After formulation, LNPs were separated by following storage conditions: time 0 (immediately after formulation), 1 h at RT, 1 day at RT, 7 days at RT, 7 days 4 °C, 7 days at −80 °C, 7 days at −20 °C and 14 days RT. For each set of conditions, LNP samples were tested for size, polydispersity index (PDI) and encapsulation efficiency (EE). Hydrodynamic diameter and PDI were measured using Dynamic Light Scattering (DLS) particle size analyzer (Anton Paar Particle Analyzer Litesizer 500) at a 90° scattering angle. RNA encapsulation efficiency was measured using the Invitrogen Quant-iT RiboGreen RNA Assay Kit (#R11490) according to manufacturer’s protocol.
Quant it ribogreen rna assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Australia, Switzerland, France, Germany
The Quant-iT RiboGreen RNA Assay Kit is a fluorescent nucleic acid stain used for the quantitation of RNA in solution. The kit provides a sensitive and accurate method for measuring RNA concentration.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
2100 bioanalyzer, by Agilent Technologies (13 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (10 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (9 mentions)
Bioanalyzer, by Agilent Technologies (8 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (7 mentions)
Rneasy plus mini kit, by Qiagen (6 mentions)
Rna 6000 nano kit, by Agilent Technologies (6 mentions)
Mirneasy mini kit, by Qiagen (5 mentions)
Tri reagent, by Merck Group (5 mentions)
Zetasizer nano z, by Malvern Panalytical (5 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (5 mentions)
Rneasy micro kit, by Qiagen (5 mentions)
Proteinase k, by Qiagen (4 mentions)
Rna 6000 pico kit, by Agilent Technologies (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (4 mentions)
2100 bioanalyser, by Agilent Technologies (4 mentions)
Allprep dna rna protein mini kit, by Qiagen (4 mentions)
234 protocols using quant it ribogreen rna assay kit
1
Lipid Nanoparticle Stability Evaluation
2
Lipid Nanoparticle Encapsulation of circRNAs
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The circRNAs were encapsulated with lipid nanoparticles (LNPs) according to a previously described process (Ickenstein and Garidel, 2019 (link)). First, the circRNA was diluted with PNI Formulation Buffer (Precision NanoSystems, #NWW0043) to a final concentration of 170 μg/ml. Then, the lab-prepared or commercial LNP (Precision NanoSystems) were mixed with the circRNA solution at the volume ratio of 1:3 through the Ignite NxGen Cartridge (Precision NanoSystems, #NIT0002) using NanoAssemblr Ignite (Precision NanoSystems). Then the LNP-circRNA formulations were diluted 40-fold with 1×PBS buffer (pH 7.2∼7.4) and concentrated by ultrafiltration with Amicon® Ultra Centrifugal Filter Unit (Millipore). The concentration and encapsulation rate of circRNAs were measured by the Quant-it RiboGreen RNA Assay Kit (Invitrogen, #R11490). The size of LNP-circRNA particles was measured using dynamic light scattering on a Malvern Zetasizer Nano-ZS 300 (Malvern). Samples were irradiated with a red laser, and scattered light was detected. The results were analyzed to obtain an autocorrelation function using the software Zetasizer V7.13.
3
RNA-Seq Analysis of FGF2, Fulvestrant, and Palbociclib Treatments
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CAMA1 cells were plated in full media ± 2 ng/mL FGF2 (Sigma) and then treated with DMSO or 1 μM fulvestrant plus 1 μM palbociclib ± 1 μM lucitanib for 6 h. Cells were harvested and RNA was purified using a RNA purification kit (Maxwell, Promega). Total RNA was quantified using the Quant-iT Ribo-Green RNA Assay Kit (Invitrogen) and normalized to 4 ng/mL; 200 ng of each sample were used for library preparation in an automated variant of the Illumina Tru Seq RNA Sample Preparation protocol (Revision A, 2010). This method uses oligo(dT) beads to select mRNA from the total RNA sample and is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant cDNA went through library preparation (end repair, base “A” addition, adapter ligation, and enrichment) using Broad Institute–designed indexed adapters for multiplexing. After enrichment, libraries were quantitated with qPCR using the KAPA LibraryQuantification Kit for Illumina Sequencing Platforms and pooled equimolarly. The entire process was performed in a 96-well format with all pipetting done by either the Agilent Bravo or PerkinElmer JANUS Mini liquid handlers.
4
Transcriptomic Analysis of Mouse Skeletal Muscle
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Snap frozen gastrocnemius muscles of male C57BL/6JRj mice were pulverized and lysed in RLT buffer (Qiagen) and then treated with proteinase K (Qiagen). DNAse treatment and RNA extraction was performed with an automated iColumn 24 (AccuBioMed) using an AccuPure Tissue RNA Mini Kit (AccuBioMed). RNA purity and integrity were examined on a Bioanalyzer (Agilent), while RNA concentration was determined using a Quant-iT™ RiboGreen™ RNA assay kit and Qubit fluorometer (Invitrogen). Libraries were prepared with TruSeq Stranded mRNA HT Sample Prep Kit. Stranded, paired-end sequencing with 101 base pair read length was performed on an Illumina HiSeq2500 platform.
