Rnasein

Manufactured by Promega

Sourced in United States, United Kingdom

RNasein is a laboratory product developed by Promega for effective inactivation of RNase enzymes. It provides a reliable and efficient means to prevent RNA degradation during RNA-based experiments.

Automatically generated - may contain errors

Lab products found in correlation

Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (6 mentions) Tapestation 2200, by Agilent Technologies (4 mentions) Random primer, by Promega (4 mentions) Igepal ca 630, by Merck Group (2 mentions) Rlt plus buffer, by Qiagen (2 mentions) M cvipi reaction buffer, by New England Biolabs (2 mentions) M cvipi, by New England Biolabs (2 mentions) Rnase free dnase, by Qiagen (2 mentions) M mlv reverse transcriptase, by Promega (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Green go taq flexi reaction buffer, by Promega (2 mentions) Taq polymerase, by Promega (2 mentions) Mgcl2, by Promega (2 mentions) Oligo dt, by Bioneer (2 mentions) Tripure reagent, by Roche (2 mentions) Complete protease inhibitor cocktail, by Roche (2 mentions) Flag m2 magnetic beads, by Merck Group (2 mentions) Turbo dnase, by Thermo Fisher Scientific (2 mentions) Rnase a, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

RNasein Plus (7 citations) RNaseIN Plus RNase inhibitor (4 citations)

24 protocols using rnasein

Microscopy and Fractionation of Fibroblasts

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For microscopy analysis, fibroblasts were grown on cover slips and permeabilzed in cytoskeleton buffer (CSK) with 0.5% Triton X-100 for 3 minutes. Cells were washed once with cytoskeleton buffer and then incubated for 5 minutes at 37°C with either RNase A (ThermoFisher, 0.1 mg/ml) or RNase inhibitor (RNasein, Promega) in CSK buffer. Treated nuclei were immediately fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature and analyzed as described above.
Analysis of protein diffusion from nuclear pellets was performed on 5 million human fibroblasts harvested and washed in PBS buffer. Nuclei were permeabilzed in 0.5 ml cytoskeleton buffer (CSK) with 0.5% Triton X-100 for 3 minutes and pelleted by centrifugation at 4°C for 5 min at 800 g. The supernatant was recovered as the cytoplasmic fractions. Nuclei were washed once in CSK lacking detergent resuspended in CSK. A portion was saved as the nuclear fraction. Nuclei were then incubated for 5 minutes at 37°C with either RNase A (ThermoFisher, 0.1 mg/ml) or RNase inhibitor (RNasein, Promega) in 0.5 ml CSK buffer and again pelleted by centrifugation at 4°C for 5 min at 800 g. The supernatant and pellets were recovered for analysis by western blot.

Creamer K.M., Kolpa H.J, & Lawrence J.B. (2021). Nascent RNA scaffolds contribute to chromosome territory architecture and counter chromatin compaction. Molecular cell, 81(17), 3509-3525.e5.

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In Vitro Transcription and Processing of crRNA Array

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DNA template for in vitro transcription of a crRNA array was amplified from pBA439-HAK by PCR using a forward primer with T7 promoter sequence and a reverse primer that included poly-T to terminate transcription. 0.5 μg of DNA template were transcribed in 100 μL reaction using 30 μg T7 RNAP, 2 mM NTPs, 40 U RNaseIN (Promega) in 50 mM Tris-HCl pH 7.5, 15 mM MgCl₂, 5 mM DTT, and 2 mM spermidine at 37°C for 2 h. The transcription reaction was treated with 2 U of DNase I (ThermoFisher Scientific) at 37°C for 30 min and clean-up using QIAGEN RNA easy kit according to the manufacturer’s protocol. In vitro crRNA array processing was carried out in 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5 mM MgCl₂, 20 U RNaseIN (Promega), 0.1% BSA for 30 min at 37°C, stopped by adding 1% SDS, 2× TBE-Urea gel loading buffer and denatured for 10 min at 95°C. Samples were then put on ice for 10 min before running them on an 12% TBE^– 8 M Urea polyacrylamide gel in 1× TBE buffer at 200 V for 40 min. Gel staining was carried out in 1× SYBR Gold in 1× TBE for 5 min and imaged on a gel doc system (Syngene).

