen™ , by Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008) based on the single-step RNA isolation modified method reported by Chomczynski and Sacchi [ , ]. .. The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses.

    Polymerase Chain Reaction:

    Article Title: piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues
    Article Snippet: Mosquito Tissue Dissection and RNA Isolation DENV-2-positive mosquitoes were identified by reverse-transcription PCR (RT-PCR) with DENV-2-specific primers at 9 days post-infection (dpi). .. Midguts were placed into RNase-free 1.5 mL microcentrifuge tubes containing 1 mL of RNAlater (Life Technologies, Thermo Fisher Scientific, Walthman, MA, USA) and were then flash frozen in a dry ice/ethanol bath and stored at −80 °C until subsequent RNA extraction.

    Article Title: Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose
    Article Snippet: qPCR DNase treated (DNase I, RNase free; Fermentas) total RNA (5 μg) was reversely transcribed with the RevertAid™ First Strand cDNA Kit (Fermentas) according to the manufacturer’s protocol with a combination (1:1) of the provided oligo-dT and random hexamer primers. .. Determination of the PCR efficiency was performed using triplicate reactions from a dilution series of cDNA, and the amplification efficiency then calculated from the given slopes in the realplex v2.2 software.

    Article Title: Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum
    Article Snippet: .. The pipette contents were expelled into RNase-free 0.25 ml PCR tubes containing 5 μ l of DEPC-treated H2 0, 1 μ l of Superase RNase inhibitor (Ambion), 1 μ l of 0.1 M dithiothreitol, and 1 μ l of oligo-dT12–18 primer (Invitrogen), briefly centrifuged, and stored at −20°C. .. Multiplex PCR was performed with two rounds of amplification as described by with the modification of including the first-round PCR pairs for VMAT1 and VMAT2 in the first PCR round and amplifying these two signals separately in the second round with the corresponding nested primer sets.

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. RNA integrity was tested by PCR amplification of the β -actin housekeeping gene.

    Nested PCR:

    Article Title: Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum
    Article Snippet: The pipette contents were expelled into RNase-free 0.25 ml PCR tubes containing 5 μ l of DEPC-treated H2 0, 1 μ l of Superase RNase inhibitor (Ambion), 1 μ l of 0.1 M dithiothreitol, and 1 μ l of oligo-dT12–18 primer (Invitrogen), briefly centrifuged, and stored at −20°C. .. The nested PCR strategy for vesicular monoamine transporter (VMAT) isoforms was chosen to increase specificity.

    Mouse Assay:

    Article Title: Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury
    Article Snippet: .. Quantitative RT-PCR Knees from WT mice (n = 8) and Mig-6 −/− mice (n = 7) were dissected under RNase-free conditions and homogenized in 1 ml of TRIzol reagent (Life Technologies, Carlsbad, CA, USA) with a Fast Prep-24 tissue and cell homogenizer (MP Biomedicals, Solon, OH, USA). .. RNA was chloroform-precipitated and extracted with an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions.

    RNA Extraction:

    Article Title: piRNA Profiling of Dengue Virus Type 2-Infected Asian Tiger Mosquito and Midgut Tissues
    Article Snippet: .. Midguts were placed into RNase-free 1.5 mL microcentrifuge tubes containing 1 mL of RNAlater (Life Technologies, Thermo Fisher Scientific, Walthman, MA, USA) and were then flash frozen in a dry ice/ethanol bath and stored at −80 °C until subsequent RNA extraction. .. Total RNA from each group was extracted with Trizol® Reagent, according to the manufacturer’s protocol.

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: .. RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. The isolated RNA was dissolved in RNA storage buffer (Ambion, Grand Island, NY, USA) and stored at −80 o C until used.

    Article Title: ABA Alleviates Uptake and Accumulation of Zinc in Grapevine (Vitis vinifera L.) by Inducing Expression of ZIP and Detoxification-Related Genes
    Article Snippet: Paragraph title: RNA Extraction and Analysis of Transcriptional Level by Real-Time PCR ... All RNA samples were digested to remove genomic DNA and reverse-transcribed in a 20 mL reaction mixture for cDNA synthesis using a DNase I, RNase-free, and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States), respectively, following the manufacturer’s manuals.

    Software:

    Article Title: Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose
    Article Snippet: qPCR DNase treated (DNase I, RNase free; Fermentas) total RNA (5 μg) was reversely transcribed with the RevertAid™ First Strand cDNA Kit (Fermentas) according to the manufacturer’s protocol with a combination (1:1) of the provided oligo-dT and random hexamer primers. .. Determination of the PCR efficiency was performed using triplicate reactions from a dilution series of cDNA, and the amplification efficiency then calculated from the given slopes in the realplex v2.2 software.

