polymerase  (New England Biolabs)


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  • 95
    Name:
    E coli Poly A Polymerase
    Description:
    E coli Poly A Polymerase 500 units
    Catalog Number:
    m0276l
    Price:
    292
    Size:
    500 units
    Category:
    DNA Polymerases
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    Structured Review

    New England Biolabs polymerase
    E coli Poly A Polymerase
    E coli Poly A Polymerase 500 units
    https://www.bioz.com/result/polymerase/product/New England Biolabs
    Average 95 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    polymerase - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Luciferase:

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control
    Article Snippet: To generate each 3′ region, a 5′-phosphate–bearing RNA oligonucleotide (IDT) consisting of the barcode segment followed by a 10 nt poly(A) segment was extended with E. coli poly(A)-polymerase (NEB), with ATP concentration and reaction time adjusted to yield of tails of the desired length. .. The 5′ region of the standards was synthesized by in vitro transcription of a template containing Renilla luciferase sequence followed by that of a modified HDV ribozyme .

    Synthesized:

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control
    Article Snippet: Tail-length standards The common 5′ region of each standard and the unique 3′ region, consisting of the standard-specific barcode and poly(A)-tail , were synthesized separately and then ligated together to make full-length standards. .. To generate each 3′ region, a 5′-phosphate–bearing RNA oligonucleotide (IDT) consisting of the barcode segment followed by a 10 nt poly(A) segment was extended with E. coli poly(A)-polymerase (NEB), with ATP concentration and reaction time adjusted to yield of tails of the desired length.

    Article Title: RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response
    Article Snippet: 10 μL T7 reactions were carried out at 37 °C for 2 h. Reactions were then treated with 2 U RNase-free DNaseI (NEB) for 15 min at 37 °C to remove DNA template, and then polyadenylated with 5 U E. coli Poly-A Polymerase, 10× buffer, and 10 mM ATP (NEB) for 1 h at 37 °C. .. Synthesized mRNAs were clean and concentrated with RNA Clean and Concentrator-25 Kit from Zymo Research.

    Quantitative RT-PCR:

    Article Title: Maternal high fat diet during pregnancy and lactation alters hepatic expression of insulin like growth factor-2 and key microRNAs in the adult offspring
    Article Snippet: Paragraph title: Measurement of miRNAs using real time RT-PCR ... For poly(T) adaptor RT-PCR, ~100 ng total RNA was added to a reaction containing 2.5 units E. Coli Poly A polymerase (New England Biolabs Ltd. Herts.

    Article Title: MicroRNAs of the mir-17~92 cluster regulate multiple aspects of pancreatic tumor development and progression
    Article Snippet: Paragraph title: qRT-PCR ... DNA-free RNA was then polyadenylated (New England Biolabs #M0276) and subsequently reverse transcribed (Invitrogen #18080) using a pool of specially designed primers at a concentration of 50 uM to generate cDNA copies of polyadenylated miRNAs.

    Article Title: Identification of new miRNA biomarkers associated with HER2-positive breast cancers
    Article Snippet: Paragraph title: Real-time RT-PCR analysis of miRNA expression ... RNA was first poly-adenylated using E.coli poly(A) polymerase from New England Biolabs.

    Article Title: cAMP-inducible coactivator CRTC3 attenuates brown adipose tissue thermogenesis
    Article Snippet: .. For mature miRNA detection, total RNA was polyadenylated with Escherichia coli poly(A) polymerase according to the manufacturer’s instruction (New England Biolab) and then used for reverse transcription and qRT-PCR with specific primers ( ). .. Relative mRNA expression was calculated by 2−ΔΔCT methods.

    Article Title: Genetic Heterogeneity of Breast Cancer Metastasis May Be Related to miR-21 Regulation of TIMP-3 in Translation
    Article Snippet: Paragraph title: 2.4. Real-Time RT-qPCR ... Small RNA was added poly-A tail by poly-A polymerase (NEB, M0276) before reverse transcription using primers in as before ( ).

    Real-time Polymerase Chain Reaction:

    Article Title: Maternal high fat diet during pregnancy and lactation alters hepatic expression of insulin like growth factor-2 and key microRNAs in the adult offspring
    Article Snippet: Measurement of miRNAs using real time RT-PCR Two qPCR methods were used in the validation of microarray: the stem-loop RT-PCR method [ ], using miRNA specific primers purchased from Applied Biosystems and polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using sequence complementary to the poly(T) adapters during RT reactions [ ]. .. For poly(T) adaptor RT-PCR, ~100 ng total RNA was added to a reaction containing 2.5 units E. Coli Poly A polymerase (New England Biolabs Ltd. Herts.

    Article Title: Evaluation of Placental mir-155-5p and Long Non-coding RNA sONE Expression in Patients with Severe Pre-eclampsia
    Article Snippet: At first, reverse transcription reactions contained7.5 µltotal RNA,0.5 µl Poly adenine polymerase (PAP)(NEB# M0276L,USA), 1µl PAP Buffer (10x)and 1µl dNTPs (10 mM). .. The 10□l reaction mixture was initiallyincubated at 37°C for 20-30 min, then 65°C for 20 min. After this step, Thermo Scientific kit (K1622, USA) was used by adding 2 µlRT Adaptor to 5 µl template RNA synthesizedduring the first step, 5 µl nuclease free water,1µlRiboLock RNase Inhibitor (20 U/□l), 2 µl dNTPs (10mM) and1 µlRevertAid M- MuLV RT (200 U/ΜL).Then the mixture was incubated at 25°C for 5 minat 42°C for 1h and finally70°C for 5 min. After performing gradient PCR to find the appropriate temperature , standard curve was plotted for each primer till final analysis was done according to a Livak, s formula (2^-Δct ).Real-time quantitative PCR was performe-dusing a Corrbetreal-time PCR detection system (Rotor gene, Corbett 6000(2.1.0), Qiagen, Germany).