5
RNA Extraction and cDNA Synthesis Protocol
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Swimbladder and rete mirabile tissue was homogenized in Precellys tubes CKMix (No. KT03961-1-009.2), with a Precellys 24 homogenizer (Bertin Technologies, France) (2 × 30 s at 6000 rpm, 45 s break). Total RNA was isolated using the Qiagen miRNeasy® Mini Kit (No. 217004; Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Quality of extracted RNA was assessed by electrophoresis on a 1.5% agarose-gel, confirming the integrity of 18S and 28S RNA-bands. RNA concentration was determined using the Quant-iT™ RiboGreen® RNA Assay Kit (No. R11490; Invitrogen by Thermo Fisher Scientific Inc., Waltham, MA, USA) and a VICTOR™ X4 Multilabel Plate Reader (PerkinElmer, Inc., Waltham, MA, USA). A total of 450 ng RNA was applied for first strand cDNA synthesis. The RNA was preincubated with 2.5 μl Random Hexamer Primer (No. SO142, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 70 °C for 5 min before the RT reaction was set up using RiboLock™ RNase Inhibitor, 10 mM dNTP Mix, and RevertAid H Minus Reverse Transcriptase in 50 μl approaches (No. EO0381, No. R0191, and No. EP0451, respectively, Thermo Fisher Scientific Inc., Waltham, MA, USA). For each tissue sample, a cDNA synthesis without reverse transcriptase (noRT) was performed to detect DNA contaminations in the final qPCR reaction.
6
Femur RNA Extraction and Gene Expression
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The bone marrow and the cortical fraction of left femur were separated by flushing with a syringe and then immediately frozen in liquid nitrogen. Total RNA was extracted by TRI Reagent (Sigma, St Louis, MO, USA) and then purified with a Qiagen pure tissue kit (RNeasy Plus Mini Kit, Qiagen, Valencia, CA, USA). The RNA quality was assessed by Experion automated electrophoresis station (Bio‐Rad, Hercules, CA, USA), followed by Ribogreen assay (Quant‐iT RiboGreen RNA Assay Kit, Invitrogen, Life Technologies, Eugene, OR, USA) for accurate RNA quantification. Total RNA reverse transcription was performed with iScript cDNA synthesis kit (Bio‐Rad). Quantitative real‐time (qRT) polymerase chain reaction (PCR) was conducted on CFX96 RealTime System (Bio‐Rad) with LightCycler FastStart DNA Master plus SYBRgreen I (Roche Diagnostics, Basel, Switzerland). The mRNA expression levels were normalized to the housekeeping Hprt1 gene expression using the ∆Ct method.(23 ) Primer sequences are shown in Supplemental Table S3 .
7
Plant Total RNA Extraction Protocol
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All samples were homogenised using mortars with liquid nitrogen. Then, RNA extraction was performed using 100 mg of ground tissue with the FavorPrep Plant Total RNA Purification Mini Kit (Favorgen, Biotech Corp, Ping-Tung, Taiwan), according the manufacturer’s instructions. RNA purity was determined through electrophoresis gel and spectrophometrically by 260/280 ratio, using a nanoplate within the microplate reader. RNA quantification was carried out by fluorescence using the Quant-iT RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA) in a QFX Fluorometer (DeNovix, Wilmington, DE, USA).
8
Nucleic Acid Extraction from Cultured Cells and Tissues
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Cultured cells were harvested by trypsin-EDTA solution and washed. Breast cancer tissue samples were grounded to powder by mortar and pestle under liquid nitrogen. Nucleic acids and protein were isolated using Allprep DNA/RNA/protein Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Nucleic acids were quantified using Quanti-iTTM PicoGreenTM dsDNA Assay Kit (InvitrogenTM, Carlsbad, CA, USA) and Quant-iT RiboGreen RNA Assay Kit (Invitrogen) in Infinite M200 microplate reader (Tecan Group Ltd., Männendorf, Switzerland). RNA integrity was checked by Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Assay Kit (Agilent Technologies, Santa Clara, Inc., CA, USA).
9
Immunoprecipitation and RNA Extraction
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ARC-enriched microdissections were ice-cold homogenized in 0.25 ml homogenization buffer (50 mM Tris, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/mL Promega RNasin, 1 mg/mL heparin, 100 μg/mL cycloheximide, Sigma protease inhibitor mixture at pH 7.5). After clearing, 20 μL was separated as input and stored at -80°C until further processing. 2 μl of mouse monoclonal anti-HA antibody (HA.11, ascites fluid; Covance) was added to the remaining homogenate and allowed to rotate for 2 h at 4°C. After incubation, 200 μl of Dynabeads protein G magnetic beads (Invitrogen) was added and incubated for 2h at 4°C with rotation. Immunoprecipitates (IPs) were washed 3 times for 10 min with gentle rotation at 4°C in high-salt buffer (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 100 μg/mL cycloheximide at pH 7.5). After final wash, samples were placed in a magnet on ice and Qiagen RLT buffer was added to the remaining pellets and input samples. Total RNA was prepared according to manufacturer’s instructions using the RNeasy-plus Mini kit (Qiagen) and quantified with the Quant-iT RiboGreen RNA assay kit (Invitrogen). RNA integrity was assessed on a 2100 Bioanalyzer device (Agilent Technologies) using the RNA 6000 Pico kit (Agilent Technologies).
10
RNA Extraction and Quality Control for Microarray
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Total RNA was extracted from six samples [three F1 (SJL × B6wt) and three F1 (SJL × B6Tlr2−) mice] in total using TRIZOL® reagent (Life Technologies Inc., Carlsbad, CA, USA) and then purified with RNeasy Micro columns (Qiagen, Venlo, Netherlands), as described by the manufacturers. The quality of all samples was strictly controlled to verify the RNA integrity before use in microarray experiments. RNA quantity and purity were evaluated spectrophotometrically by Nanodrop 1000 (Thermo Scientific, Waltham, MA, USA) and then by Quant-iT™ Ribogreen® RNA Assay kit (Invitrogen, Waltham, MA, USA), while the quality was assessed by the Agilent 2100 bioanalyzer RNA Pico 6000 kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Only samples with good RNA yield and no RNA degradation (28S:18S >1.7 and RNA integrity number >6) were retained for further experiments.
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