Singsuksawat E., Onnome S., Posiri P., Suphatrakul A., Srisuk N., Nantachokchawapan R., Praneechit H., Sae-kow C., Chidpratum P., Sa-ngiamsuntorn K., Hongeng S., Avirutnan P., Duangchinda T, & Siridechadilok B. (2021). Potent programmable antiviral against dengue virus in primary human cells by Cas13b RNP with short spacer and delivery by VLP. Molecular Therapy. Methods & Clinical Development, 21, 729-740.

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Cytoplasmic RNA Extraction from HeLa Cells

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A total of 3.10⁵ electroporated HeLa cells were lysed in 100 μl of RLNa buffer (10 mM Tris–HCl (pH 8), 10 mM NaCl, 3 mM MgCl₂, 1 mM DTT, 0.5% NP40 and 10 U/ml of RNaseIN (Promega Co., Madison, WI, USA). The lysate was cleared to remove membrane debris. Tris-Reagent (Sigma-Aldrich, St Louis, MO, USA) was added to the lysate and cytoplasmic RNAs were extracted following the protocol provided by the manufacturer. Cytoplasmic RNAs were treated with RQ1 DNase (Promega Co., Madison, WI, USA) to avoid DNA contamination and purified with isopropanol and washed with 70% EtOH.

Mengardi C., Limousin T., Ricci E.P., Soto-Rifo R., Decimo D, & Ohlmann T. (2017). microRNAs stimulate translation initiation mediated by HCV-like IRESes. Nucleic Acids Research, 45(8), 4810-4824.

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RNA-tagging for FMRP Interactome

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The RNA-tagging technique was adapted from Yang et al. (Yang et al., 2005 (link)). Briefly, 5-day old adult heads were lysed (50mM Tris-HCl (pH 8.1), 10mM EDTA, 150mM NaCl, 1%SDS), diluted in 50mM Tris-HCl (pH 8.1), 10mM EDTA, 50mM NaCl, cleared by 12,000rpm, then IPed with anti-Flag-M2 agarose (Sigma). Fractionated lysates (below) were precipitated with the 6A15 anti-dFMRP mAb (Abcam). Precipitates were eluted with Elution buffer (EB: 50mM Tris-HCl (pH 7.0), 10 mM EDTA, 1.3% SDS). All buffers contain RNaseIN (Promega) and cOmplete protease inhibitor (Roche).

Bienkowski R.S., Banerjee A., Rounds J.C., Rha J., Omotade O.F., Gross C., Morris K.J., Leung S.W., Pak C., Jones S.K., Santoro M.R., Warren S.T., Zheng J.Q., Bassell G.J., Corbett A.H, & Moberg K.H. (2017). The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile-X protein ortholog in Drosophila neurons. Cell reports, 20(6), 1372-1384.

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Testis RNA Extraction and cDNA Synthesis

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RNA was extracted from the whole snap-frozen testis sample from each fetus as described previously [22 (link)–24 (link)]. The RNA was quantified, and the quality assessed spectrophotometrically using a Nanodrop ND-1000 (Labtech International Ltd., Heathfield, East Sussex, U.K.) and electrophoretically using a Tapestation 2200 (Agilent Technologies, Edinburgh, U.K.) (RNA Integrity Number Equivalent (RINe); 9.048 ± 0.092). Extracted RNA was stored at – 80 °C until required.
Complementary DNA (cDNA) was prepared from 0.3 μg of RNA with SuperScript III reverse transcriptase (Life Technologies, Paisley, Renfrewshire, U.K.) following the manufacturer’s instructions. Each reaction contained 250 ng random primers (Promega, Chilworth, Hampshire, U.K.) and 40 units RNaseIn (Promega). Negative controls without reverse transcriptase were included to check for genomic contamination and all cDNA was stored at – 20 °C until required.

