Rnase inhibitor

Manufactured by Enzynomics

The RNase inhibitor is a laboratory reagent designed to prevent the degradation of ribonucleic acid (RNA) by ribonuclease (RNase) enzymes. It functions by binding to and inhibiting the activity of RNase, thereby preserving the integrity of RNA samples.

Automatically generated - may contain errors

Lab products found in correlation

M mlv reverse transcriptase, by Promega (5 mentions) Direct zol rna miniprep kit, by Zymo Research (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Rnase inhibitor, by Thermo Fisher Scientific (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Rnalater solution, by Thermo Fisher Scientific (1 mentions) Trizol reagent, by Promega (1 mentions) Topreal qpcr 2 premix, by Enzynomics (1 mentions) Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Lightcycler 480 sybr green 1 master kit, by Roche (1 mentions) Dynabeads protein g, by Thermo Fisher Scientific (1 mentions) Sybr green fluorescent dye, by GeNet Bio (1 mentions) Ariamx pcr system, by Agilent Technologies (1 mentions) Control rabbit igg, by Merck Group (1 mentions) Goldhotstart taq pcr master mix, by Bioneer (1 mentions) Dynabead, by Thermo Fisher Scientific (1 mentions) Whatman 3mm paper, by GE Healthcare (1 mentions) Hybond xl membrane, by GE Healthcare (1 mentions) Hybond ecl membrane, by GE Healthcare (1 mentions)

8 protocols using rnase inhibitor

Total RNA Extraction and cDNA Synthesis

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Total RNA from ND7/23 cells was extracted using Trizol reagent (Ambion, USA) according to the manufacturer’s protocol. Six-week old male Sprague Dawley rat (Orient Bio, Korea) dorsal root ganglia (DRG) cDNA was used as positive control templates to validate the specificity of some PCR primers. Fresh rat cadavers were used to dissect DRG. Animals were delivered on the day of experiment and sacrificed via CO₂ inhalation for another study as approved by Seoul National University IACUC (SNU-160202-3-2, Dr. Seog Bae Oh). Tissue was collected in RNAlater solution (Ambion, USA), washed using Diethylpyrocarbonate (DEPC)-PBS and homogenized in Trizol reagent and RNA extracted.
mRNA was reverse transcribed (RT) in 6μl reaction containing M-MLV reverse transcriptase (200 units, Promega, USA), 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl₂, 10mM dithiothreitol, dNTPs each at 0.5mM and RNase inhibitor (25 units, Enzynomics, Korea). Complementary DNA (cDNA) was synthesized from 500ng of total RNA, freshly isolated, with reverse transcriptase primed with (40pmole) oligo-dT or gene-specific primers. The cDNA was stored at -20°C until use.

Lee J., Kim S., Kim H.M., Kim H.J, & Yu F.H. (2019). NaV1.6 and NaV1.7 channels are major endogenous voltage-gated sodium channels in ND7/23 cells. PLoS ONE, 14(8), e0221156.

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qRT-PCR Analysis of Purified Total-cells RNAs

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Total-cells RNAs purified with TRIzol reagent (Life Technologies) were subjected to DNA digestion using 0.05 U/μL DNase I (Thermo Fischer Scientific) supplemented with 0.4 U/μL RNase inhibitor (Thermo Scientific) at 37 °C for 45 min. The purified RNA was incubated with 6 U/μL RevertAid reverse transcriptase (Thermo Fisher Scientific) supplemented with 1 U/μL RNase inhibitor at 37 °C for 2 h. Then, qRT-PCR analyses were performed with TOPreal qPCR 2× PreMIX (Enzynomics) and gene-specific oligonucleotides on a LightCycler 480 SYBR Green I Master Kit (Roche) according to the messenger ribonucleic acid (MIQE) guidelines (Bustin et al., 2009 (link)). The gene-specific oligonucleotide sequences used in this study are listed in Supplementary Table S1.

Shin M.K., Chang J., Park J., Lee H.J., Woo J.S, & Kim Y.K. (2024). Nonsense-mediated mRNA decay of mRNAs encoding a signal peptide occurs primarily after mRNA targeting to the endoplasmic reticulum. Molecules and Cells, 47(4), 100049.