    Article Title: Identification of new miRNA biomarkers associated with HER2-positive breast cancers
    Article Snippet: RNA was first poly-adenylated using E.coli poly(A) polymerase from New England Biolabs. .. For the quantitative polymerase chain reaction (qPCR), 2μl of cDNA was mixed with 10μl of RT2 SYBR® Green ROX qPCR Mastermix from Qiagen, 6μl of RNase-free water, 1μl of a universal reverse primer (5′GCG AGC ACA GAA TTA ATA CGA C3′) and a forward primer specific to the miRNA.

    Article Title: cAMP-inducible coactivator CRTC3 attenuates brown adipose tissue thermogenesis
    Article Snippet: Relative mRNA expression was determined with SYBR green master mix (Roche) by LightCycler 480 qPCR machine (Roche). .. For mature miRNA detection, total RNA was polyadenylated with Escherichia coli poly(A) polymerase according to the manufacturer’s instruction (New England Biolab) and then used for reverse transcription and qRT-PCR with specific primers ( ).

    Article Title: MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells
    Article Snippet: miRNA and piRNA quantification cDNA synthesis was carried out according to [ ] with the following modifications: 100 ng of total RNA was polyadenylated and reverse transcribed in the same reaction tube with E. coli Poly(A) Polymerase (M0276S, NEB) and Superscript II (Invitrogen) and the 5'-CAGGTCCAGT15 VN primer. .. Primers for quantitative PCR analysis are listed in .

    Article Title: Genetic Heterogeneity of Breast Cancer Metastasis May Be Related to miR-21 Regulation of TIMP-3 in Translation
    Article Snippet: Small RNA was added poly-A tail by poly-A polymerase (NEB, M0276) before reverse transcription using primers in as before ( ). .. Real-time quantitative PCR was performed on Roche LightCycler 2.0 with SYBR Premix Ex Taq (DRR041A, Takara, Japan).

    Microarray:

    Article Title: Engagement of circular RNA HECW2 in the nonautophagic role of ATG5 implicated in the endothelial-mesenchymal transition
    Article Snippet: Paragraph title: MicroRNA microarray assay ... The assay was performed with 4 to 5 μg of the total RNA samples, which were 3’-extended with a poly (A) tail using a poly (A) polymerase (NEB, M0276L).

    Article Title: Maternal high fat diet during pregnancy and lactation alters hepatic expression of insulin like growth factor-2 and key microRNAs in the adult offspring
    Article Snippet: Measurement of miRNAs using real time RT-PCR Two qPCR methods were used in the validation of microarray: the stem-loop RT-PCR method [ ], using miRNA specific primers purchased from Applied Biosystems and polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using sequence complementary to the poly(T) adapters during RT reactions [ ]. .. For poly(T) adaptor RT-PCR, ~100 ng total RNA was added to a reaction containing 2.5 units E. Coli Poly A polymerase (New England Biolabs Ltd. Herts.

    Incubation:

    Article Title: Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages
    Article Snippet: Fragmented RNA was de-phosphorylated using 1 μL T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) and 5 μL 5x PNK buffer (0.5 M MES, 50 mM MgCl2 , 50 mM mercaptoethanol, 1.5 M NaCl, pH 5.5–5.8) supplemented with 1 µ/μL SUPERase-In for 45 min at 37°C, an additional 1 μL T4 polynucleotide kinase was added to the reaction, followed by incubation for 45 min, and subsequent heat-inactivation for 5 min at 70°C and ethanol precipitation overnight with glycogen. .. Poly(A)-tailing reaction was performed using 3.75 µ E. coli poly(A)-polymerase (New England Biolabs) in 10x poly(A)-polymerase buffer supplemented with ATP (50:1 molar ratio to RNA) and 1 µ/μL SUPERase-In for 30 min at 37°C.

    Article Title: Evaluation of Placental mir-155-5p and Long Non-coding RNA sONE Expression in Patients with Severe Pre-eclampsia
    Article Snippet: At first, reverse transcription reactions contained7.5 µltotal RNA,0.5 µl Poly adenine polymerase (PAP)(NEB# M0276L,USA), 1µl PAP Buffer (10x)and 1µl dNTPs (10 mM). .. The 10□l reaction mixture was initiallyincubated at 37°C for 20-30 min, then 65°C for 20 min. After this step, Thermo Scientific kit (K1622, USA) was used by adding 2 µlRT Adaptor to 5 µl template RNA synthesizedduring the first step, 5 µl nuclease free water,1µlRiboLock RNase Inhibitor (20 U/□l), 2 µl dNTPs (10mM) and1 µlRevertAid M- MuLV RT (200 U/ΜL).Then the mixture was incubated at 25°C for 5 minat 42°C for 1h and finally70°C for 5 min. After performing gradient PCR to find the appropriate temperature , standard curve was plotted for each primer till final analysis was done according to a Livak, s formula (2^-Δct ).Real-time quantitative PCR was performe-dusing a Corrbetreal-time PCR detection system (Rotor gene, Corbett 6000(2.1.0), Qiagen, Germany).

