Quant it ribogreen assay

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The Quant-iT RiboGreen Assay is a fluorescent nucleic acid stain used for the sensitive quantitation of RNA in solution. It provides a simple, accurate, and rapid method for determining the concentration of RNA in purified samples or in cell culture media.

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Lab products found in correlation

Agilent 2100 bioanalyzer rna 6000 pico chip, by Agilent Technologies (7 mentions) Rneasy minelute cleanup kit, by Qiagen (4 mentions) Dnase 1, by Thermo Fisher Scientific (4 mentions) Tbs 380 fluorometer, by Promega (4 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions) Globinclear kit, by Thermo Fisher Scientific (3 mentions) Paxgene blood rna kit, by Qiagen (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Allprep dna rna ffpe kit, by Qiagen (2 mentions) Nanosight ns300, by Malvern Panalytical (2 mentions) Nano zs zetasizer, by Malvern Panalytical (2 mentions) Nano z, by Malvern Panalytical (2 mentions) Amicon ultra 15 ml centrifugal filter units, by Merck Group (2 mentions) Triton x 100, by Merck Group (2 mentions) Zetasizer nano, by Malvern Panalytical (1 mentions) Cholesterol, by Merck Group (1 mentions) 1 2 distearoyl sn glycero 3 phosphocholine dspc, by Avanti Polar Lipids (1 mentions) Zetasizer, by Malvern Panalytical (1 mentions) C12 200, by WuXi AppTec (1 mentions) Paxgene blood rna tube, by Preanalytix (1 mentions)

Spelling variants of this product

RiboGreen (90 citations) RiboGreen assay (57 citations) Quant-IT RiboGreen (49 citations) Quant-iTTM RiboGreen RNA Assay Kit (17 citations) Quant-iTTM Ribogreen Assay (2 citations)

34 protocols using quant it ribogreen assay

Lipid Nanoparticle Encapsulation of Purified mRNA

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Purified N1-methyl-pseudouridine mRNA was formulated in LNP as previously described.⁷⁵ (link) In brief, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, a PEG lipid, and an ionizable cationic lipid dissolved in ethanol were rapidly mixed with an aqueous acidic solution containing mRNA using an in-line mixer. The ionizable lipid and LNP composition are described in the international patent application WO2017075531(2017). The post in-line solution was dialyzed with PBS to remove the ethanol and displace the acidic solution. Subsequently, LNP was measured for size (60-65 nm) and polydispersity (PDI < 0.075) by dynamic light scattering (Malvern Nano ZS Zetasizer). Encapsulation efficiencies were >97% as measured by the Quant-iT Ribogreen Assay (Invitrogen).

Hoffmann M.A., Yang Z., Huey-Tubman K.E., Cohen A.A., Gnanapragasam P.N., Nakatomi L.M., Storm K.N., Moon W.J., Lin P.J., West AP J.r, & Bjorkman P.J. (2023). ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines. Cell, 186(11), 2380-2391.e9.

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Optimization of siRNA-Loaded Lipid Nanoparticles

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Four siRNAs with the lowest predicted off-target potentials and 100% homology with mouse RAB27A gene sequence (NM_001301230.1) were selected for synthesis and screening. Single-strand RNAs were purchased from Horizon Discovery (Louis, MO, USA). Mouse macrophage line IC-21 or melanoma YUMM1.7 cells were transfected with siRNA against RAB27A using Lipofectamine RNAiMAX reagent according to the manufacturer’s protocols. The expression of Rab27a protein was examined 24 h after transfection by western blotting. siRNA sequence 5′-GUACAGAGCCAAUGGGCCA-3′ showed best knockdown efficiency and was selected for further studies. Lipid nanoparticles were prepared with C12–200 ionizable lipid, cholesterol, DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine) and C14-PEG2000 at M ratios of 35:46.5:16:2.5 using microfluidic mixing as previously described.^{77 (link),100 (link)} C12–200 LNP was tested on a Zetasizer Nano (Malvern Instruments, Malvern, UK) to obtain the hydrodynamic size, polydispersity index (PDI) and zeta potential. siRNA concentration and encapsulation efficiency were determined by a Quant-iT RiboGreen assay (Invitrogen, MA, USA).

Altier C., Suyemoto M, & Lawhon S.D. (2000). Regulation of Salmonella enterica Serovar Typhimurium Invasion Genes by csrA. Infection and Immunity, 68(12), 6790-6797.

