Approximately 30mg of tissue were homogenized (OMNI-TH homogenizer with dispersible tips) and total RNA was extracted thereof using the RNA Blood Mini Kit from Qiagen (Hilden, Germany) according to the manufacturer's instructions. DNase treatment was included. RNA concentration and quality were determined by spectrophotometry (NanoDrop ND-1000) and gel electrophoresis. One microgram of total RNA was reverse transcribed in a 20μl reaction comprising 20pmol CDS-primer (5′-Tn=30VN-3′), 100nM of each dNTP, 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl2 10mM DTT, 20U RNasin und 200U SuperScriptII (GibcoBRL). To allow optimal annealing, primer and RNA were incubated at 70°C for 10 min and cooled down on ice. The remaining reagents were then added in form of a master mix and cDNA synthesis was done at 42°C for one hour. The reaction was stopped at 70°C for 15 minutes and stored at −80°C until PCR analyses.
Rnasin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
RNasin is a ribonuclease (RNase) inhibitor that helps protect RNA from degradation during laboratory procedures. It is a recombinant protein that binds to and inhibits the activity of RNase enzymes, thereby preserving the integrity of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Mgcl2, by Thermo Fisher Scientific (7 mentions)
Cycloheximide (chx), by Merck Group (6 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (5 mentions)
Rnasin, by Promega (4 mentions)
Moloney murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Rneasy minelute cleanup kit, by Qiagen (3 mentions)
Itaq sybr green supermix with rox, by Bio-Rad (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Gradient fractionator, by Brandel Inc (3 mentions)
Ultracentrifuge, by Beckman Coulter (3 mentions)
Ua 5 detector, by Teledyne (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Rt buffer, by Thermo Fisher Scientific (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Random primer, by Thermo Fisher Scientific (2 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
72 protocols using rnasin
1
Total RNA Extraction and cDNA Synthesis
2
RNA to cDNA Reverse Transcription
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1.0 μg RNA was heat-denatured (65°C, 10 min), chilled on ice, and subjected to random hexadeoxynucleotide primed reverse transcription using the first strand cDNA synthesis kit (Pharmacia, Freiburg, Germany). Reverse transcription (final volume 15 μl) was conducted at 37°C for 60 min in the presence of 0.2 μM of random hexanucleotide primer and 40 U RNase inhibitor (RNAsin, Gibco, Germany). Following synthesis of the completed first strand cDNA the resulting RNA-cDNA double-stranded helix was heat-denatured (95°C, 5 min) to provide cDNA as a template for polymerisation.
3
RNA Extraction and RNA-Seq Library Preparation
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RNA was isolated as described earlier, using a Trizol extraction on mycelium ground in liquid nitrogen, followed by DNase treatment and purification over RNeasy RNA purification columns, according to the instructions of the manufacturer (Qiagen).
Synthesis of cDNA was performed using 1 μg of RNA, poly dT primers, Promega RNasin (ribonuclease inhibitor) and Gibco Superscript II RNase H− Reverse transcriptase, according to instructions of Gibco.
Of 2 μg of total RNA of each biological replicate, polyadenylated RNA was amplified and ligated to adapters to make a library suitable for multiplex illumina paired-end sequencing. Each sample was barcoded and sequenced in 8 different lanes. After de-multiplexing, total reads of the different lanes were combined. This rendered one file of reads per biological replicate.
Synthesis of cDNA was performed using 1 μg of RNA, poly dT primers, Promega RNasin (ribonuclease inhibitor) and Gibco Superscript II RNase H− Reverse transcriptase, according to instructions of Gibco.
Of 2 μg of total RNA of each biological replicate, polyadenylated RNA was amplified and ligated to adapters to make a library suitable for multiplex illumina paired-end sequencing. Each sample was barcoded and sequenced in 8 different lanes. After de-multiplexing, total reads of the different lanes were combined. This rendered one file of reads per biological replicate.
4
Quantitative RT-PCR Analysis of Yeast RNA
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RNA was extracted from 10 ml of yeast cells grown as described above using the hot phenol method. After treatment with RQ1 ribonuclease (RNase)–free deoxyribonuclease (Promega) and heat inactivation, a fraction of the RNA (500 ng) was reverse transcribed with M-MLV (Moloney murine leukemia virus) reverse transcriptase (Promega) and oligo d(T)15 primer in the presence of the RNase inhibitor RNasin (80 U/ml; Fermentas) according to the manufacturer’s instructions. Real-time PCR quantification was carried out as described below using 1/50 of the complementary DNA product and primer pairs specific for the indicated genes (see table S3). The values were normalized to those obtained for the 18S ribosomal RNA or a control mRNA, as indicated in the figures to account for differences in total RNA across samples.