Stenhouse C., Cortes-Araya Y., Donadeu F.X, & Ashworth C.J. (2022). Associations between testicular development and fetal size in the pig. Journal of Animal Science and Biotechnology, 13, 24.

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Single-cell Multiomics of Early Embryo

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Single-cells were flow-sorted (E6.5 and E7.5 stages, using a BD Influx or BD Aria III) or manually picked when cell numbers were too low (E4.5, E5.5). Cells were isolated into 96 well PCR plates containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl⁻¹ RNasein (Promega), 0.1% IGEPAL CA-630 (Sigma)). Samples were incubated for 15 minutes at 37°C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at -80°C prior to processing. Poly-A RNA was captured on oligo-dT conjugated to magnetic beads and amplified cDNA was prepared according to the G&T-seq^{32 (link)} and Smartseq2 protocols^{33 (link)}. The lysate containing gDNA was purified on AMPureXP beads before bisulfite-seq libraries were prepared according to the scBS-seq protocol^{34 (link)}.
A subset of embryo cells were processed for scRNA-seq only (1,419 cells after QC). These followed the same protocol but we discarded the gDNA after separation.
A full step-by-step protocol for scNMT-seq is available online: dx.doi.org/10.17504/protocols.io.6jnhcme.

Argelaguet R., Clark S.J., Mohammed H., Stapel L.C., Krueger C., Kapourani C.A., Imaz-Rosshandler I., Lohoff T., Xiang Y., Hanna C.W., Smallwood S., Ibarra-Soria X., Buettner F., Sanguinetti G., Xie W., Krueger F., Göttgens B., Rugg-Gunn P.J., Kelsey G., Dean W., Nichols J., Stegle O., Marioni J.C, & Reik W. (2019). Multi-omics profiling of mouse gastrulation at single cell resolution. Nature, 576(7787), 487-491.

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RNA Extraction and cDNA Synthesis from Placental Samples

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RNA was extracted from 20 to 50 μg of snap-frozen placental samples [61 (link)], with the addition of a DNase treatment step (RNase-free DNase, Qiagen), as per the manufacturer's instructions. The RNA was quantified, and the quality assessed spectrophotometrically using a Nanodrop ND-1000 (Labtech International Ltd) and electrophoretically using a Tapestation 2200 (Agilent Technologies). The mean A260/A280 and RNA Integrity Number Equivalent (RINe) for samples within each GD are detailed in Supplementary Table S3. Extracted RNA was stored in a freezer at –80°C until required. In the case of a sample having a RINe value below the ranges detailed in Supplementary Table S3, the sample was excluded from the analyses.
Complementary DNA (cDNA) was prepared from 0.3 μg of RNA with SuperScript III reverse transcriptase (Life Technologies) following the manufacturer's instructions. Each reaction contained 250 ng random primers (Promega) and 40 units RNaseIn (Promega). Negative controls without reverse transcriptase were included to check for genomic contamination. Reverse transcription was performed in duplicate for each sample and pooled, and the cDNA was stored at –20°C until required.

Stenhouse C., Hogg C.O, & Ashworth C.J. (2018). Associations between fetal size, sex and placental angiogenesis in the pig. Biology of Reproduction, 100(1), 239-252.