    Article Title: Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System
    Article Snippet: A total of 150 ng mRNA was first fragmented to ~ 100 nt by RNA Fragmentation Reagent (ThermoFisher; AM8740) at 70°C for 8 min. After the reaction was stopped by 100 mM EDTA, the RNA mixtures were then directly diluted to 450 μl CLIP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl (pH 7.4)) with 5 g anti-m6 A polyclonal antibody (Synaptic Systems; 202003) and incubated at 4 °C for 2 hr. .. After dephosphorylation with 10 U FastAP (ThermoFisher; EF0652) at 10 min at room temperature and polyadenylation with 5U E. coli Poly(A) Polymerase (NEB, M0276S) for 15 min at room temperature on beads, the RNA was then eluted by treatment with proteinase K (Thermo Scientific; 25530049) at 37°C for 1 hr.

    Article Title: MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells
    Article Snippet: miRNA and piRNA quantification cDNA synthesis was carried out according to [ ] with the following modifications: 100 ng of total RNA was polyadenylated and reverse transcribed in the same reaction tube with E. coli Poly(A) Polymerase (M0276S, NEB) and Superscript II (Invitrogen) and the 5'-CAGGTCCAGT15 VN primer. .. Incubation was performed at 37°C for 10 min, then at 42°C for 50 min and finally at 70°C for 15mn (heat inactivation).

    Expressing:

    Article Title: Evaluation of Placental mir-155-5p and Long Non-coding RNA sONE Expression in Patients with Severe Pre-eclampsia
    Article Snippet: Only samples with two clearly detectable ribosomal RNA bands (28S and 18S) and a higher than 2:1 ratio-were evaluated by nanodrop for purityof intact RNA, and were used for reverse transcription. cDNA synthesis and quantitative real- time reverse transcription (RT) PCR for miR-155 expression analysis Expression of miR-155was assessed byquant-itative real-time RT–PCR(SYBR green method), and thesmall nuclear RNA U6was used as aninternal control. .. At first, reverse transcription reactions contained7.5 µltotal RNA,0.5 µl Poly adenine polymerase (PAP)(NEB# M0276L,USA), 1µl PAP Buffer (10x)and 1µl dNTPs (10 mM).

    Article Title: Identification of new miRNA biomarkers associated with HER2-positive breast cancers
    Article Snippet: Paragraph title: Real-time RT-PCR analysis of miRNA expression ... RNA was first poly-adenylated using E.coli poly(A) polymerase from New England Biolabs.

    Article Title: cAMP-inducible coactivator CRTC3 attenuates brown adipose tissue thermogenesis
    Article Snippet: Relative mRNA expression was determined with SYBR green master mix (Roche) by LightCycler 480 qPCR machine (Roche). .. For mature miRNA detection, total RNA was polyadenylated with Escherichia coli poly(A) polymerase according to the manufacturer’s instruction (New England Biolab) and then used for reverse transcription and qRT-PCR with specific primers ( ).

    Modification:

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control
    Article Snippet: To generate each 3′ region, a 5′-phosphate–bearing RNA oligonucleotide (IDT) consisting of the barcode segment followed by a 10 nt poly(A) segment was extended with E. coli poly(A)-polymerase (NEB), with ATP concentration and reaction time adjusted to yield of tails of the desired length. .. The 5′ region of the standards was synthesized by in vitro transcription of a template containing Renilla luciferase sequence followed by that of a modified HDV ribozyme .

    Article Title: Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System
    Article Snippet: After dephosphorylation with 10 U FastAP (ThermoFisher; EF0652) at 10 min at room temperature and polyadenylation with 5U E. coli Poly(A) Polymerase (NEB, M0276S) for 15 min at room temperature on beads, the RNA was then eluted by treatment with proteinase K (Thermo Scientific; 25530049) at 37°C for 1 hr. .. The m6 A CLIP library was prepared using a modified SMART-seq2 protocol without tagmentation.

    Gel Purification:

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control
    Article Snippet: To generate each 3′ region, a 5′-phosphate–bearing RNA oligonucleotide (IDT) consisting of the barcode segment followed by a 10 nt poly(A) segment was extended with E. coli poly(A)-polymerase (NEB), with ATP concentration and reaction time adjusted to yield of tails of the desired length. .. After gel-purification of the 5′ HDV self-cleavage product, the 2′, 3′-cyclic phosphate at its 3′ end was removed with T4 polynucleotide kinase (NEB; 3000 µl reaction containing 30,000 U enzyme and 100 mM MES-NaOH, pH 5.5, 10 mM MgCl2 , 10 mM β-mercaptoethanol, 300 mM NaCl, 37°C, 6 h).

    other:

    Article Title: An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries
    Article Snippet: Poly(A)-tailing with Escherichia coli poly(A) polymerase One single bead (pre-swollen in water for 3 h) was manually picked and transferred to 20 µl of the Poly(A)-tailing reaction mixture prepared according to the manufacturer (New England Biolabs).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Maternal high fat diet during pregnancy and lactation alters hepatic expression of insulin like growth factor-2 and key microRNAs in the adult offspring
    Article Snippet: .. For poly(T) adaptor RT-PCR, ~100 ng total RNA was added to a reaction containing 2.5 units E. Coli Poly A polymerase (New England Biolabs Ltd. Herts. ..

    Article Title: Evaluation of Placental mir-155-5p and Long Non-coding RNA sONE Expression in Patients with Severe Pre-eclampsia
    Article Snippet: Only samples with two clearly detectable ribosomal RNA bands (28S and 18S) and a higher than 2:1 ratio-were evaluated by nanodrop for purityof intact RNA, and were used for reverse transcription. cDNA synthesis and quantitative real- time reverse transcription (RT) PCR for miR-155 expression analysis Expression of miR-155was assessed byquant-itative real-time RT–PCR(SYBR green method), and thesmall nuclear RNA U6was used as aninternal control. .. At first, reverse transcription reactions contained7.5 µltotal RNA,0.5 µl Poly adenine polymerase (PAP)(NEB# M0276L,USA), 1µl PAP Buffer (10x)and 1µl dNTPs (10 mM).

    Article Title: Identification of new miRNA biomarkers associated with HER2-positive breast cancers
    Article Snippet: RNA was first poly-adenylated using E.coli poly(A) polymerase from New England Biolabs. .. Poly-adenylated RNA was then used for reverse transcription and the reverse-transcription polymerase chain reaction (RT-PCR).

    Recombinant:

    Article Title: The oncomiR miR-197 is a novel prognostic indicator for non-small cell lung cancer patients
    Article Snippet: In brief, 500 ng of RNA was polyadenylated in the presence of ATP (800 μ M ) by 1 U of E. coli Poly(A) Polymerase in the reaction buffer supplied by the manufacturer (New England Biolabs Inc., Ipswich, MA, USA) at 37 °C for 60 min, followed by an enzyme inactivation step at 65 °C for 10 min. .. Immediately after that, the polyadenylated RNA was reverse transcribed into first-strand cDNA in a 20 μ l reaction containing the reaction buffer of the manufacturer, 100 U M-MLV reverse transcriptase in the reaction, 20 U RNaseOUT recombinant ribonuclease inhibitor (Invitrogen, Carlsbad, CA, USA) and 0.25 μ M poly(T) adapter (5′-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3′) at 37 °C for 60 min, followed by an enzyme inactivation step at 70 °C for 15 min.

    Article Title: Assessing the clinical value of microRNAs in formalin-fixed paraffin-embedded liposarcoma tissues: Overexpressed miR-155 is an indicator of poor prognosis
    Article Snippet: Polyadenylation of total RNA and reverse transcription One μg of total RNA per sample was polyadenylated with the addition of 800 μM ATP and 1 U of E. coli Poly(A) Polymerase in the reaction buffer supplied by the manufacturer (New England Biolabs Inc., Ipswich, MA, USA) at 37°C for 60 min, followed by a reaction termination step at 65 °C for 10 min. .. Subsequently, the polyadenylated RNA was reverse transcribed with 100 U M-MLV reverse transcriptase (Invitrogen, USA) in the reaction buffer supplied by the manufacturer, in the presence of 20 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen, USA), and 0.25 μM poly(T) adapter(5’-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3’) at 37°C for 60 min. An enzyme inactivation step followed at 70 °C for 15 min.

    In Vivo:

    Article Title: Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages
    Article Snippet: For the following RNA samples: two replicates of the four day 0 in vivo wound samples, day 1 wound samples, day 4 wound samples, and day 14 wound samples, RNA library preparation was performed as previously described ( ). .. Poly(A)-tailing reaction was performed using 3.75 µ E. coli poly(A)-polymerase (New England Biolabs) in 10x poly(A)-polymerase buffer supplemented with ATP (50:1 molar ratio to RNA) and 1 µ/μL SUPERase-In for 30 min at 37°C.

    RNA Sequencing Assay:

    Article Title: Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages
    Article Snippet: Paragraph title: RNA-Seq ... Poly(A)-tailing reaction was performed using 3.75 µ E. coli poly(A)-polymerase (New England Biolabs) in 10x poly(A)-polymerase buffer supplemented with ATP (50:1 molar ratio to RNA) and 1 µ/μL SUPERase-In for 30 min at 37°C.

    Isolation:

    Article Title: The oncomiR miR-197 is a novel prognostic indicator for non-small cell lung cancer patients
    Article Snippet: Paragraph title: Tissue homogenisation, total RNA isolation, polyadenylation and reverse transcription ... In brief, 500 ng of RNA was polyadenylated in the presence of ATP (800 μ M ) by 1 U of E. coli Poly(A) Polymerase in the reaction buffer supplied by the manufacturer (New England Biolabs Inc., Ipswich, MA, USA) at 37 °C for 60 min, followed by an enzyme inactivation step at 65 °C for 10 min.

    Article Title: MicroRNAs of the mir-17~92 cluster regulate multiple aspects of pancreatic tumor development and progression
    Article Snippet: After RNA isolation with TRIzol reagent (Invitrogen #15596), genomic contaminants were removed using DNAse (Life Technologies #AM1907). .. DNA-free RNA was then polyadenylated (New England Biolabs #M0276) and subsequently reverse transcribed (Invitrogen #18080) using a pool of specially designed primers at a concentration of 50 uM to generate cDNA copies of polyadenylated miRNAs.

    Article Title: cAMP-inducible coactivator CRTC3 attenuates brown adipose tissue thermogenesis
    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen), and 1–2 μg of RNA was used to make cDNA with Transcriptor first-strand cDNA synthesis kit according to the manufacturer’s instruction (Roche). .. For mature miRNA detection, total RNA was polyadenylated with Escherichia coli poly(A) polymerase according to the manufacturer’s instruction (New England Biolab) and then used for reverse transcription and qRT-PCR with specific primers ( ).

    Article Title: Genetic Heterogeneity of Breast Cancer Metastasis May Be Related to miR-21 Regulation of TIMP-3 in Translation
    Article Snippet: Small RNA and total RNA of breast tissue were extracted by mirVana miRNA Isolation Kit (AM1560, ABI, USA). .. Small RNA was added poly-A tail by poly-A polymerase (NEB, M0276) before reverse transcription using primers in as before ( ).