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RNA Immunoprecipitation and qPCR Analysis

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Cells were lysed with RIP buffer [150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40 and 100 U/ml RNase inhibitor (Enzynomics, Daejeon, Korea)] containing a protease inhibitor cocktail (Roche Applied Science). Cell lysates containing 600 μg of protein were incubated with the anti-RBFOX2 antibody (Bethyl Laboratories) or control rabbit IgG (Sigma-Aldrich) for 4 h at 4°C, and the lysate was centrifuged at 10,000 × g for 10 min. Dynabeads^® Protein G (50 μl; Invitrogen, CA, USA) was washed three times with RIP buffer and incubated with the cell lysate for 2 h at 4°C, after which the beads were washed six times with RIP buffer. Total RNA was extracted using a Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA, USA) and reverse-transcribed using M-MLV reverse transcriptase (Promega) with random hexamers. The primer set for the TEAD1 and TEAD2 intronic region used in this experiment is shown in Supplementary Table S3. RIP-qPCR was performed using SYBR Green fluorescent dye (GENET BIO) and the AriaMx PCR system (Agilent Technologies). Normalization was performed using β-actin as an internal control.

Choi S., Lee H.S., Cho N., Kim I., Cheon S., Park C., Kim E.M., Kim W, & Kim K.K. (2022). RBFOX2-regulated TEAD1 alternative splicing plays a pivotal role in Hippo-YAP signaling. Nucleic Acids Research, 50(15), 8658-8673.

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DJ-1 Minigene Splicing Assay

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For the minigene assay, SK-N-SH cells were transfected with 50 ng of DJ-1 minigene vector with 2 μg of PTB or PSF expression vector for 36 h. Total RNA was extracted using a GeneAll Hybrid-R™ RNA extraction kit (GeneAll, Seoul, Korea). Reverse transcription was performed with 1 μg of total RNA, random hexamer, M-MLV reverse transcriptase (Promega), and RNase inhibitor (Enzynomics, Daejeon, Korea). RT-PCR was performed using GoldHotStart Taq PCR Master Mix (Bioneer) followed by agarose gel electrophoresis. The PCR band intensity was measured using ImageJ software. The DJ-1 E6 exclusion ratio was calculated using the PCR band intensity normalized to the PCR product size. The primers used in RT-PCR reactions were as follows: DJ-1 minigene E6 splicing pattern 5′-GAT CAC TCT CGG CAT GGA CG-3′ and 5′-GCT TGT AAG AAT CAG GCC GTC-3′ and endogenous DJ-1 E6 splicing pattern 5′-CCA TAT GAT GTG GTG GTT CTA CC-3′ and 5′-GCT TGT AAG AAT CAG GCC GTC-3′.

Cho N., Joo J., Choi S., Kang B.G., Lee A.J., Youn S.Y., Park S.H., Kim E.M., Masliah E., Ko Y., Cha S.S., Jung I, & Kim K.K. (2023). A novel splicing variant of DJ-1 in Parkinson's disease induces mitochondrial dysfunction. Heliyon, 9(3), e14039.

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Rbfox2 Protein-RNA Interaction Profiling

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Cells were lysed with RIP buffer [150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, and 100 U/mL RNase inhibitor (Enzynomics, Daejeon, Korea)] supplemented with a protease inhibitor cocktail (Roche Applied Science). Cell lysates containing 600 μg of protein were incubated with anti-Rbfox2 antibody for 4 h at 4 °C. The lysate was centrifuged at 10,000 × g for 10 min, and the supernatant was precleared by interaction with Dynabeads (Protein G; Invitrogen, Carlsbad, CA, USA) for 2 h at 4 °C with constant shaking. The Dynabead Protein G/antibody/protein complexes were washed six times with RIP buffer, and total RNA was extracted using a Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA). The RNA was then reverse transcribed by M-MLV reverse transcriptase (Promega, Madison, WI, USA) with random hexamers. The primers for reverse transcription (RT)-PCR were as follows: RB1-F (5′-ACAGATTTGTCCTTCCCGTG-3′) and RB1-R (5′-CCATGATTCGATGCTCACAT-3′). The RT-PCR end products were visualized by 1.8% agarose gel electrophoresis.

Choi S., Sa M., Cho N., Kim K.K, & Park S.H. (2019). Rbfox2 dissociation from stress granules suppresses cancer progression. Experimental & Molecular Medicine, 51(4), 49.

