Exosomes were isolated from 3 ml urine using the exosome precipitation reagent ExoQuick-TC (System Biosciences; Mountain View, CA). The modified exosome precipitation protocol from Alvarez et al. [34 (link),35 (link)] was used. Samples were centrifuged for 15 min at 3,000 x g and 1/3 volume ExoQuick TC (System Biosciences) was added to the supernatant. After overnight precipitation at 4°C samples were centrifuged for 30 min at 10,000 g at room temperature and the resulting exosome pellet was resuspended with 200μl exosome buffer and subsequently lysed for 5 min with 150μl lysis buffer at RT. Exosomal miRNAs were isolated using SeraMir Exosome RNA Amplification Kit (System Biosciences) according to manufacturer´s protocol. In the independent confirmation cohort urinary exosomal miRNAs were isolated using the exoRNeasy Serum/Plasma Maxi Kit (QIAGEN, Hilden, Germany) according to manufacturer´s instructions. Quality control was performed on the Agilent 2100 Bioanalyzer, using the RNA 6000 Nano Kit (Agilent Technologies Inc, Waldbronn, Germany) and miRNA concentration was measured using Quant-IT micro RNA assay kit (Life Technologies) according to manufacturer´s instructions.
Quant it micro rna assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Quant-IT micro RNA assay kit is a laboratory instrument designed to quantify the concentration of microRNA molecules in a sample. It provides a sensitive and accurate method for measuring the abundance of these small, non-coding RNA species.
Automatically generated - may contain errors
Lab products found in correlation
Seramir exosome rna amplification kit, by System Biosciences (3 mentions)
Exoquick tc, by System Biosciences (3 mentions)
Exorneasy serum plasma maxi kit, by Qiagen (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Rna 6000 nano kit, by Agilent Technologies (2 mentions)
Universal cdna synthesis kit 2, by Qiagen (1 mentions)
Biotek synergy ht multidetection microplate reader, by Agilent Technologies (1 mentions)
Quant it, by Thermo Fisher Scientific (1 mentions)
C57bl 6j female mice, by Jackson ImmunoResearch (1 mentions)
5 protocols using quant it micro rna assay kit
1
Urinary exosomal miRNA isolation and analysis
2
Plasma miRNA Profiling and Validation
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All cDNAs from the profiling and validation step were reverse-transcribed using the Universal cDNA synthesis kit II from Exiqon according to the manufacturer’s protocol. For the profiling phase, 4 μl of RNA was added to the reverse transcription (RT) mix containing 4 μl 5x reaction buffer, 2 μl enzyme mix, 1 μl UniSp6 spike-in and 9 μl H2O.
For the validation phase, the miRNA concentrations were first assessed using the Quant-iT microRNA assay kit from Life Technologies following the manufacturer’s instructions. Briefly, 16 μl of plasma RNA was added to 200 μl of working solution, and the fluorescence was read on a microplate reader (Biotek Synergy HT Multi-Detection Microplate Reader, Biotek Instruments Inc.). All miRNAs were then diluted to 5 ng/μl, and 8 μl of the diluted miRNAs were reverse transcribed with minor modifications to the original protocol. The H2O volume was adjusted to 5 μl to obtain a final volume of 20 μl. We performed several tests to define the optimal volume of diluted miRNAs for RT.
For the validation phase, the miRNA concentrations were first assessed using the Quant-iT microRNA assay kit from Life Technologies following the manufacturer’s instructions. Briefly, 16 μl of plasma RNA was added to 200 μl of working solution, and the fluorescence was read on a microplate reader (Biotek Synergy HT Multi-Detection Microplate Reader, Biotek Instruments Inc.). All miRNAs were then diluted to 5 ng/μl, and 8 μl of the diluted miRNAs were reverse transcribed with minor modifications to the original protocol. The H2O volume was adjusted to 5 μl to obtain a final volume of 20 μl. We performed several tests to define the optimal volume of diluted miRNAs for RT.