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses. .. Real-Time Quantitative Polymerase Chain Reaction (qPCR) was conducted using the StepOne™ detection system, EXPRESS SYBR® GreenER™ , and ROX™ qPCR SuperMix Universal, and sequence detector software v2.3 (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses. .. Real-time quantitative polymerase chain reaction (qPCR) was conducted using the StepOne detection system, EXPRESS SYBR® GreenER™ qPCR SuperMix Universal, and sequence detector software (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Real-time Polymerase Chain Reaction:

    Article Title: Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties
    Article Snippet: Paragraph title: Quantitative real-time PCR ... In a final volume of 20 μL, 1 μg of RNase-free and DNase-treated total RNA was mixed with 5 × First-Strand buffer, 500 μM dNTPs, 500 nM OdT-T71 (5'-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG GGA GAT24 ), 2 mM DTT, 40 U RNaseOut (Invitrogen) and SuperScriptIII (200 U/μL).

    Article Title: Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury
    Article Snippet: Total RNA was extracted under RNase-free conditions using an RNA isolation kit (RNAqueous Micro Kit; Ambion) according to the manufacturer's instructions. .. Real-time RT-PCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) using TaqMan gene expression assays for murine cGKI (Mm00440954_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1), purchased from Applied Biosystems.

    Article Title: Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose
    Article Snippet: .. qPCR DNase treated (DNase I, RNase free; Fermentas) total RNA (5 μg) was reversely transcribed with the RevertAid™ First Strand cDNA Kit (Fermentas) according to the manufacturer’s protocol with a combination (1:1) of the provided oligo-dT and random hexamer primers. ..

    Article Title: Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance
    Article Snippet: Complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L, with oligo (dT) 18 primer (100 ng/μ L), RNase free, DEPC treated water, and SuperScript Reverse Transcriptase III kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until used for expression analyses. .. Real-Time Quantitative Polymerase Chain Reaction (qPCR) was conducted using the StepOne™ detection system, EXPRESS SYBR® GreenER™ , and ROX™ qPCR SuperMix Universal, and sequence detector software v2.3 (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Article Title: Low CD36 and LOX-1 Levels and CD36 Gene Subexpression Are Associated with Metabolic Dysregulation in Older Individuals with Abdominal Obesity
    Article Snippet: The complementary DNA synthesis (cDNA) was performed with 2 μ g of each total RNA sample using a reaction size of 20 μ L with oligo (dT) 18-primer (100 ng/μ L), RNase-free, DEPC treated water and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) kit (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) and stored at −20°C until being used for expression analyses. .. Real-time quantitative polymerase chain reaction (qPCR) was conducted using the StepOne detection system, EXPRESS SYBR® GreenER™ qPCR SuperMix Universal, and sequence detector software (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404) was used for data analysis.

    Article Title: ABA Alleviates Uptake and Accumulation of Zinc in Grapevine (Vitis vinifera L.) by Inducing Expression of ZIP and Detoxification-Related Genes
    Article Snippet: Paragraph title: RNA Extraction and Analysis of Transcriptional Level by Real-Time PCR ... All RNA samples were digested to remove genomic DNA and reverse-transcribed in a 20 mL reaction mixture for cDNA synthesis using a DNase I, RNase-free, and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States), respectively, following the manufacturer’s manuals.

    Multiplex Assay:

    Article Title: Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum
    Article Snippet: Paragraph title: Single-cell multiplex reverse transcription-PCR analysis ... The pipette contents were expelled into RNase-free 0.25 ml PCR tubes containing 5 μ l of DEPC-treated H2 0, 1 μ l of Superase RNase inhibitor (Ambion), 1 μ l of 0.1 M dithiothreitol, and 1 μ l of oligo-dT12–18 primer (Invitrogen), briefly centrifuged, and stored at −20°C.

    Laser Capture Microdissection:

    Article Title: HuR Function and Translational State Analysis Following Global Brain Ischemia and Reperfusion
    Article Snippet: Paragraph title: Microdissection of CA1, CA3 and forebrain ... Consumables were RNase-free and containers and surfaces were treated with RNaseZap (Invitrogen, Carlsbad, CA) and rinsed x5 with sterile water.