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Quantifying MMP-1 and TIMP-1 Expression

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Total RNA from human dermal fibroblasts was extracted with 1 mL of TRIzol (Invitrogen) per the manufacturer instructions, and 5 μg samples were reverse transcribed in 40-μL of reaction mixture containing 8 μL 5× M-MLV RT (Murine Leukemia Virus Reverse Transcriptase) buffer, 3 μL dNTPs (10 mM), 0.45 μL RNasein (40 U/μL), 0.3 μL M-MLV reverse transcriptase (200 U/μL; Promega, Madison, WI, USA), and 1.5 μL of oligo dT (50 pM/μL; Bioneer, Daejeon, Korea). Single stranded cDNA was amplified by PCR using 4 μL of 5× Green Go-Taq® Flexi reaction buffer, 0.4 μL dNTPs (10 mM), 0.1 μL Taq polymerase (5 U/μL), 1.2 μL MgCl₂ (25 mM; Promega), and 0.4 μL each (20 pM/μL) of specific sense and anti-sense primers for MMP-1, TIMP-1 or β-actin. PCR primer sequences are described in Table 1. PCR conditions were 28 cycles of 94°C for 60 sec, 50°C for 60 sec, and 72°C for 60 sec. PCR products were run on a 1.2% agarose gel with a β-actin internal control to evaluate the relative expression of MMP-1 and TIMP-1.

Park M.A., Sim M.J, & Kim Y.C. (2017). Anti-Photoaging Effects of Angelica acutiloba Root Ethanol Extract in Human Dermal Fibroblasts. Toxicological Research, 33(2), 125-134.

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RNA Extraction and cDNA Preparation

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RNA was extracted from 20 to 50 μg of tissue from snap-frozen samples as previously described [43 (link)], with the addition of a DNase treatment step (RNase-free DNase, Qiagen, Manchester, U.K.). The RNA was quantified spectrophotometrically using a Nanodrop ND-1000 (Labtech International Ltd., Heathfield, U.K.), and the quality assessed electrophoretically using a Tapestation 2200 (Agilent Technologies, Edinburgh, U.K.) (Supplementary Table 2). If the RINe value obtained was lower than the desired ranges (Supplementary Table 2), the sample was excluded from the analyses.
Complementary DNA (cDNA) was prepared from 0.3 μg of RNA with SuperScript III reverse transcriptase (Life Technologies, ThermoFisher Scientific, Altrincham, U.K.). Each reaction contained 250 ng random primers (Promega, Southampton, U.K.) and 40 units RNaseIn (Promega, Southampton, U.K.). Negative controls without reverse transcriptase were included to check for genomic contamination. Reverse transcription was performed in duplicate for each sample and pooled.

Stenhouse C., Hogg C.O, & Ashworth C.J. (2018). Associations between fetal size, sex and both proliferation and apoptosis at the porcine feto-maternal interface. Placenta, 70, 15-24.

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Isolation and Analysis of FTO-Bound RNA

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Cells expressing FLAG-tagged FTO were grown to 90% confluence, washed with ice cold PBS and lysed in lysis buffer (LB) containing 150 mM NaCl, 50 mM Tris pH 7.6, 1% Triton X-100, EDTA-free Complete Protease Inhibitor Cocktail (Roche), 1 mM DTT, RNase In (Promega). Lysates were sonicated 6 × 10 s at 7% amplitude, incubated with Turbo DNase (Fermentas) for 15 min at 37°C and cleared by centrifugation. Supernatants were subsequently pre-cleared with magnetic beads without antibodies for 1 h at 4°C. FLAG M2 Magnetic beads (Sigma) were incubated with 10 μg of yeast tRNA for 1 h at 4°C. The pre-cleared extracts were applied on the pre-blocked FLAG beads and incubated for 2 h at 4°C. Beads were washed three times with LB and the bound RNA was extracted with the TriPure reagent (Roche) according to manufacturer's instructions. The isolated RNA was treated with the Turbo DNase (Fermentas) and used as a template for cDNA synthesis by Superscript III reverse transcriptase (Invitrogen) with random hexamer priming. The cDNA was subsequently analyzed by semi-quantitative or real-time qPCR.

Bartosovic M., Molares H.C., Gregorova P., Hrossova D., Kudla G, & Vanacova S. (2017). N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3′-end processing. Nucleic Acids Research, 45(19), 11356-11370.

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