Purified GST-HuD and His-ZBP1 proteins were probed with Dynabead Protein G (Life Technologies) conjugated with anti-GST and anti-His antibody (Life technologies). Beads conjugated with mouse IgG were used to see the background binding. Beads were washed with binding buffer (Promega) and incubated with 1 μg of total RNA isolated from rat dorsal root ganglion (DRG) in binding buffer (10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 1 mM DTT, 400 U of RNaseOUT™ and 5% glycerol) at 4°C for 30 min. After washing, beads were incubated in TRIzol (Life Technologies) and extracted by phenol–chloroform to isolate RNA. RNA was precipitated with ethanol using a glycogen carrier.
Rnaseout
Manufactured by Promega
Sourced in Australia, United States
RNaseOut is a recombinant ribonuclease inhibitor protein that protects RNA from degradation by RNase enzymes during in vitro experiments. It has a high affinity for a wide range of RNase enzymes, effectively inhibiting their activity.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Rnaseout, by Thermo Fisher Scientific (2 mentions)
S 300 column, by GE Healthcare (2 mentions)
Ribo m7g cap analogue, by Promega (2 mentions)
Random hexamer, by Promega (2 mentions)
Binding buffer, by Promega (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Superscript 2 rt, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
First strand buffer, by Thermo Fisher Scientific (1 mentions)
Trizol, by Qiagen (1 mentions)
Dntps, by New England Biolabs (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (1 mentions)
Facsdiva software v8, by BD (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
9 protocols using rnaseout
1
RNA-Protein Interaction Assay using GST-HuD and His-ZBP1
2
Monitoring Promoter Regulation in M. pneumoniae
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To monitor promoter regulation, reporter chimeras with YFP-Venus were built. As YFP seems to be a very stable protein in M. pneumoniae, gene expression was followed by retro-transcription (RT) followed by real time or quantitative PCR (qPCR). Briefly, RNA was purified as above, and 1 μg was hybridized to 2 μg random hexamers (Invtrogen) by heating to 65°C for 5 min and quick chilling on ice in a 11 μl total volume. Retrotranscription was performed by adding 4 μl 5X first-strand buffer, 2 μl 0.1 M DTT, 1 μl RNase OUT (40 units/μl, Promega), 1 μl 10 mM dNTP mix, and 1 μl SuperScript II RT (200 units, Invitrogen), and incubating for 50 min at 42°C before inactivation at 70°C for 15 min. A 2x GoTaq qPCR mastermix was used (Promega) with 0.5 ng cDNA per 10 μl reaction and 0.5 μM oligos, and run on a Lightcycler 480 (Roche). Oligonucleotides can be found in Online Table 3. Ribosomal RNA (16S) was used as a reference.
3
In Vitro Transcription of Labeled RNA
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A transcription reaction containing 40 mM Tris (pH 7.5), 20 mM MgCl2, 10 mM NaCl, 2 mM spermidine HCl, 10 mM DTT, 4 mM NTPs, 1 μg DNA template (PCR product), 5% RNaseOut (Invitrogen), and 5% T7 polymerase (1:20 dilution) was incubated at 37 °C for 4 h. DNA fragments were removed using an S-300 column (GE Healthcare). The product was extracted by phenol-chloroform and precipitated by ethanol. The pellet was dissolved in the desired amount of TE buffer and stored at –20 °C. For radiolabelled RNA, A transcription reaction containing 40 mM Tris (pH 7.5), 6 mM MgCl2, 10 mM NaCl, 2 mM spermidine HCl, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.05 mM GTP, 50 ng DNA template (PCR product), 4% RNaseOut, 10% T7 (1:20 dilution), 1 mM Ribo m7G Cap analogue (Promega) and 0.33 μM [α-32P] GTP (10 mCi/ml, 3000 Ci/mmol) (Pelkin Elmer) was incubated at 37 °C for 1.5 h. Samples were then purified via a denaturing gel.