3
Exosomal miRNA Isolation and Quantification
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Exosomal miRNAs were isolated from 300 µL plasma using the exoRNeasy Serum/Plasma Maxi Kit (QIAGEN, Hilden, Germany) according to manufacturer’s instructions. Quality control was performed on the Agilent 2100 Bioanalyzer, using the RNA 6000 Nano Kit (Agilent Technologies Inc, Waldbronn, Germany) and miRNA concentration was measured using Quant-IT microRNA assay kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions.
4
Synthesis and Characterization of CNP-miR146a
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CNP-miR146a was synthesized as previously described [33 (link)]. In brief, CNPs were synthesized via chemical hydrolysis, and then conjugated to miR146a using 1′-1′ carbonyldiimidazole (CDI) chemistry to covalently bind the miR146a amino group to the CNP hydroxyl group. The CNP-miR146a concentration was assessed by Quant-iT™ (ThermoFisher Quant-iT™ microRNA Assay Kit, Waltham, MA, USA) to determine the miR146a concentration, and inductively coupled plasma mass spectrometry (ICP-MS) to determine the cerium concentration. The conjugated CNP-miR146a was diluted in sterile phosphate buffer saline (PBS) to a concentration of 11.4 ng/uL and stored at −20 °C.
5
Lung Injury Treatment with CNP-miR146a Nanoparticles
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All animal studies described were approved by the Institutional Animal Care and Use Committee (IACUC, protocol #00230) at the University of Colorado Denver | Anschutz Medical Campus. All mice were acclimated for at least one week prior to experimentation.
Eight- to ten-week-old C57BL/6 J female mice (Jackson Laboratory) were injured intratracheally (IT) with lipopolysaccharide (LPS, O55:B5 #203A, List Labs). Each mouse received 50 μg LPS dissolved in 50 μL sterile phosphate buffered saline (PBS) at time zero. Control mice were administered 50 μL IT PBS. Four hours later, a cohort of LPS-injured mice were treated with 0.5 ng/kg IT CNP-miR146a nanoparticles dissolved in PBS. CNP-miR146a concentration was assessed by Quant-iT™ to determine miR146a concentration (ThermoFisher Quant-iT™ microRNA Assay Kit) and inductively coupled plasma mass spectrometry (ICP-MS) to determine cerium concentration. Control mice were administered 50 μL of IT PBS. The following groups were utilized for the study: control (PBS + PBS), injured and untreated (LPS + PBS), and injured and treated (LPS + CNP-miR146a). In our prior work we have shown that the conjugate therapeutic (CNP-miR146a) is a more effective lung injury treatment than CNP or miR146a alone.12 Therefore, in the current study we focus on a comprehensive evaluation of CNP-miR146a effects.
Eight- to ten-week-old C57BL/6 J female mice (Jackson Laboratory) were injured intratracheally (IT) with lipopolysaccharide (LPS, O55:B5 #203A, List Labs). Each mouse received 50 μg LPS dissolved in 50 μL sterile phosphate buffered saline (PBS) at time zero. Control mice were administered 50 μL IT PBS. Four hours later, a cohort of LPS-injured mice were treated with 0.5 ng/kg IT CNP-miR146a nanoparticles dissolved in PBS. CNP-miR146a concentration was assessed by Quant-iT™ to determine miR146a concentration (ThermoFisher Quant-iT™ microRNA Assay Kit) and inductively coupled plasma mass spectrometry (ICP-MS) to determine cerium concentration. Control mice were administered 50 μL of IT PBS. The following groups were utilized for the study: control (PBS + PBS), injured and untreated (LPS + PBS), and injured and treated (LPS + CNP-miR146a). In our prior work we have shown that the conjugate therapeutic (CNP-miR146a) is a more effective lung injury treatment than CNP or miR146a alone.12 Therefore, in the current study we focus on a comprehensive evaluation of CNP-miR146a effects.
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