RNA immunoprecipitation was performed with cytoplasmic extracts. Briefly, K562 cells or freshly isolated bone marrow mononuclear cells derived from BCR/ABL+ mice were lysed in ice cold lysis buffer (20 mM Tris [pH7.5], 150 mM NaCl, 1% Triton X-100 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate) supplemented with Protease Inhibitor Cocktail (Roche) and 40 U/ml Protector RNase Inhibitors (Roche) for 20 min. The lysates were then centrifuged for 15 min at 12,000g and supernatant was collected. About 1 mg protein extract was incubated with 10 μg mouse anti-HNRNPA1 monoclonal antibody (9H10, Abcam) or 10 μg mouse immunoglobulin G [IgG] (Abcam) for 12 hr at 4 oC at a vertical shaking table. After that, 30 μl protein A sepharose (Abcam) was added for another 2 hr, followed by three washes with ice cold lysis buffer. Co-immunoprecipitated RNA was extracted using Tripure Isolation Reagent (Roche). miRNA qPCR analysis was performed with SYBR green mix (Roche) after reverse transcription using miRcute kit (Tiangen). The miRNA fold enrichment in immunoprecipitated samples was expressed as percentage against input and compared to IgG control.
Protector rnase inhibitor
Manufactured by Roche
Sourced in Germany, United States, Switzerland, Denmark
The Protector RNase Inhibitor is a laboratory product designed to protect RNA from degradation by RNase enzymes. It functions by inhibiting the activity of RNase enzymes, thereby preserving the integrity of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Transcriptor reverse transcriptase, by Roche (13 mentions)
Lightcycler 480, by Roche (5 mentions)
Dntps, by Roche (5 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (5 mentions)
Rneasy plant mini kit, by Qiagen (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
High pure rna isolation kit, by Roche (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Transcriptor buffer, by Roche (3 mentions)
Klenow enzyme, by Roche (3 mentions)
Salmon sperm dna, by Roche (3 mentions)
Platinum sybr green qpcr udg mix, by Thermo Fisher Scientific (3 mentions)
Oligo dt 15 primer, by Roche (3 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (3 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Random hexamer, by Thermo Fisher Scientific (2 mentions)
65 protocols using protector rnase inhibitor
1
RNA Immunoprecipitation Analysis of miRNA
2
Biotinylated miRNA Interactome Profiling
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Biotinylated miRNA pull-down was performed with cytoplasmic extracts. Briefly, 20 nmol biotinylated single stand miRNA oligonucleotides (GenePharma, Shanghai, China) were re-suspended in 100 μL lysis buffer supplemented with Protease Inhibitor Cocktail (Roche) and 40 U/ml Protector RNase Inhibitors (Roche) and incubated with 400 μL protein extract (about 1mg) for 4 hr at 4 oC on a vertical shaking table. Streptavidin sepharose beads (Cell Signaling Technology), pre-washed three times with lysis buffer, were added to the mixture and incubated for an additional 2 hr at 4 oC. The beads were then washed three times with 1 mL lysis buffer each. Beads were mixed with protein loading buffer and heated at 95 oC for 10 min to allow collection of bound proteins for western blot or mass spectrometry (MS) analysis.
3
CRISPR-Mediated Gene Editing in LCLs
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FA-55 LCL were washed once in PBS and centrifuged at 200× g for 10 min. Per condition, 1e6 LCL were resuspended in 16 μL of Amaxa SE Nucleofector Solution (Lonza, Basel, Switzerland, #PBC1-00675) and combined with either 1 μg of B2M Ex.1 SD or FANCA Ex.4 sgRNAs (Synthego, Menlo Park, CA, USA), 1.5 μg of ABE7.10max mRNA (TriLink Biotechnologies, San Diego, CA, USA) or 1.5 μg of ABE8e mRNA (TriLink Biotechnologies, San Diego, CA, USA), and 0.1 μg of eGFP mRNA (TriLink Biotechnologies, San Diego, CA, USA, #L-7201-1000), and 1.5 μL of Protector RNase Inhibitor (1:50, 40 U/μL, Roche, Basel, Switzerland, #03335399001). The final 20 μL reaction suspension was incubated at RT for 5 min and then added to 20 μL P3 Amaxa Nucleocuvettes (Lonza, Basel, Switzerland, #PBC1-00675) and electorporated via protocol EO-117 in an Amaxa 4D-Nucleofector (Lonza, Basel, Switzerland). LCLs were allowed to recover in antibiotic-free LCL medium under normoxic conditions (21% O2, 5% CO2, 37 °C) for 20 min, followed by culturing in complete LCL medium for 5 days under normoxic conditions (21% O2, 5% CO2, 37 °C).
4
IFN-α Gene Expression Analysis by qRT-PCR
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IFN-α gene expression was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) of five IFN-α-stimulated-genes: ADAR, ISG15, IFIT1, Mx1 and OAS1 [31 (link)]. Total RNA was extracted from peripheral blood mononuclear cells (obtained after Ficoll preparation and storage with Protector RNAse inhibitor (Roche applied biosciences), using the High pure RNA isolation kit (Roche applied biosciences). RNA extraction quality control was measured with the Agilent 2100 bioanalyzer in combination with Agilent RNA 6000 Nano kit (RNA sample quality was considered if the obtained RIN was >7). A total of 300 ng of RNA was reverse transcribed into cDNA using the Transcriptor Reverse Transcriptase kit (Roche applied biosciences). PCR was performed (RT2 SYBR Green qPCR MasterMix, SA Biosciences) in 96-wells plates using LightCycler 480 (Roche Diagnostics) to determine the expression levels of ADAR, ISG15, IFIT1, Mx1 and OAS1 target genes. IFN-α gene expression measurements were expressed as fold change which is calculated as a ratio of averages from control and test sample values.
