Total m6A was measured in 1 μg of total RNA extracted from the uterus using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Shimadzu Corporation, Kyoto, Japan). RNA was heated at 95 °C for 5 min, followed by 2 min of cooling on ice. Subsequently incubated with 1 μL of S1 nuclease (Takara, Okasa, Japan) for 4 h at 37 °C. Then, 1 μL of alkaline phosphatase (Takara, Okasa, Japan) was added, and the reaction was incubated for 1 h at 37 °C. The reaction mixture was extracted with chloroform, freeze-dried at −40 °C for 24 h, and then dissolved in 100 μL of ultra-pure water post-centrifugation. HPLC separation was performed using a C18 column (Shimadzu Corporation, Kyoto, Japan) with a flow rate of 0.2 mL/min at 35 °C. Solvent A was 0.1% (vol/vol) formic acid in water, and solvent B was 0.1% (v/v) formic acid in methanol. A gradient of 5 min of 5% B, 10 min of 5–30% B, 5 min of 30–50% B, 3 min of 50–5% B, and 17 min of 5% B was used.
Alkaline phosphatase
Manufactured by Takara Bio
Sourced in Japan, China
Alkaline phosphatase is an enzyme that catalyzes the hydrolysis of phosphate esters in an alkaline environment. It is commonly used in various laboratory applications to facilitate biochemical reactions and analyses.
Automatically generated - may contain errors
Lab products found in correlation
S1 nuclease, by Takara Bio (6 mentions)
Phosphodiesterase 1, by Merck Group (6 mentions)
Nuclease p1, by Merck Group (4 mentions)
Nuclease p1, by Takara Bio (4 mentions)
Dnase 1, by Takara Bio (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Exionlc ad, by Thermo Fisher Scientific (2 mentions)
6500 triple quadrupole, by Thermo Fisher Scientific (2 mentions)
Exonuclease 1, by Takara Bio (2 mentions)
Safranin o, by Merck Group (2 mentions)
Oil red o, by Merck Group (2 mentions)
Dna polymerase, by Takara Bio (2 mentions)
T4 dna ligase, by Takara Bio (2 mentions)
Qtrap 6500, by AB Sciex (2 mentions)
C18 column, by Shimadzu (1 mentions)
C18 reverse phase column, by GL Sciences (1 mentions)
8 ohdg dna damage elisa kit, by Cell Biolabs (1 mentions)
Versamax, by Molecular Devices (1 mentions)
8 ohdg, by Cell Biolabs (1 mentions)
13c10 15n5 datp, by Merck Group (1 mentions)
34 protocols using alkaline phosphatase
1
Quantification of m6A RNA Modification
2
Quantification of Modified Ribonucleosides
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Total RNAs isolated from WT (wild type), tit1Δ, trm140Δ, trm141Δ, and trm140Δ trm141Δ cells were digested with 2.5 U of Nuclease P1 (Sigma-Aldrich) and 0.2 U alkaline phosphatase (Takara) in 5 mM ammonium acetate pH 5.3 and 20 mM HEPES-KOH pH 7.0 for 3 h at 37°C. Nucleotides were separated by C18 reverse-phase column (GL Science) and directly injected to Agilent 6460 Triple Quadrupole Mass spectrometry. MRM parameters for m3C and m5C were as follows: m3C/m5C: precursor ion, m/z 258, product ion, m/z 126.