    Spectrophotometry:

    Article Title: Oxidant-Induced Activation of cGMP-Dependent Protein Kinase Iα Mediates Neuropathic Pain After Peripheral Nerve Injury
    Article Snippet: Total RNA was extracted under RNase-free conditions using an RNA isolation kit (RNAqueous Micro Kit; Ambion) according to the manufacturer's instructions. .. DNase was treated for 15 min to minimize genomic DNA contamination and quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). cDNA was synthesized from 200 ng of RNA using random hexamers of the Verso cDNA kit (Thermo Scientific).

    Article Title: Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury
    Article Snippet: Quantitative RT-PCR Knees from WT mice (n = 8) and Mig-6 −/− mice (n = 7) were dissected under RNase-free conditions and homogenized in 1 ml of TRIzol reagent (Life Technologies, Carlsbad, CA, USA) with a Fast Prep-24 tissue and cell homogenizer (MP Biomedicals, Solon, OH, USA). .. RNA concentration was calculated and integrity was analyzed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively, and potential DNA contamination was removed by RNase-free DNase treatment (amplification grade DNaseI; Sigma-Aldrich). cDNA was made with a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA) on 0.5 μg of RNA.

    Article Title: Skip the Alignment: Degenerate, Multiplex Primer and Probe Design Using K-mer Matching Instead of Alignments
    Article Snippet: The pellet was washed with 70% ethanol and then centrifuged at max g for 10 min at 4°C, air dried briefly at room temperature, dissolved in RNase-free, DEPC treated water (Ambion, Austin, TX) and stored at −80°C until needed. .. The concentration of extracted RNA was determined for the samples using a ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE).

    Article Title: ABA Alleviates Uptake and Accumulation of Zinc in Grapevine (Vitis vinifera L.) by Inducing Expression of ZIP and Detoxification-Related Genes
    Article Snippet: The quantity and quality of the RNA were analyzed with an ND-1000 Spectrophotometer NanoDrop (Thermo Fisher Scientific, Wilmington, DE, United States). .. All RNA samples were digested to remove genomic DNA and reverse-transcribed in a 20 mL reaction mixture for cDNA synthesis using a DNase I, RNase-free, and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, United States), respectively, following the manufacturer’s manuals.

    Concentration Assay:

    Article Title: Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury
    Article Snippet: Quantitative RT-PCR Knees from WT mice (n = 8) and Mig-6 −/− mice (n = 7) were dissected under RNase-free conditions and homogenized in 1 ml of TRIzol reagent (Life Technologies, Carlsbad, CA, USA) with a Fast Prep-24 tissue and cell homogenizer (MP Biomedicals, Solon, OH, USA). .. RNA concentration was calculated and integrity was analyzed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively, and potential DNA contamination was removed by RNase-free DNase treatment (amplification grade DNaseI; Sigma-Aldrich). cDNA was made with a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA) on 0.5 μg of RNA.

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. RNA concentration was determined by absorbance reading at 260 nm with the NanoDrop spectophotometer (Wilmington, DE, USA) at the time of CK-19mRNA analysis.

    Article Title: Skip the Alignment: Degenerate, Multiplex Primer and Probe Design Using K-mer Matching Instead of Alignments
    Article Snippet: The pellet was washed with 70% ethanol and then centrifuged at max g for 10 min at 4°C, air dried briefly at room temperature, dissolved in RNase-free, DEPC treated water (Ambion, Austin, TX) and stored at −80°C until needed. .. The concentration of extracted RNA was determined for the samples using a ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE).

    Preserving:

    Article Title: Super-resolution measurement of distance between transcription sites using RNA FISH with intronic probes
    Article Snippet: For RNA FISH, preserving RNA integrity is an additional major issue; RNA is prone to hydrolysis both spontaneously and enzymatically. .. Gloves should be used and changed frequently, glassware and metalware should be baked (230 °C for 2 h) or treated with RNaseZap reagent (Invitrogen), plastic consumables should be from previously unopened containers or certified RNase-free, and water and buffers should be treated with diethylpyrocarbonate (DEPC), except for reagents containing amine groups (e.g., Tris, HEPES) which should be treated with RNAsecure reagent (Invitrogen).

    Staining:

    Article Title: A novel environment-evoked transcriptional signature predicts reactivity in single dentate granule neurons
    Article Snippet: The nucleic acid stain Hoechst 33342 (5 µM, Life Technologies) was included in the media to facilitate visualization of the nuclei for quantification but excluded for sorting. .. For RNA-seq experiments, tools and solutions were made RNAse-free and RNAse inhibitors were used (Ambion #AM2684 at 1:1000 in both isolation and storage buffers).