5
Transcriptional Analysis of retS Mutant
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Overnight PAK and PAK ΔretS cultures were subcultured in LB medium with a starting OD600 of 0.1 and incubated at 37°C with shaking for 6 h. Cells were then harvested into RNAlater stabilization solution (Ambion), and RNA was extracted using an RNeasy extraction kit (Qiagen). To remove DNA, a Turbo DNA-free kit (Applied Biosystems) was used, and the RNA was repurified using an RNeasy kit, following the supplier's indications. cDNA was synthesized from 200 ng of RNA template by adding 20 U of Protector RNase inhibitor from Roche, 10 pmol of Pd(N)6 random hexamer oligonucleotides from Amersham, and 10 pmol deoxynucleoside triphosphates from Bioline to the reaction mix. Quantitative real-time reverse transcription-PCR (qRT-PCR) was performed on an ABI 7300 real-time PCR system using ABI SYBR green PCR master mix.
6
Total RNA Isolation and Real-Time qRT-PCR
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Total RNA from cultured cells was isolated using TRI reagent solution (Ambion, TX, USA) and purified with a Nucleospin RNA cleanup kit (Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. DNase treatment was performed separately using RQ1 DNase (Promega, WI, USA) in the presence of Protector RNase inhibitor (Roche, Basel, Switzerland). First strand cDNA was synthesized using up to 2 μg of total RNA and the High Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA, USA). Gene expression was analyzed by real-time qRT-PCR in a StepOnePlus Real-Time PCR System (Applied Biosystems) using FastStart Universal SYBR Green Master (ROX) (Roche). All protocols were performed according to the manufacturer's instructions. Primers sequences and conditions were described before53 (link) and are indicated in the Supplementary Table 1 . mRNA expression levels were calculated according to the formula: relative expression of gene= 2^(Ct internal reference-Ct gene), using 18S mRNA expression as internal reference.
7
NASBA-Based RNA Amplification and Detection
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For target RNA amplification with NASBA, 2.01 μL 3X reaction buffer (Life Sciences, NEC-1-24;), 0.99 μL 6X nucleotide mix (Life Sciences, NEC-1-24), 0.03 μL Protector RNase Inhibitor (Roche), 0.12 μL of each NASBA primer (12.5 mM, IDT), 0.15 μL nuclease-free water (Thermo Fisher, 10977015), and 1.2 μL target RNA were mixed at 4°C and heated to 65°C for 2 min, followed by a 10-minute incubation at 41°C. 1.5 μL Enzyme Mix (Life Sciences, NEC-1-24) was then added to the reaction for a final volume of 6 μL. After mixing, the reaction was incubated at 41°C for 2 hours. For a 35 μL two-pot reaction, 5 μL of the NASBA amplified RNA product was combined with 0.7 μL of RNA aptaswitch and 10X DFHBI-1T dye mix. This reaction was incubated and measured at 37 °C for an additional 2 hr. The 10X DFHBI-1T dye mix consists of 40 μM DFHBI-1T (Lucerna, 410), 40 mM HEPES (Gibco™, 15630080), pH 7.4, 100 mM KCl (Invitrogen™, AM9640G), and 5 mM MgCl2 (Invitrogen™, AM9530G).
8
Isolation and Analysis of Total RNA from S. oneidensis
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Total RNA was isolated from a 0.5 ml late-exponential phase culture (OD600nm = 0.9) of WT S. oneidensis MR-1 using the RNeasy Plus Mini kit in combination with the RNAprotect Bacteria Reagent (Qiagen) (yield: 33 μg total RNA). One microgram of total RNA sample was treated with RNase-free DNase I and Protector RNase Inhibitor (Roche). First strand cDNA was synthesized using random hexamer primers and the Transcriptor First Strand cDNA Synthesis Kit (Roche). Target transcripts were PCR amplified using HotGoldStar polymerase (Eurogentech) (100 ng DNA template; 4 μM primers). PCR products were resolved on an agarose gel.
9
Real-Time qPCR for Inflammation and Cancer
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Total RNA was treated with DNase (TURBO DNA-free, Life Technologies) according to the instructions of the supplier. The treated RNA was then analysed on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA) and only used if the RNA integrity number was above 7. Reverse transcription was carried out using 250 ng of RNA, Transcriptor Reverse Transcriptase, primer ‘random’, PCR nucleotide mix and Protector RNase inhibitor (all Roche) as per instructions of the manufacturer. Real-time quantitative PCR (RT-qPCR) was carried out on a LightCycler 480 (Roche) targeting several genes published as being putatively involved in inflammation and cancer progression using primers and probes designed with the Universal ProbeLibrary Assay Design Center (Roche). For primer sequences, see online supplementary table S2. The master mix comprised 5 µL SensiMix II Probe No-ROX mix (BIOLINE, London, UK), 2 µM forward and reverse primer, 2.5 µL DNA and water to 10 µL. The program employed was 95°C for 10 min, 95°C for 10 , 60°C for 45 s, 72°C for 1 s, with the last three steps repeated 45 times. The relative expression of each gene was calculated using the 2−ΔΔCt method.23 (link) The expression was normalised to the average expression of all control individuals. The housekeeping gene used was β-actin.
10
RNA Quantification by RNase Protection
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RNase protection assay was performed according to Perche et al.78 (link) Samples containing 2 μg mRNA were incubated with 5 U of RNase A/T1 Mix (Thermo scientific) for 2 h at 37°C. The RNase was then inactivated with Protector RNase Inhibitor (Roche) before complex dissociation using sulfated dextran (10% of final volume). Then, samples were analyzed on a 1% agarose-formaldehyde gel containing ethidium bromide. Gels were imaged using a Gene Flash imager (Syngene, Cambridge, UK).
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