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RNA-Antibody Binding Assay

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RNA was renatured by heating for 5 min at 65°C, chilling on ice for 10 min, and slowly returning to room temperature. RNA–antibody complexes were formed in 100 μL PBS containing 0.6 nM RNA, 2 nM–100 nM antibodies, and 2 units/μL RNase inhibitor (Enzynomics) for 30 min at room temperature. The reaction mixture was applied to a dot-blot apparatus in which Hybond ECL membrane (GE Healthcare) and Hybond-XL membrane (GE Healthcare) were layered with Whatman 3 MM paper in between. In this process, RNAs that passed through the Hybond ECL membrane without binding to the antibody were immobilized on the Hybond-XL membrane. The Hybond-XL membrane was washed two times with PBS and hybridized with 5′-³²P–labeled antisense oligonucleotide probe (5′-TTTGAGGGAAGTTACGCTTAT for BC200 RNA, 5′-GGGAGAACGGGGTCTCGC for truncated BC200 RNA derivatives, or 5′-GGGAATCTCCGAGATGCCGCC for Escherichia coli 6S RNA as an irrelevant RNA), exposed to the imaging plate, and analyzed on the Phosphor-image analyzer. The amount of radiolabeled RNA on each filter was quantified with ImageJ software (NIH), and plots of bound fractions of RNA versus concentration of antibody were obtained with Origin software (OriginLab). Binding constants (K_d) were calculated by fitting data to the Hill equation.

Jung E., Lee J., Hong H.J., Park I, & Lee Y. (2014). RNA recognition by a human antibody against brain cytoplasmic 200 RNA. RNA, 20(6), 805-814.

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SARS-CoV-2 N gene detection protocol

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All natural oligonucleotides and M-MLV reverse transcriptase were purchased from Bioneer (South Korea) and Cosmogenetech (South Korea). Vent (exo-) DNA polymerase was purchased from New England BioLabs (USA). Lambda exonuclease, RNase Inhibitor, dATP, dCTP, and dGTP were purchased from Enzynomics (South Korea). A TwistAmp® Basic for RPA reaction kit was purchased from Twist DX (UK). A QIAquick nucleotide removal kit for DNA purification was purchased from QIAGEN (Germany). The fluorescence of a sample in a quartz cuvette (path length: 1 cm) was recorded at room temperature using a PF-6500 spectrofluorometer (JASCO). The N gene of the SARS-CoV-2 sequence was downloaded from GenBank (RefSeq: NC_045512.2). For the verification with human specimen, the spiked sample was used.

Choi M.H., Lee J, & Seo Y.J. (2021). Combined recombinase polymerase amplification/rkDNA–graphene oxide probing system for detection of SARS-CoV-2. Analytica Chimica Acta, 1158, 338390.

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Quantitative Analysis of Differentiation Markers

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The cell pellet was obtained after being differentiated for 14 days. Total RNA from hASC were purified by Rneasy Mini Kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. cDNA was synthesized from 1 μg of total RNA by reverse transcription using dNTP mix, Rnase inhibitor (Enzynomics Co., Ltd., Daejeon, Korea), M-MLV Reverse Transcriptase, M-MLV RT 5× Buffer (Promega Corporation, Madison, WI, USA) and random oligo primers. qRT-PCR was performed using SYBR Green 2× Mastermix kit (Messenger of biotechnology, Hanam, Korea) on a BioRad CFX96 Real-Time PCR Detection System instrument (Bio-Rad Laboratories, Hercules, CA, USA) under the following conditions: 10 min at 95 °C, and then 40 cycles of 15 s at 95 °C for denaturation and 60 s at 60 °C for annealing and elongation. Specificity of products was verified by melting curve analysis. The delta-delta-C_t method was employed to determine the relative gene expression level of gene of interest normalized to the house-keeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers used for differentiation markers and Nrf2 pathway genes are listed in Table 1.

Han G.E., Kang H.T., Chung S., Lim C., Linton J.A., Lee J.H., Kim W., Kim S.H, & Lee J.H. (2018). Novel Neohesperidin Dihydrochalcone Analogue Inhibits Adipogenic Differentiation of Human Adipose-Derived Stem Cells through the Nrf2 Pathway. International Journal of Molecular Sciences, 19(8), 2215.

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