3
Quantification of DNA Oxidative Damage
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We evaluated the concentration of 8-hydroxydeoxyguanosine (8-OHdG) using an 8-OHdG DNA damage ELISA kit (Cell Biolabs, San Diego, CA, USA), according to manufacturer’s instructions. Briefly, isolated DNA was denatured at 95 °C for 5 min and immediately transferred to ice. A total of 10 units of nuclease P1 and 20 mM sodium acetate (pH 5.2) were added to total DNA, and digested DNA to nucleosides for 2 h at 37 °C. Next, alkaline phosphatase (Takara, Japan) added for 15 min at 37 °C, followed by incubation for 15 min at 50 °C in 100 mM Tris buffer (pH 7.5). The reaction mixtures were centrifuged for 5 min at 6000× g and the supernatants were extracted for assay analysis. Briefly, 50 µL of each sample is treated in wells and incubated for 10 min at room temperature. An amount of 50 µL of anti-8-OHdG antibody is treated in each well and incubated for 1 h at room temperature on orbital shaker. Each well is rinsed 3 times, 100 µL secondary antibody is added in each well and incubated for 1 h at room temperature. After incubation, 100 µl of substrate solution buffer is added in each well for 20 min at room temperature and then the reaction is stopped by adding stop solution. The absorbance of 8-OHdG was measured using a microplate reader at 450 nm (VERSA max™, Molecular Devices, San Jose, CA, USA).
4
Quantifying 8-Hydroxydeoxyguanosine (8-OHdG) via ELISA
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The ELISA kits for quantitating 8-hydroxydeoxyguanosine (8-OHdG) were purchased from Cell Biolabs Inc. The assay was performed following instructions of the manual. Briefly, DNA was isolated from tissues and then incubated at 95 °C for 5 min and rapidly chilled on ice. DNA samples were digested to nucleosides by incubating the denatured DNA with 10 units of nuclease P1 for 2 h at 37 °C in 20 mM sodium acetate (pH 5.2), treated with 10 units of alkaline phosphatase (Takara Bio Inc.) for 15 min at 37 °C, and then incubated at 50 °C for 15 min in 100 mM Tris buffer (pH 7.5). The reaction mixture was centrifuged for 5 min at 6000× g and the supernatant were used for the assay. Simply, 50 µL of unknown samples or standards were incubated at room temperature for 10 min, then 50 µL of the diluted anti-8-OHdG antibody was added and incubated at room temperature for 1 h on an orbital shaker. After washing three times, 100 µL of diluted secondary antibody-enzyme conjugate was added and incubated at room temperature for 1 h before washing three times. Substrate solution (100 µL) was added to each well and incubated at room temperature for 20 min. The enzyme reaction was stopped by adding 100 µL of stop solution, and absorbance was measured using the microplate reader at 450 nm.
5
Quantitative Metabolic Analysis of Nucleotides
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All NTPs were purchased from TriLink BioTechnologies (San Diego, CA) and Sigma-Aldrich (Beijing, China) (Table S1 and Fig. S1 in ESI† ). The stable isotopic ATP and dATP (13C10,15N5-ATP and 13C10,15N5-dATP) were purchased from Sigma-Aldrich (Beijing, China). Quinoline-8-carbaldehyde, activated manganese dioxide (MnO2), l -methionine-(methyl-D3) (D3-Met) and phosphodiesterase I were purchased from Sigma-Aldrich (Beijing, China). S1 nuclease and alkaline phosphatase were from Takara Biotechnology Co., Ltd (Dalian, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime (Shanghai, China).
6
RNA Digestion and Mass Spectrometric Analysis
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Cells were harvested, and total RNA was isolated using TRIzol (Invitrogen). RNA (10 μg) was digested with a mixture of 2.5 U of nuclease P1 (Wako) in 5 mM ammonium acetate pH 5.3, 0.2 U alkaline phosphatase (TaKaRa) and 20 mM HEPES–KOH pH 7.0. The samples were then incubated for 3 h at 37°C and subjected to mass spectrometric analysis. Two microliters of digested RNA was then subjected to ultrahigh-pressure liquid chromatography coupled with a quadrupole-Orbitrap mass spectrometry (Q Exactive, Thermo Fisher). 5-taurinomethyl-2-thiouridine (τm5s2U: m/z 398.0686), 5-taurinomethyl-uridine (τm5U: m/z 382.0915), 2-thiouridine (s2U: m/z 261.0540), 5-methylaminomethyl-2-thiouridine (mnm5s2U: m/z 304.0962), and 5-methylaminomethyluridine (mnm5U: m/z 288.1190) were detected using full mass scan mode at a mass resolution of 70 000 and a mass tolerance of 5 ppm.
