To concentrate and cleanup RNA samples, sodium acetate–ethanol or isopropanol precipitation was carried out. 3 M sodium acetate (pH 5.4, Life Technologies) was added to make up 1/10th of the sample volume. 1–2 μl of GlycoBlue (Invitrogen) was used as co-precipitant. Depending on the sample volume either 3 volumes of ice-cold 100% Ethanol (<300 μl) or 1 volume of ice-cold 100% Isopropanol was added. After thorough mixing, samples were precipitated at –80°C for at least 30 min and spun for at least 60 min in the pre-cooled 4°C centrifuge at 18213 g. RNA pellets were washed with at least 250 μl of ice-cold 80% ethanol and spun for at least 4 min. After aspiration by pipetting RNA pellets dried at room temperature for 3–5 min and were resuspended in 10 mM Tris, pH 7.0. To clean up samples further and remove traces of short DNA oligos post DNase treatment, we purified RNA samples by RNA Clean & Concentrator-5 kit (Zymo Research). If this kit was used prior to DNase treatment, elution was done with 85 μl DEPC-water. Otherwise, samples were eluted into 10 mM Tris, pH 7.0. For DNase treatment, 5 μl 10× Turbo DNase buffer and 5 μl 2U/μl TurboDNase (Thermo Fisher Scientific) were added to 85 μl of sample and incubated for 20–30 min at 37°C. RNA was cleaned up either with the RNA Clean & Concentrator-5 kit (Zymo Research) or by Ethanol precipitation.
Rna clean concentrator 5 kit
Manufactured by Zymo Research
Sourced in United States, Germany
The RNA Clean & Concentrator-5 kit is a laboratory product designed to purify and concentrate RNA samples. It utilizes a silica-based membrane to selectively bind RNA molecules, allowing for efficient removal of contaminants and concentration of the desired RNA material.
Automatically generated - may contain errors
Lab products found in correlation
Turbo dnase, by Thermo Fisher Scientific (17 mentions)
Rneasy mini kit, by Qiagen (9 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Trizol, by Thermo Fisher Scientific (9 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions)
Dnase 1, by New England Biolabs (7 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (7 mentions)
Direct zol rna miniprep kit, by Zymo Research (5 mentions)
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (4 mentions)
Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (4 mentions)
Tri reagent, by Merck Group (3 mentions)
Ribo zero rrna removal kit, by Illumina (3 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Quick rna microprep kit, by Zymo Research (3 mentions)
Nebnext ultra 2 rna library prep kit, by New England Biolabs (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Trizol ls, by Thermo Fisher Scientific (3 mentions)
Rna 6000 pico kit, by Agilent Technologies (3 mentions)
Paxgene blood rna extraction kit, by Qiagen (3 mentions)
176 protocols using rna clean concentrator 5 kit
1
RNA Clean-up and Concentration Protocol
2
RNA Extraction and Quantification Protocol
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Samples corresponding to 1 ml of culture at an OD600 of 0.4 grown for dead/live staining, as described above, were collected and centrifuged at 10,000g for 3 min. The resulting pellets were frozen in liquid nitrogen and stored −80 °C until further processing. Total RNA was extracted using 1.5 ml of Trizol and 1.5 ml of ethanol with the Direct-zol RNA Miniprep kit (R2051, Zymo), according to the manufacturer’s instructions. The RNA was further purified using the RNA Clean & Concentrator-5 kit (R1013, Zymo). A total of 0.5 µg of RNA from each sample in 5 µl was combined with 2.5 µl of RNA loading dye, heated to 70 °C for 10 min and subsequently chilled on ice for 2 min. The RNA High-Range ladder that was used was also heat-treated. The denatured samples (5 µl) and the leader (3 µl) were loaded onto a 1% TBE gel. The gel was run for 40 min at 120 V. Next, the gel was stained for 30 min in ethidium bromide, washed for 10 ml and imaged. Gel images were analysed using GelAnalyzer v.19.1 (www.gelanalyzer.com ).
3
In Vitro miRNA Methylation Assay
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The methylation assay is carried out in a reaction mixture (total volume: 20 μl) containing 25 mM Tris–HCl (pH8.0), 50 mM KCl, 2.5 mM MgCl2, 0.05 mM EDTA, 2.5% glycerol, 5 mM DTT, 20 μM AdoMet, 10μM synthetic miR-21-5p and 5 μM recombinant HENMT1 proteins. The genes encoding full-length HENMT1 was cloned into the pET15b vector with an N-terminal His tag and transfected into BL21 (DE3) Escherichia coli cells. Recombinant HENMT1 protein was generated and purified as previously described (22 (link)). The mixtures were incubated at 37°C for 40 min. The RNAs were then extracted by RNA Clean & Concentrator-5 kit (Zymo Research) according to the manufacturer's instructions.
4
RNA Extraction and Purification
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TriZOL was used for RNA extraction according to the manufacturer’s instructions. All steps were performed on ice or 4 °C. The RNA was resuspended in DNase/RNase free H2O. For obtaining ultrapure RNA without any residual phenol-chloroform contamination, the Zymo RNA Clean & Concentrator-5 kit was used.
5
Cappable-seq RNA Extraction and Sequencing
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RNA was extracted from 5 mL cultures prepared as detailed above. Cultures were centrifuged for 10 min at 3,700 g, and the pellet was thoroughly resuspended in 1 mL TRI Reagent® (Sigma-Aldrich). Total RNA was then extracted using the Direct-zol RNA MiniPrep Kit (Zymo Research) with the recommended DNase I treatment, according to manufacturer's instructions. Cappable-seq was carried out as described elsewhere (41) , with the following modifications. Approximately 10 μg of clean RNA was used for each assay. The very first RNA clean-up step was performed using Agencourt® RNAClean® XP Beads (Beckman) to ensure maximum elimination of unincorporated DTB-GTP. The removal of 3' phosphates from fragmented RNA was performed using the Thermo Scientific™ T4 Polynucleotide Kinase (Thermo Fisher Scientific) and its supplied ATP-free buffer. For RNA-seq samples, 800 ng of total RNA was fragmented in 5X RNA Fragmentation Buffer (200 mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM MgOA) by incubating at 95°C for 7 minutes and quenching immediately on ice. RNA was then purified using the RNA Clean & Concentrator-5 Kit (Zymo Research), according to manufacturer's instructions. The quality and concentration of RNA before and after fragmentation were evaluated using a 2100 Bioanalyzer instrument (Agilent Technologies). Replicates are detailed in Supplementary Table S2.
6
Quantifying m6A RNA Modifications
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Real-time qPCR was performed to assess the relative abundance of the selected mRNA in m6A antibody IP samples and input samples. Briefly, total RNA was isolated with an RNeasy mini kit. With 500 ng RNA kept as an input sample, the remaining RNA was used for m6A-immunoprecipitation. Hundred microgram of RNA was diluted into 500 μl IP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris, pH 7.4, 100 U RNase inhibitor) and incubated with m6A antibody (Synaptic Systems, Goettingen, Germany). The mixture was rotated at 4 °C for 2 h, then Dynabeads® Protein A (Thermo Fisher Scientific, Waltham, MA) coated with BSA was added into the solution and rotated for an additional 2 h at 4 °C. After washing by IP buffer with RNase inhibitors four times, the m6A IP portion was eluted with elution buffer (5 mM Tris-HCL pH 7.5, 1 mM EDTA pH 8.0, 0.05% SDS, and 4.2 μl Proteinase K (20 mg/ml)18 (link). The final eluted mRNA was concentrated with an RNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA). The same amount of the concentrated IP RNA or input RNA from each sample was used for the cDNA library. The mRNA expression was determined by the number of amplification cycles (Cq). The relative m6A levels in genes were calculated by the m6A levels (m6A IP) normalized using the expression of each gene (Input).
7
Cytokine and Chemokine Expression in MIO-M1 Cells Treated with SLP
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Expression of proinflammatory cytokines (IL-6, TNF-α) and chemokines (CCL2, CXCL-1) from MIO-M1 cells treated with SLP were measured by real-time quantitative PCR. MIO-M1 cells were seeded (2 × 106 cells/well) into a 6-well plate and treated with 10 μg/mL SLP from WT B. thuringiensis, 10 μg/mL SLP extract control (SLP fraction from ΔslpA B. thuringiensis), or PBS for 10 hours. Untreated cells were the negative control. Total RNA was isolated using a QIAGEN RNeasy Mini Kit (QIAGEN, Hilden, Germany). DNA was removed by using a TURBO DNA-free Kit (Invitrogen, Carlsbad, CA, USA). Using an RNA Clean & Concentrator-5 Kit (Zymo Research, Irvine, CA, USA), 50 μL RNA was purified. The concentration and purity were confirmed on a Nanodrop. All procedures were performed following the manufacturer's instructions. Real-time quantitative PCR was performed with the Applied Biosystems 7500, using an iTaq Universal SYBR Green One-Step Kit (Bio-Rad, Hercules, CA, USA) and specific primers (Table ) following the manufacturer's instructions.75 (link) PCR amplification was performed in triplicate and water was used to replace RNA in each run as negative control. Relative gene expression was determined using ΔCT method using GAPDH as a reference housekeeping gene.75 (link)
8
RNA-Seq Library Preparation and Sequencing
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Total RNA was depleted of ribosomal RNA using the Ribo-Zero rRNA Removal Kit (Illumina). rRNA-depleted RNA was size selected (>200 nucleotides) to remove 5S rRNA and tRNA using RNA Clean & Concentrator-5 Kit (Zymo Research). Sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). All cDNA and PCR products were purified with AMPure XP beads. Samples were sequenced on an Illumina NextSeq 500 (High Output Kit, single-end, 75 cycles).
9
Cfo-Nos Riboprobe Synthesis and WMISH
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DIG-labelled riboprobes for Cfo-Nos with 761 bp length were generated. Linearized DNA template (amplified from plasmid DNA with T7/SP6 primers) was used to synthesize sense and antisense DIG-labelled riboprobes with T7 and SP6 RNA polymerases (Life Technologies, Carlsbad, CA, USA), respectively. Riboprobes were purified with RNA Clean & Concentrator-5 kit (ZymoResearch, Irvine, CA, USA), and their concentrations were measured on a NanoDrop ND-1000 Spectrophotometer. WMISH was performed as previously described by Perry and colleagues [26 (link)], and specimens were stored at 4 °C in 80% glycerol/20% 1× PBS, until imaged.
10
Sequence-Independent Viral RNA Extraction
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Approximately 140 μL of VTM was passed through a 0.22μm filter (Dot Scientific, Burton, MI, USA). Total nucleic acid was extracted using the Qiagen QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), substituting carrier RNA with linear polyacrylamide (Invitrogen, Carlsbad, CA, USA) and eluting in 30 μL of nuclease free H2O. The sample was treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 30 min and concentrated to 8μL using the RNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA). The full protocol for nucleic acid extraction and subsequent cDNA generation is available at https://www.protocols.io/view/sequence-independent-single-primer-amplification-o-bckxiuxn .
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