    Polymerase Chain Reaction:

    Article Title: MicroRNAs of the mir-17~92 cluster regulate multiple aspects of pancreatic tumor development and progression
    Article Snippet: DNA-free RNA was then polyadenylated (New England Biolabs #M0276) and subsequently reverse transcribed (Invitrogen #18080) using a pool of specially designed primers at a concentration of 50 uM to generate cDNA copies of polyadenylated miRNAs. .. PCR for miRNAs was performed as follows: 1) denaturation at 94°C for 15 seconds, 2) annealing at 55°C for 30 seconds, and 3) extension at 70°C for 34 seconds, for a total of 40 cycles.

    Article Title: Evaluation of Placental mir-155-5p and Long Non-coding RNA sONE Expression in Patients with Severe Pre-eclampsia
    Article Snippet: At first, reverse transcription reactions contained7.5 µltotal RNA,0.5 µl Poly adenine polymerase (PAP)(NEB# M0276L,USA), 1µl PAP Buffer (10x)and 1µl dNTPs (10 mM). .. The 10□l reaction mixture was initiallyincubated at 37°C for 20-30 min, then 65°C for 20 min. After this step, Thermo Scientific kit (K1622, USA) was used by adding 2 µlRT Adaptor to 5 µl template RNA synthesizedduring the first step, 5 µl nuclease free water,1µlRiboLock RNase Inhibitor (20 U/□l), 2 µl dNTPs (10mM) and1 µlRevertAid M- MuLV RT (200 U/ΜL).Then the mixture was incubated at 25°C for 5 minat 42°C for 1h and finally70°C for 5 min. After performing gradient PCR to find the appropriate temperature , standard curve was plotted for each primer till final analysis was done according to a Livak, s formula (2^-Δct ).Real-time quantitative PCR was performe-dusing a Corrbetreal-time PCR detection system (Rotor gene, Corbett 6000(2.1.0), Qiagen, Germany).

    RNA Extraction:

    Article Title: Evaluation of Placental mir-155-5p and Long Non-coding RNA sONE Expression in Patients with Severe Pre-eclampsia
    Article Snippet: RNA extraction RNA was extracted using the miRNeasy Mini Kit (Qiagen Inc, Valencia,CA) according to manufacturer’s instructions. .. At first, reverse transcription reactions contained7.5 µltotal RNA,0.5 µl Poly adenine polymerase (PAP)(NEB# M0276L,USA), 1µl PAP Buffer (10x)and 1µl dNTPs (10 mM).

    Article Title: Identification of new miRNA biomarkers associated with HER2-positive breast cancers
    Article Snippet: Real-time RT-PCR analysis of miRNA expression RNA extraction from this point was done using TRIzol® protocol [ ]. .. RNA was first poly-adenylated using E.coli poly(A) polymerase from New England Biolabs.

    Purification:

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control
    Article Snippet: To generate each 3′ region, a 5′-phosphate–bearing RNA oligonucleotide (IDT) consisting of the barcode segment followed by a 10 nt poly(A) segment was extended with E. coli poly(A)-polymerase (NEB), with ATP concentration and reaction time adjusted to yield of tails of the desired length. .. To narrow the tail-length distribution, extension products were sequentially purified on two denaturing polyacrylamide gels, excising products with tails of the desired length range and reducing the variability of tailed RNA to be mostly within ~5–25 nt, depending on the length of tail added ( ).

    Article Title: Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages
    Article Snippet: Poly(A) RNA was fragmented using RNA Fragmentation Reagents (Ambion) for 10 min at 70°C and purified by running through a Micro Bio-Spin P-30 column (Bio-Rad, Irvine, CA) according to the manufacturer’s instructions. .. Poly(A)-tailing reaction was performed using 3.75 µ E. coli poly(A)-polymerase (New England Biolabs) in 10x poly(A)-polymerase buffer supplemented with ATP (50:1 molar ratio to RNA) and 1 µ/μL SUPERase-In for 30 min at 37°C.

    Article Title: Genetic Heterogeneity of Breast Cancer Metastasis May Be Related to miR-21 Regulation of TIMP-3 in Translation
    Article Snippet: Small RNA was added poly-A tail by poly-A polymerase (NEB, M0276) before reverse transcription using primers in as before ( ). .. For negative control, template was replaced by purified non-reverse-transcripted RNA.

    Sequencing:

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control
    Article Snippet: To generate each 3′ region, a 5′-phosphate–bearing RNA oligonucleotide (IDT) consisting of the barcode segment followed by a 10 nt poly(A) segment was extended with E. coli poly(A)-polymerase (NEB), with ATP concentration and reaction time adjusted to yield of tails of the desired length. .. The 5′ region of the standards was synthesized by in vitro transcription of a template containing Renilla luciferase sequence followed by that of a modified HDV ribozyme .

    Article Title: Maternal high fat diet during pregnancy and lactation alters hepatic expression of insulin like growth factor-2 and key microRNAs in the adult offspring
    Article Snippet: Measurement of miRNAs using real time RT-PCR Two qPCR methods were used in the validation of microarray: the stem-loop RT-PCR method [ ], using miRNA specific primers purchased from Applied Biosystems and polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using sequence complementary to the poly(T) adapters during RT reactions [ ]. .. For poly(T) adaptor RT-PCR, ~100 ng total RNA was added to a reaction containing 2.5 units E. Coli Poly A polymerase (New England Biolabs Ltd. Herts.

    De-Phosphorylation Assay:

    Article Title: Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System
    Article Snippet: .. After dephosphorylation with 10 U FastAP (ThermoFisher; EF0652) at 10 min at room temperature and polyadenylation with 5U E. coli Poly(A) Polymerase (NEB, M0276S) for 15 min at room temperature on beads, the RNA was then eluted by treatment with proteinase K (Thermo Scientific; 25530049) at 37°C for 1 hr. .. The eluted RNA was extracted with Trizol reagent (ThermoFisher; 15596018) and recovered by RNA Clean and Concentrator-5 spin columns (Zymo; R1015).