7
RNA Nucleoside Quantification by LC-MS/MS
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Briefly, adding buffer S1 nuclease (Takara Biotechnology, Dalian, Liaoning, China), Alkaline Phosphatase (Takara Biotechnology) and Phosphodiesterase I (Sigma‐Aldrich) into 1 μg RNA, then the mixture was incubated at 37°C. After the RNA was digested into nucleosides completely, the mixture was extracted with chloroform (Sigma‐Aldrich). The resulting aqueous layer was collected for analysis with LC‐MS/MS. The sample extracts were analyzed using an UltraPerformanceLiquid (UPLC)‐MS/MS system (UPLC, ExionLC™ AD, Applied Biosystems 6500 Triple Quadrupole; oster City, CA, USA). The analytical conditions were as follows, LC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 μm; Waters, Milford, MA, USA); solvent system, water (2 mmol/L NH4HCO3): methanol (2 mmol/L NH4HCO3); gradient program, 95:5V/V at 0 min, 95:5V/V at 1 min, 5:95 V/V at 9 min, 5:95 V/V at 11 min, 95:5 V/V at 11.1 min, 95:5 V/V at 14 min; flow rate, 0.30 mL/min; temperature, 40°C; injection volume: 10 μL. The effluent was alternatively connected to an Electrospray Ionization‐triple quadrupole‐linear ion trap. A specific set of multiple reaction monitoring transitions were monitored for each period according to the metabolites eluted within this period.
8
Synthesis of Double-Stranded cDNA from RNA
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RNase H (TaKaRa) was added to the obtained reverse-transcribed products to degrade free RNA. To synthesize double-strand cDNA (dscDNA), anchored random primers were added and incubated at 65°C for 5 min and then placed on ice for 5 min for denaturation (Hameed et al., 2020 (link)). After this, 1 μL of Klenow fragment (TaKaRa), 1 μL of 10 mM dNTPs (TaKaRa), 2 μL of 10 × Klenow buffer (TaKaRa), and 6 μL of ddH2O (TaKaRa) were added and incubated at 37°C for 60 min, followed by an incubation at 75°C for 10 min. To remove phosphates and the free single-strand nucleic acid in the dscDNA reaction, 0.5 μL of Exonuclease I (TaKaRa), 1 μL of alkaline phosphatase (TaKaRa), 5 μL of 10 × phosphatase buffer (TaKaRa), and 24 μL of DEPC H2O (TaKaRa) were added and incubated at 37°C for 60 min, followed by an incubation at 75°C for 10 min.
9
Efg1 Binding to U14 snoRNA and 18S-h10
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U14 snoRNA and the helix 10 region of 18S rRNA (18S-h10, 5′-UACGCAUGGCCUUGUGCUGGCGA-3′) were prepared by in vitro transcription and dephosphorylated with alkaline phosphatase (Takara). An 11-nt RNA (5′-CCAUGAGUGUU-3′) was purchased from Takara. The RNAs were 5′-32P-labeled with T4 polynucleotide kinase (NEB) and purified with MicroSpin G-25 columns (GE Healthcare). U14 and 18S-h10 were annealed by heating at 95°C and slowly cooling down to room temperature. RNAs (∼0.2 nM) were mixed with 2-fold serial dilutions (0.03–8000 nM) of Efg1 in 20 μl of binding buffer (25 mM HEPES pH 7.6, 200 mM KCl, 2 mM MgCl2, 0.01% NP40, 10% glycerol). The reactions were incubated at room temperature for 30 min and resolved in a native polyacrylamide gel run in Tris-glycine buffer (pH 8.3) at room temperature. The gels were dried and visualized with a Typhoon PhosphorImager (GE Healthcare).
10
Multilineage Differentiation of MSCs
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Specific StemPro differentiation media (Gibco) were used for the osteogenic, chondrogenic and adipogenic induction of undifferentiated MSC cultures in vitro. Safranin O (Sigma), Oil Red O (Sigma), Alkaline Phosphatase (Takara Bio Inc.), and Alizarin Red (Sigma) stainings were performed for the determination of the outcome of the differentiation assays [18 (link), 19 ].
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