    Fluorescence In Situ Hybridization:

    Article Title: Super-resolution measurement of distance between transcription sites using RNA FISH with intronic probes
    Article Snippet: For RNA FISH, preserving RNA integrity is an additional major issue; RNA is prone to hydrolysis both spontaneously and enzymatically. .. Gloves should be used and changed frequently, glassware and metalware should be baked (230 °C for 2 h) or treated with RNaseZap reagent (Invitrogen), plastic consumables should be from previously unopened containers or certified RNase-free, and water and buffers should be treated with diethylpyrocarbonate (DEPC), except for reagents containing amine groups (e.g., Tris, HEPES) which should be treated with RNAsecure reagent (Invitrogen).

    Hood:

    Article Title: Direct comparison study of DNA methylation markers in EpCAM-positive circulating tumour cells, corresponding circulating tumour DNA, and paired primary tumours in breast cancer
    Article Snippet: .. All RNA preparation and handling steps took place in a dedicated area in a laminar flow hood, under RNase-free conditions. mRNA was isolated from the total RNA by use of the Dynabeads mRNA purification Kit (Invitrogen, USA) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, USA) and was used for CK-19 expression studies in CTCs as previously described [ ]. .. Isolation of ctDNA from plasma Peripheral blood in EDTA was collected and processed immediately for plasma isolation.

    Article Title: Clinical relevance of circulating CK-19mRNA-positive tumour cells before front-line treatment in patients with metastatic breast cancer
    Article Snippet: .. RNA extraction and real-time RT-PCR assay for CK-19mRNA-positive cells Peripheral blood mononuclear cells were obtained by Ficoll-Hypaque gradient-density centrifugation within 2–4 h from vein puncture, and total RNA isolation was carried out in a laminar flow hood under RNAse-free conditions by using Trizol (Invitrogen, Grand Island, NY, USA; ). .. The isolated RNA was dissolved in RNA storage buffer (Ambion, Grand Island, NY, USA) and stored at −80 o C until used.

    Article Title: HuR Function and Translational State Analysis Following Global Brain Ischemia and Reperfusion
    Article Snippet: Consumables were RNase-free and containers and surfaces were treated with RNaseZap (Invitrogen, Carlsbad, CA) and rinsed x5 with sterile water. .. Procedures were performed under a laminar flow hood whenever possible, and all solutions were prepared with DEPC-treated water.

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    Thermo Fisher rnase free
    IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: <t>RNase</t> A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and <t>DNase</t> I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.
    Rnase Free, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 22 article reviews
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    Overview of <t>RT-RamDA</t> and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of <t>RNase</t> H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Rnase Free Water, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and DNase I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.

    Journal: Oncotarget

    Article Title: Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

    doi: 10.18632/oncotarget.8420

    Figure Lengend Snippet: IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and DNase I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.

    Article Snippet: qRT-PCR analysis 1 μg total RNA was subjected to DNase I treatment in a 30 μL reaction volume using DNase I, RNase-free (Thermo Scientific) following the manufacturer's protocol. cDNA was synthesized from 10 μL of the DNase treated RNA using the High-Capacity cDNA Reverse Transcription Kit (LifeTechnologies) following the manufacturer's protocol.

    Techniques: Binding Assay, Irradiation, Transfection, Plasmid Preparation, Luciferase, Construct, Purification, Isolation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Lysis, Immunoprecipitation, Marker, Expressing, Mutagenesis, Incubation, Activation Assay, Blocking Assay, RNA Binding Assay

    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: A mixture containing 2 μL of conventional RT mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18 (Thermo Fisher), 8 pmol random hexamers (TaKaRa), and 1.5× PrimeScript enzyme mix in RNase-free water) or 2 μL of RT-RamDA mix (1.5× PrimeScript buffer, 0.6 pmol oligo(dT)18, 8 pmol random hexamers or NSRs, 0.2 U of DNase I Amplification Grade (Thermo Fisher), 100 ng of T4 gene 32 protein (Roche), and 1.5× PrimeScript enzyme mix in RNase-free water) was added to 1 μL of diluted, denatured template RNA.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: To eliminate genomic DNA contamination, 1 μL of genomic DNA digestion mix (0.5× PrimeScript Buffer, 0.2 U of DNase I Amplification Grade, 1: 5 000 000 ERCC RNA Spike-In Mix I (Thermo Fisher) in RNase-free water) was added to 1 μL of the denatured sample.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay