DsrA sRNA was 5' end labeled with γ-[32P]-ATP using T4 polynucleotide kinase (NEB) and purified using a Chroma TE-30 spin column (Takara). Radiolabeled DsrA (2 nM) was incubated with Hfq protein (70 nM) in a 40 μL reaction containing: 50 mM Tris-HCl pH 7.5, 20 mM MgCl2, 100 mM NaCl, 100 mM KCl, 40 μM NTPs (GTP, ATP, UTP, CTP), 2 U RNasin Plus, 20% glycerol (v/v). Unlabeled rpoS301 TECs stalled in absence of CTP were assembled in 40 μL (total volume) containing 25 nM DNA template, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 25 nM T7 RNAP, 2 μM GTP, 2 μM ATP, 0.5 μM UTP, 1 U/μL RNasin Plus (Promega). Stalled TECs were incubated at RT for 5 minutes and heparin was added to a final concentration of 1 mg/mL to inhibit reinitiation. Hfq-DsrA binding was monitored during transcription by adding the Hfq-DsrA-NTP restart mixture to the stalled TECs in a 1:1 ratio. In the control lanes, 2 nM radiolabeled DsrA was incubated in annealing buffer 50 mM Tris-HCl pH 7.5, 20 mM MgCl2, 100 mM NaCl, 100 mM KCl, 1 U/μL RNasin Plus (Promega) with 25 nM rpoS RNA and/or 35 nM Hfq as indicated. Aliquots were taken out and immediately loaded onto a pre-run 8% native polyacrylamide gel at 15W for 1.5 hr at 4°C in 1X TBE. Gels were dried, exposed overnight to a phosphorescence storage screen, and imaged (GE Typhoon).
Rnasin plus
Manufactured by Promega
Sourced in United States, Germany, United Kingdom
RNasin Plus is a ribonuclease (RNase) inhibitor that protects RNA from degradation during in vitro experiments. It functions by binding and inhibiting RNase activity, thereby preserving the integrity of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Mirvana kit, by Thermo Fisher Scientific (4 mentions)
M mlv reverse transcriptase, by Promega (4 mentions)
M mulv reverse transcriptase, by New England Biolabs (4 mentions)
Dithiothreitol (dtt), by Merck Group (4 mentions)
First strand buffer, by Thermo Fisher Scientific (4 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Rnase free bsa, by Gemini Bio (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Oligo dt primer, by Roche (2 mentions)
Cfx96 system, by Bio-Rad (2 mentions)
Typhoon fla 7000 phosphoimager, by GE Healthcare (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
129 protocols using rnasin plus
1
Monitoring Hfq-DsrA Binding During Transcription
2
Nuclei Isolation from Cryopreserved Tissue
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Upon thawing, tissue was immediately incubated in 1% PFA (with 1 µL mL−1 RNasin Plus (Promega, cat. #N2611)) for 5 min at 4°C. Nuclei were prepared by Dounce homogenizing in Homogenization Buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris buffer, pH 8.0, 1 µM DTT, 1x Protease Inhibitor (Promega, cat. #6521), Hoechst 33342 10 µg mL−1 (Thermo Fisher Scientific, cat. #H3570), 0.1% Triton X-100, 1 µL mL−1 RNasin Plus). Sample was then overlaid on top of a 20% sucrose solution (25 mM KCl, 5 mM MgCl2, 10 mM Tris buffer, pH 8.0) and spun at 500 xg for 12 min at 4°C.
3
3D-SIM Microscopy of Fixed Cells
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Cells were grown on 18 × 18 mm No. 1.5H precision cover slips (Marienfield) for 3D-SIM microscopy and treated as described above. Coverslips were washed twice with PBS and fixed using 3% formaldehyde (pH 7) in PBS for 10 min at room temperature. A stepwise exchange of PBST.5 was carried out, before cells were permeabilized using 0.2% Triton X-100 PBS for 10 min at room temperature, then blocked for 30 min (2% BSA/0.5% fish skin gelatine/PBST.5, 2U/μL RNAsin Plus (Promega) at room temperature). Coverslips were washed twice in PBST.5, then once in 2× SSC before an overnight incubation at 37°C with FISH probe/hybridization buffer (prepared as above) in a humid chamber. The following day coverslips were washed with 2× SSC as detailed above, then stained with 2 μg/mL DAPI in PBST.5 for 10 min at room temperature. Coverslips were washed again with PBS, then milliQ water, before mounting as above but using Vectashield mounting medium (no DAPI), and imaged within a week using the DeltaVision OMX V3 Blaze system (GE Healthcare).
4
Radiolabeled RNA Synthesis Protocol
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Reagents were sourced from the following: DNA primers from Integrated DNA Technologies, NTPs from New England Biolabs, CpA from TriLink, RNasin Plus from Promega, Streptavidin-coated M280 Dyna Beads from Invitrogen, and 800 Ci/mmol [α-32P]CTP from PerkinElmer Life Science and MP Biomedicals.
5
Nuclear and Cytoplasmic RNA Isolation
6
Cell Washing and Resuspension Protocol
7
miRNA-939 Transfection and LPS Treatment
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Transfections were performed following the manufacturer’s protocol for RNAiMax transfection reagent using either miR-939-5p mimic (MC 12517) or negative control mimic (4464058) (Life Technologies) with the following modifications. For each well of a 6-well plate, 7.5 μl RNAiMax reagent was diluted in 150 μl of serum-free media, and 30 pmol of miR-939 or control mimic was diluted in 150 μl of serum-free media individually. The dilutions were combined and incubated at room temperature for 15 minutes. This transfection complex (300 μl) was added to 0.5 × 106 cells/well in 1.7 ml serum containing media in 6-well plates and incubated for 6 hours at 37 °C, after which the media was changed. After 24 hours, cells were treated with 1 μg/ml lipopolysaccharide (LPS) in complete culture media. Cells were collected by centrifugation at 135 × g for 5 minutes at 4 °C and the conditioned media was stored at 4 °C. The cell pellet was washed with 1× phosphate-buffered saline and resuspended in either RNA lysis buffer (mirVana kit; Life Technologies) containing 0.5 U/μl RNAsin Plus (Promega; Madison, WI) or radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Thermo Scientific; Waltham, MA).
8
Cytoplasmic and Nuclear RNA Extraction
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Ten million cells were lysed in 200 μl of hypotonic lysis buffer (10 mM Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 1× protease inhibitor cocktail (Sigma Aldrich), and 300 U/ml RNasin Plus (Promega Corp.)) for 6 min on ice. Lysate was centrifuged at 1300 g for 5 min to separate the nuclei from the crude cytoplasmic fraction. The supernatant was re-centrifuged 5 min at 20 000 g, and the purified cytoplasmic fraction was transferred to a new eppendorf tube before three volumes of TRIzol® was added for cytoplasmic RNA extraction. Pelleted nuclei were washed once with lysis buffer and 300 μl of TRIzol® was added for RNA extraction. RNA pellets were resuspended in RNase-free water and quantified by NanoDrop™. Following DNaseI treatment, 1 μg of RNA from each fraction was used to generate cDNA using random hexamer priming and the reverse transcriptase SuperScript® III (Thermo Fisher Scientific). The amount of transcripts present in either the cytoplasm or the nucleus is shown as a percentage of the total quantity of RNA extracted from each fraction.
9
Detecting Ricin-Induced Truncated cDNA
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Example 1
Detection of Ricin-Induced Truncated cDNA Following Incubation of Different Amounts of Ricin in Reticulocyte Lysate, Using a Common Reverse Transcriptase.
Purified ricin [Gal et al., 2014, Toxicol. Rep. 1:495-504] was added to reticulocyte lysates at final concentrations of 0.5-50 ng/ml and incubated at 37° C. for 2 hours. Following tagging and reverse transcription with the reverse transcriptase MLV RT, the RT products were amplified and analyzed by qRT-PCR [For performing the RT reaction, the following components were added to the PCR tube: 2 μl of dNTPs (5 mM), 1 μl of DTT (100 mM), 1 μl of RNasin plus (Promega), 2 μl of BSA (10 mg/ml), 2 μl of MLV buffer (×10) and 1.5 μl of RT-MLV enzyme. The PCR tube was introduced into a PCR thermal cycler device with the following program: 45 min at 40° C., 2 min at 4° C. No DMSO was used in the denaturation and annealing step that precedes the RT reaction]. As shown in
10
Transfection of miR-939-5p Mimics
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Transfections were performed following the manufacturer’s protocol for RNAiMax transfection reagent (Life Technologies) using either miR-939-5p mimic (MC 12517), custom synthesized EXOmut-miR-939 mimic (Assay ID CTMFW3V, Thermofisher Scientific), or negative control mimic (4464058) (Life Technologies) with the following modifications. For each well of a 6-well plate, 7.5 µl RNAiMax reagent was diluted in 150 µl of serum-free media, and 30 pmol of miR-939 or control mimic was diluted in 150 µl of serum-free media individually. The dilutions were combined and incubated at room temperature for 15 min. This transfection complex (300 µl) was added to 0.5 × 106 cells/well in 1.7 ml serum containing media in 6-well plates and incubated for 6 h at 37°C, after which the media was replaced with complete RPMI and incubated for 24 h. Cells were collected by centrifugation at 135 × g for 5 min at 4°C. The cell pellet was washed with 1× phosphate-buffered saline and resuspended in RNA lysis buffer (mirVana kit; Life Technologies) containing 0.5 U/µl RNAsin Plus (Promega; Madison, WI).
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