    Chromatin Immunoprecipitation:

    Article Title: Engagement of circular RNA HECW2 in the nonautophagic role of ATG5 implicated in the endothelial-mesenchymal transition
    Article Snippet: The assay was performed with 4 to 5 μg of the total RNA samples, which were 3’-extended with a poly (A) tail using a poly (A) polymerase (NEB, M0276L). .. The hybridization was performed overnight on a μParaflo microfluidic chip using a microcirculation pump (Atactic Technologies, Houston, USA).

    In Situ:

    Article Title: Engagement of circular RNA HECW2 in the nonautophagic role of ATG5 implicated in the endothelial-mesenchymal transition
    Article Snippet: The assay was performed with 4 to 5 μg of the total RNA samples, which were 3’-extended with a poly (A) tail using a poly (A) polymerase (NEB, M0276L). .. The detection probes were created via in situ synthesis using photogenerated reagent chemistry.

    Hybridization:

    Article Title: Engagement of circular RNA HECW2 in the nonautophagic role of ATG5 implicated in the endothelial-mesenchymal transition
    Article Snippet: The assay was performed with 4 to 5 μg of the total RNA samples, which were 3’-extended with a poly (A) tail using a poly (A) polymerase (NEB, M0276L). .. The hybridization was performed overnight on a μParaflo microfluidic chip using a microcirculation pump (Atactic Technologies, Houston, USA).

    SYBR Green Assay:

    Article Title: Identification of new miRNA biomarkers associated with HER2-positive breast cancers
    Article Snippet: RNA was first poly-adenylated using E.coli poly(A) polymerase from New England Biolabs. .. For the quantitative polymerase chain reaction (qPCR), 2μl of cDNA was mixed with 10μl of RT2 SYBR® Green ROX qPCR Mastermix from Qiagen, 6μl of RNase-free water, 1μl of a universal reverse primer (5′GCG AGC ACA GAA TTA ATA CGA C3′) and a forward primer specific to the miRNA.

    Article Title: cAMP-inducible coactivator CRTC3 attenuates brown adipose tissue thermogenesis
    Article Snippet: Relative mRNA expression was determined with SYBR green master mix (Roche) by LightCycler 480 qPCR machine (Roche). .. For mature miRNA detection, total RNA was polyadenylated with Escherichia coli poly(A) polymerase according to the manufacturer’s instruction (New England Biolab) and then used for reverse transcription and qRT-PCR with specific primers ( ).

    Negative Control:

    Article Title: Genetic Heterogeneity of Breast Cancer Metastasis May Be Related to miR-21 Regulation of TIMP-3 in Translation
    Article Snippet: Small RNA was added poly-A tail by poly-A polymerase (NEB, M0276) before reverse transcription using primers in as before ( ). .. For negative control, template was replaced by purified non-reverse-transcripted RNA.

    Agarose Gel Electrophoresis:

    Article Title: Evaluation of Placental mir-155-5p and Long Non-coding RNA sONE Expression in Patients with Severe Pre-eclampsia
    Article Snippet: RNA quality was assessed by agarose gel electrophoresis. .. At first, reverse transcription reactions contained7.5 µltotal RNA,0.5 µl Poly adenine polymerase (PAP)(NEB# M0276L,USA), 1µl PAP Buffer (10x)and 1µl dNTPs (10 mM).

    In Vitro:

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control
    Article Snippet: To generate each 3′ region, a 5′-phosphate–bearing RNA oligonucleotide (IDT) consisting of the barcode segment followed by a 10 nt poly(A) segment was extended with E. coli poly(A)-polymerase (NEB), with ATP concentration and reaction time adjusted to yield of tails of the desired length. .. The 5′ region of the standards was synthesized by in vitro transcription of a template containing Renilla luciferase sequence followed by that of a modified HDV ribozyme .

    Article Title: RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response
    Article Snippet: Linearized DNA was in vitro transcribed using HiScribe T7 High Yield RNA Synthesis Kit (NEB), with 3′-O-Me-m7 GpppG anti-reverse cap analog (ARCA) or ApppG cap (NEB) added at eight times the concentration of GTP, for a capping efficiency of ~ 90%. .. 10 μL T7 reactions were carried out at 37 °C for 2 h. Reactions were then treated with 2 U RNase-free DNaseI (NEB) for 15 min at 37 °C to remove DNA template, and then polyadenylated with 5 U E. coli Poly-A Polymerase, 10× buffer, and 10 mM ATP (NEB) for 1 h at 37 °C.

    Homogenization:

    Article Title: The oncomiR miR-197 is a novel prognostic indicator for non-small cell lung cancer patients
    Article Snippet: Paragraph title: Tissue homogenisation, total RNA isolation, polyadenylation and reverse transcription ... In brief, 500 ng of RNA was polyadenylated in the presence of ATP (800 μ M ) by 1 U of E. coli Poly(A) Polymerase in the reaction buffer supplied by the manufacturer (New England Biolabs Inc., Ipswich, MA, USA) at 37 °C for 60 min, followed by an enzyme inactivation step at 65 °C for 10 min.