5
Quantifying miRNA Expression in Macrophages
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For cDNA preparation, 1 μg total RNA (kept equal for each amplification) was subjected to reverse transcription using 20U M-MLV reverse transcriptase (Fermantas, Germany), 1X RT buffer, 20 mM dNTPs (New England Biolabs, USA), 20U RNasin (Fermentas, Germany), 0.1 M DTT with DEPC treated water, and 100 ng of random hexamers (Fermentas, Germany). The expression levels were quantified on ABI7500 Fast system as per manufacturer instructions (Applied Biosystem) using 5 pmol/μl of specific primers with snoRNA142 being taken as endogenous control. Briefly, 20 μl of real time mix contained 10 μl of Power SYBER green master mix (Applied Biosystem), 1 μl cDNA, 6 μl MilliQ water and 1.5 μl of forward, and reverse primers. PCR conditions were set with an initial incubation of 50°C for 2 min, followed by denaturation at 95°C for 10 min, and 40 cycles at 95°C for 15 s, 60°C for 1 min, and 72°C for 40 s. The abundance/ decline of miRNA were normalized to geometric average of endogenous control snoRNA142 for ΔCt. The fold change (ΔCt) was calculated as the difference between infected groups vs. non-infected RAW 264.7 macrophages. The mRNA expression levels were quantified at 24 h post infection.
6
Single-cell sorting of HA-specific CD8+ T cells
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For the detection of HA533-541-specific CD8+ T cells, splenocytes were stained with a live/dead-aqua (LifeTechnologies), anti-CD8 APC (BD Biosciences), linage markers (anti-CD14 FITC, anti-CD19 FITC, anti-CD335 FITC, anti-F4/80 FITC [BD Biosciences)] and a recombinant H-2Kd-restricted MHC-I pentamer loaded with HA533-541 peptide and bound to PE-labeled streptavidin (50 µg/ml) (Proimmune) targeting the T cell receptor of HA533-541-specific CD8+ T cells (18 (link)). Pentamer positive (pent+) CD8+ T cells from vaccinated mice were single-cell sorted as lineage marker negative (CD14-, CD19-, CD335-, F4/80-), CD8+ and pent+ (S1 Figure
7
RNA Isolation and RT-qPCR Workflow
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Total RNA was prepared using TriReagent (Ambion, Germany) according to the manufacturer´s instructions. Isolated total RNA from cell samples was treated with RNase-free DNase (1 U/μg RNA) (Epicentre Biotechnologies, Madison, WI) and 0.5 μg was used for cDNA synthesis. Reverse transcription (RT) was performed in a 20 μl volume consisting of 4 μl of 10x RT buffer (Invitrogen), 4 μl of 50 mM MgCl2 (Invitrogen), 4 μl of 10 mM dNTP solution (Bioline), 2μl (20 Units) of RNAsin (Applied Biosystems), 2 μl (50 Units) of MMLV reverse transcriptase (Applied Biosystems) and 2 μl hexamers (50 μM) (Applied Biosystems). The samples were incubated at 25°C for 10 minutes for primer annealing and then incubated at 42°C for 1 hour. Finally, the samples were heated to 95°C for 5 minutes. The cDNA was diluted 1:5 and 2 μl (10 ng) were used for PCR amplification. PCR program: activation of the Taq Polymerase for 10 min at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Primer sequences are listed in S1 Table . As control, the housekeeping genes Gapdh or Papola were amplified.
8
Immunoprecipitation and RNA Isolation from Ago2 Complexes
9
Immunoaffinity Purification of FMRP-mRNA Complexes
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FL83B cells or Fmr1-WT and -KO liver were homogenized in IA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1.25 mM MgCl2, 0.5% NP40, 1 mM DTT, 5 U/mL RNasin (Invitrogen)). The lysates were centrifuged at 10,000 g for 15 min at 4 °C. The collected supernatant was then raised to 400 mM NaCl and 30 mM EDTA [37] . For each IA assay, 500 μg of proteins from FL83B cells or liver extract was incubated in the presence of 60 μg of anti-FMRP IgY#C10 antibodies or non-immune IgY immobilized on 15 μL of anti-chicken IgY agarose beads (Gallus Immunotech Inc.). Samples were incubated overnight under rotation at 4 °C and washed 4 times with buffer containing 10 mM Tris pH 7.4, 400 mM NaCl, 10 mM EDTA, 0.5% NP40. 1/5th of each assay was used for immunoblot analyses using anti-FMRP mAb1C3 antibody [36] (link) IA-captured mRNA were then phenol-extracted, precipitated with sodium acetate, and resuspended in water and subjected to reverse transcription (RT) using the SuperScript III system (Invitrogen). RT products were subjected to polymerase chain reaction (PCR), using a PCR Master Kit (Promega) and primers detailed in Table S8 . PCR products were visualized on a 2.5% TAE agarose gel and amplicon size was verified using the BenchTop DNA ladder (Promega).
10
RNA Immunoprecipitation of METTL16 in NIH3T3 Cells
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RNA Immunoprecipitation was performed as previously described with slight modifications12 (link). NIH3T3 cells were transfected with the expression vector for wild-type METTL16, or empty plasmid as a control. After 24 h of incubation, the medium was removed, and cells were lysed in RIP Lysis buffer (20 mM Tris-HCl (pH 7.4), 100 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, 0.2 U/mL RNasin (Promega) and Complete Mini Protease Inhibitor Cocktail). Lysates were aliquoted to obtain input samples. Then cell lysates were mixed with Dynabeads Protein G (Invitrogen) preincubated with anti-FLAG antibody, and incubated at 4 °C for 3 h with gentle rotation. Then the beads were immobilized, and washed five times in RIP Wash buffer (20 mM Tris-HCl (pH 7.4), 150 mM KCl, 2.5 mM MgCl2, 0.2 U/mL RNasin, and Complete Mini Protease Inhibitor Cocktail), and mixed with TRIzol (Invitrogen).
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