4
RNA Isolation and cDNA Synthesis
5
In vitro Transcription and Labeling of pri-miRNAs
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Pri-miR157c and pri-miR171b were transcribed in vitro from plasmids containing a genomic copy of each MIRNA with T7 RNA polymerase. Before the transcription, primers containing the T7 RNA promoter on the 5′end and complementary to MIR157c and MIR171b were used for a PCR reaction. The expected PCR products were purified from a 2% (w/v) agarose gel. The transcription reaction was done in a two-step protocol. An initial mix of 10 μl template DNA (∼900 ng/μl), 4 μl T7 RNA polymerase buffer and 6 μl DEPC treated water was heated at 95°C for 2 min and then left for 15 min at room temperature. After that, a second mix was composed of the following: 20 μl 100 mM NTP each, 2.5 μl RNase Out (Promega), 3 μl T7 RNA polymerase (60 U, Thermo Fisher Scientific) and 38.5 μl DPEC treated water was added. The transcription reaction was carried out for 5 h at 37°C. The transcription products were purified from a denaturing 6% (w/v) polyacrylamide gel and precipitated (Tris 0.01 M pH 8, EDTA 0.001 M pH 8, NaCl 0.3 M and ethanol). 100 pmol of pri-miR157c and pri-miR171b transcript were 5′-end-labeled with 32P-γ-ATP (10 μCi/μl), using T4 polynucleotide kinase (Thermo Fisher Scientific). The reaction product were purified from a denaturing 8% (w/v) polyacrylamide gel using autoradiography and precipitated as described below.
6
In Vitro Transcription of Labeled RNA
7
Purification of p62-containing granules
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To purify p62-gels from Huh-1 cells, we modified and used a method for purification of P-body in cells that was developed by elsewhere71 (link). Briefly, GFP-tagged p62 was expressed into Huh-1 cells. Twenty-four hour after transfection, the cells were suspended with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) containing 65 U/mL RNasin® Ribonuclease Inhibitors (Promega) and EDTA-free Protease inhibitor cocktail (Roche) and then lysed by passing 20 times through a 27Gx3/4. The lysates were incubated on ice for 20 min and then centrifuged at 200 × g for 5 min at 4 °C to delete nuclei. The resultant supernatants were supplemented with 10 mM MgSO4 and 1 mM CaCl2 and treated with 4 U/mL of RQ1 DNase (Promega) for 30 min at RT. Thereafter, the mixtures were spun at 10,000 × g for 7 min at 4 °C, and pellets were resuspended into 10 mL of lysis buffer with 80 Units of RNaseOut (Promega). From this fraction, p62-bodies were sorted on BD FACSAria™ II Cell Sorter with BD FACSDiva software v8.0 (BD BioSciences). Particles were detected according to their Forward-scattered light (FSC) and their green fluorescence using the 488 nm excitation laser and the 526/52 band pass filter.
8
RNA Reverse Transcription Protocol
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The extracted RNA was reverse transcribed using the Superscript III First Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. First, 11.5 µL aliquots of RNA were incubated together with 250 ng random hexamers (Promega, WI, USA) and 0.83 mmol/L (final concentration) dNTP (Finnzymes, Espoo, Finland) at 65 °C for 5 min. Thereafter the reactions were cooled on ice for 1 min followed by the reverse transcription reaction when 4 µL 5× First strand buffer, 40 U DTT and 200 U Superscript III enzyme were added. The RNA was protected from degradation by addition of 40 U recombinant RNase inhibitor RNaseOut (Promega, WI, USA). The reactions were incubated at 25 °C for 5 min, 50 °C for 1 h and finally inactivated at 70 °C for 15 min.
9
In Vitro Synthesis of RNA Transcripts
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Complete plasmid pAS2 (2 μg) was linearized with HindIII (3′ end of the full transcript) and incubated in a mix containing 10 mM DTT, 80 U RNase-Out, 0,5 mM each rNTP (Promega) and 40 U T7 RNA polimerase (Promega) in the buffer supplied with the enzyme, in a final volume of 100 μl. Polymerization proceeded for 3–6 h at 37 °C. Afterwards, the plasmid was digested with DNA-Turbo (Ambion), according to manufacturer’s directions, and RNA was quantified by UV spectrometry.
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