    Ethanol Precipitation:

    Article Title: Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages
    Article Snippet: Fragmented RNA was de-phosphorylated using 1 μL T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) and 5 μL 5x PNK buffer (0.5 M MES, 50 mM MgCl2 , 50 mM mercaptoethanol, 1.5 M NaCl, pH 5.5–5.8) supplemented with 1 µ/μL SUPERase-In for 45 min at 37°C, an additional 1 μL T4 polynucleotide kinase was added to the reaction, followed by incubation for 45 min, and subsequent heat-inactivation for 5 min at 70°C and ethanol precipitation overnight with glycogen. .. Poly(A)-tailing reaction was performed using 3.75 µ E. coli poly(A)-polymerase (New England Biolabs) in 10x poly(A)-polymerase buffer supplemented with ATP (50:1 molar ratio to RNA) and 1 µ/μL SUPERase-In for 30 min at 37°C.

    Random Hexamer Labeling:

    Article Title: Evaluation of Placental mir-155-5p and Long Non-coding RNA sONE Expression in Patients with Severe Pre-eclampsia
    Article Snippet: At first, reverse transcription reactions contained7.5 µltotal RNA,0.5 µl Poly adenine polymerase (PAP)(NEB# M0276L,USA), 1µl PAP Buffer (10x)and 1µl dNTPs (10 mM). .. PCRamplifi-cation of mir- 155 was initiated at 95°C for 5 min, followed by 45 cycles of 95°C for15 s, 61°C for 45s. cDNA synthesis and quantitative real-time reverse transcription (RT) PCR for lncone expression analysis Following total RNA extraction, lncone RNA was analyzed by converting 5 µl RNA sample to cDNA for each sample, using an RNAreverse transcription kit with random hexamer and oligo dTprimers (Thermo Scientific kit, K1622, USA), according to the manufacturer’s instructions.

    Spectrophotometry:

    Article Title: The oncomiR miR-197 is a novel prognostic indicator for non-small cell lung cancer patients
    Article Snippet: The quantity, quality and purity of the resulting RNA were thoroughly assessed by a NanoDrop 2000c spectrophotometer (Thermo Scientific Inc.) and an Agilent 2100 bioanalyzer. .. In brief, 500 ng of RNA was polyadenylated in the presence of ATP (800 μ M ) by 1 U of E. coli Poly(A) Polymerase in the reaction buffer supplied by the manufacturer (New England Biolabs Inc., Ipswich, MA, USA) at 37 °C for 60 min, followed by an enzyme inactivation step at 65 °C for 10 min.

    Concentration Assay:

    Article Title: Engagement of circular RNA HECW2 in the nonautophagic role of ATG5 implicated in the endothelial-mesenchymal transition
    Article Snippet: HBMECs were treated with Meth at a concentration of 100 μM. .. The assay was performed with 4 to 5 μg of the total RNA samples, which were 3’-extended with a poly (A) tail using a poly (A) polymerase (NEB, M0276L).

    Article Title: Poly(A)-tail profiling reveals an embryonic switch in translational control
    Article Snippet: .. To generate each 3′ region, a 5′-phosphate–bearing RNA oligonucleotide (IDT) consisting of the barcode segment followed by a 10 nt poly(A) segment was extended with E. coli poly(A)-polymerase (NEB), with ATP concentration and reaction time adjusted to yield of tails of the desired length. .. To narrow the tail-length distribution, extension products were sequentially purified on two denaturing polyacrylamide gels, excising products with tails of the desired length range and reducing the variability of tailed RNA to be mostly within ~5–25 nt, depending on the length of tail added ( ).

    Article Title: MicroRNAs of the mir-17~92 cluster regulate multiple aspects of pancreatic tumor development and progression
    Article Snippet: .. DNA-free RNA was then polyadenylated (New England Biolabs #M0276) and subsequently reverse transcribed (Invitrogen #18080) using a pool of specially designed primers at a concentration of 50 uM to generate cDNA copies of polyadenylated miRNAs. ..

    Article Title: RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response
    Article Snippet: Linearized DNA was in vitro transcribed using HiScribe T7 High Yield RNA Synthesis Kit (NEB), with 3′-O-Me-m7 GpppG anti-reverse cap analog (ARCA) or ApppG cap (NEB) added at eight times the concentration of GTP, for a capping efficiency of ~ 90%. .. 10 μL T7 reactions were carried out at 37 °C for 2 h. Reactions were then treated with 2 U RNase-free DNaseI (NEB) for 15 min at 37 °C to remove DNA template, and then polyadenylated with 5 U E. coli Poly-A Polymerase, 10× buffer, and 10 mM ATP (NEB) for 1 h at 37 °C.

    Staining:

    Article Title: Engagement of circular RNA HECW2 in the nonautophagic role of ATG5 implicated in the endothelial-mesenchymal transition
    Article Snippet: The assay was performed with 4 to 5 μg of the total RNA samples, which were 3’-extended with a poly (A) tail using a poly (A) polymerase (NEB, M0276L). .. An oligonucleotide tag was subsequently ligated to the poly (A) tail for fluorescent dye staining.

    Cross-linking Immunoprecipitation:

    Article Title: Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System
    Article Snippet: Antibody-RNA complexes were then precipitated with 50 l Protein A/G beads (Thermo Scientific) for 2 hr at 4 °C, followed by two stringent washes (50 mM Tris, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS) and two CLIP buffer washes. .. After dephosphorylation with 10 U FastAP (ThermoFisher; EF0652) at 10 min at room temperature and polyadenylation with 5U E. coli Poly(A) Polymerase (NEB, M0276S) for 15 min at room temperature on beads, the RNA was then eluted by treatment with proteinase K (Thermo Scientific; 25530049) at 37°C for 1 hr.

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    New England Biolabs poly a polymerase
    The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary <t>RNA</t> byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the <t>poly</t> A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) Native PAGE analysis of T7 transcripts generated using fully duplexed DNA template (1), template strand ssDNA alone (512B-3end in Supplementary Table S1 ) (2), and partially duplexed DNA template (generated by annealing 512B-3end with T7promoter_top_strand, Supplementary Table S1 ) (3). * indicates unknown ssRNA byproduct from transcription.
    Poly A Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) Native PAGE analysis of T7 transcripts generated using fully duplexed DNA template (1), template strand ssDNA alone (512B-3end in Supplementary Table S1 ) (2), and partially duplexed DNA template (generated by annealing 512B-3end with T7promoter_top_strand, Supplementary Table S1 ) (3). * indicates unknown ssRNA byproduct from transcription.

    Journal: Nucleic Acids Research

    Article Title: An origin of the immunogenicity of in vitro transcribed RNA

    doi: 10.1093/nar/gky177

    Figure Lengend Snippet: The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) Native PAGE analysis of T7 transcripts generated using fully duplexed DNA template (1), template strand ssDNA alone (512B-3end in Supplementary Table S1 ) (2), and partially duplexed DNA template (generated by annealing 512B-3end with T7promoter_top_strand, Supplementary Table S1 ) (3). * indicates unknown ssRNA byproduct from transcription.

    Article Snippet: For 3′-RACE, 3′ end of the RNA was first extended with poly A tails using the poly A polymerase (NEB).

    Techniques: Generated, Sequencing, Clear Native PAGE, Purification

    Dependence of BiFC fluorescence enhancement on target RNA expression in E. coli . (A) Time course of the difference in mean fluorescence between cells expressing RNA REX(A) – TERC(Q) and REX(A) – TERC(Qm) in the presence and absence of RNA induction by ATc. (B) RNA level in cells before and after induction with ATc. Data are given in mean ± SD of three experiments. IPTG was added at 0 h with or without ATc. Representative distribution of eGFP fluorescence of corresponding cells is given in Fig. S4B. †

    Journal: Chemical Science

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    doi: 10.1039/c5sc03946k

    Figure Lengend Snippet: Dependence of BiFC fluorescence enhancement on target RNA expression in E. coli . (A) Time course of the difference in mean fluorescence between cells expressing RNA REX(A) – TERC(Q) and REX(A) – TERC(Qm) in the presence and absence of RNA induction by ATc. (B) RNA level in cells before and after induction with ATc. Data are given in mean ± SD of three experiments. IPTG was added at 0 h with or without ATc. Representative distribution of eGFP fluorescence of corresponding cells is given in Fig. S4B. †

    Article Snippet: RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega).

    Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, RNA Expression, Expressing

    Pull-down of G-quadruplex-recognizing proteins by RNA from E. coli . Cells were lysed in the presence of RNase inhibitor or RNase A. Probe proteins associated with the indicated target RNAs were pulled down with an immobilized DNA oligomer complementary to the 5′ end of the RNA and detected by an antibody against eGFP. “NS” indicates a non-specifically stained band that can serve as a loading control.

    Journal: Chemical Science

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    doi: 10.1039/c5sc03946k

    Figure Lengend Snippet: Pull-down of G-quadruplex-recognizing proteins by RNA from E. coli . Cells were lysed in the presence of RNase inhibitor or RNase A. Probe proteins associated with the indicated target RNAs were pulled down with an immobilized DNA oligomer complementary to the 5′ end of the RNA and detected by an antibody against eGFP. “NS” indicates a non-specifically stained band that can serve as a loading control.

    Article Snippet: RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega).

    Techniques: Staining

    Dependence of BiFC fluorescence on the number of G 3 tracts in the RNA target expressed in E. coli . Distribution of (A) eGFP fluorescence or (B) forward scattering intensity or (C) relative RNA expression (mean ± SD of three experiments) was assayed and processed as in Fig. 4 .

    Journal: Chemical Science

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    doi: 10.1039/c5sc03946k

    Figure Lengend Snippet: Dependence of BiFC fluorescence on the number of G 3 tracts in the RNA target expressed in E. coli . Distribution of (A) eGFP fluorescence or (B) forward scattering intensity or (C) relative RNA expression (mean ± SD of three experiments) was assayed and processed as in Fig. 4 .

    Article Snippet: RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega).

    Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, RNA Expression

    Detection of BiFC fluorescence in E. coli cells by flow cytometry. Distribution of (A–C) eGFP fluorescence and (D–F) forward scattering intensity was collected from cells expressing G N -REX(A) and RHAU(Q)-G C probe proteins plus the indicated RNA. (G–I) Relative RNA expression (mean ± SD of three experiments) assayed by qPCR.

    Journal: Chemical Science

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    doi: 10.1039/c5sc03946k

    Figure Lengend Snippet: Detection of BiFC fluorescence in E. coli cells by flow cytometry. Distribution of (A–C) eGFP fluorescence and (D–F) forward scattering intensity was collected from cells expressing G N -REX(A) and RHAU(Q)-G C probe proteins plus the indicated RNA. (G–I) Relative RNA expression (mean ± SD of three experiments) assayed by qPCR.

    Article Snippet: RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega).

    Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, Flow Cytometry, Cytometry, Expressing, RNA Expression, Real-time Polymerase Chain Reaction

    The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) (3). * indicates unknown ssRNA byproduct from transcription.

    Journal: Nucleic Acids Research

    Article Title: An origin of the immunogenicity of in vitro transcribed RNA

    doi: 10.1093/nar/gky177

    Figure Lengend Snippet: The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) (3). * indicates unknown ssRNA byproduct from transcription.

    Article Snippet: For 3′-RACE, 3′ end of the RNA was first extended with poly A tails using the poly A polymerase (NEB).

    Techniques: Generated, Sequencing, Clear Native PAGE, Purification