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siRNA-Loaded Lipid Nanoparticle Synthesis

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siRNA-LNPs were synthesized by mixing together an siRNA-containing aqueous phase with a lipid-containing ethanol phase. The aqueous phase contained siRNA in 10 mM citrate buffer (pH 3). The ethanol phase contained ionizable lipid¹¹ (link) C12-200 (Wuxi AppTec), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids), cholesterol (Sigma), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14 PEG 2000, Avanti) at a 50:10:38.5:1.5 molar ratio and 5:1 C12-200:siRNA weight ratio. The aqueous and ethanol phases were mixed together at a 3:1 volume ratio in a microfluidic chip device using syringe pumps as previously described⁴⁷ (link) to a final siRNA concentration of 1 mg/mL. The resultant siRNA-LNPs were dialyzed overnight in a 20,000 molecular weight cut-off (MWCO) cassette against 1x PBS at 4°C. On average, siRNA-LNPs had a mean diameter of approximately 100–150 nm, with a polydispersity index between 0.1 and 0.2 as measured by Dynamic Light Scattering (ZetaSizer, Malvern Instruments). The siRNA encapsulation efficiency (approximately 60%) was determined using a modified Quant-iT Ribogreen Assay (Invitrogen) as previously described.⁴⁸ (link) This siRNA delivery system can target multiple liver cell types, including hepatocytes, monocytes, tissue macrophages, dendritic cells (DCs), and myofibroblasts/pericytes.

Vollmann E.H., Cao L., Amatucci A., Reynolds T., Hamann S., Dalkilic-Liddle I., Cameron T.O., Hossbach M., Kauffman K.J., Mir F.F., Anderson D.G., Novobrantseva T., Koteliansky V., Kisseleva T., Brenner D., Duffield J, & Burkly L.C. (2017). Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing. Molecular Therapy. Nucleic Acids, 7, 314-323.

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Femoral Blood RNA Extraction Protocol

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A volume of 2.5 mL of blood was taken from the femoral access sheath and transferred into a PAXgene blood RNA tube (PreAnalytiX, Hombrechtikon, Switzerland). Total RNA was extracted using the PAXgene Blood RNA kit (Qiagen, Venlo, Limburg, Netherlands) according to manufacturer’s instructions. Globin mRNA was removed by magnetic-bead capture with the GLOBINclear kit (Ambion, Austin, TX, USA) following manufacturer’s instructions. RNA purity and concentration were assessed by absorbance at 260 nm and were measured by the Quant-iT RiboGreen Assay (Invitrogen, Carlsbad, CA) and the Agilent 2100 BioAnalyzer RNA 6000 Pico Chip (Agilent, Las Vegas, NV), respectively.

Poppenberg K.E., Li L., Waqas M., Paliwal N., Jiang K., Jarvis J.N., Sun Y., Snyder K.V., Levy E.I., Siddiqui A.H., Kolega J., Meng H, & Tutino V.M. (2020). Whole blood transcriptome biomarkers of unruptured intracranial aneurysm. PLoS ONE, 15(11), e0241838.

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Quantifying mRNA Encapsulation in Liposomes

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To test the capsulation efficiency of the liposomes, we used the Quant-iT RiboGreen assay (Invitrogen). Therefore, the standard curve and the samples were prepared. For the standard curve, EGFP mRNA was diluted in different concentrations in water ranging from 0 to 1,000 ng. Additionally, lipoplexes were formed by mixing 1 μg of EGFP mRNA and liposomes or Lipofectamine 2000 (Invitrogen) and incubated for 20 min at RT. Afterward, the RiboGreen fluorescent dye was added to the samples and incubated for 5 min. Fluorescence of samples was measured at 530 nm using a Mithras microplate reader (Berthold Technologies), and the concentration of each sample was calculated using a standard curve.

Michel T., Luft D., Abraham M.K., Reinhardt S., Salinas Medina M.L., Kurz J., Schaller M., Avci-Adali M., Schlensak C., Peter K., Wendel H.P., Wang X, & Krajewski S. (2017). Cationic Nanoliposomes Meet mRNA: Efficient Delivery of Modified mRNA Using Hemocompatible and Stable Vectors for Therapeutic Applications. Molecular Therapy. Nucleic Acids, 8, 459-468.

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Quantitative Gene Expression Analysis in HKCs

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Total RNA was extracted from HKCs using the PrepEase RNA spin kit (Affymetrix, Santa Clara, CA) according to the manufacturer's direction. RNA concentration was measured with Quant-it RiboGreen assay (Invitrogen). 1 μg of RNA was reverse-transcribed to cDNA using iScript cDNA synthesis kit (Bio-Rad). Real-time PCR was then performed using the iQ SYBR Green Supermix (Bio-Rad) with the iCycler iQ real-time PCR detection system (Bio-Rad). Real-time data were collected for 40 cycles of 95 °C, 10 s; 57 °C, 45 s; and 75 °C, 30s. Primers are designed with software provided by Invitrogen and synthesized by Integrated DNA Technology (Coralville, CA). Relative expression of the gene of interest was estimated by the ΔΔCt method using β2-microglobulin as a reference gene. Samples were analyzed in triplicate, and experiments were repeated at least three times. Primers used were as follows: αSMA, 5’-AGCAGGCCAAGGGGCTATATAA-3’ (forward) and 5′-CGTAGCTGTCTTTTTGTCCCATT-3’ (reverse); COL1A1, 5’-CAATGCTGCCCTTTCTGCTCCTTT-3’ (forward) and 5’-CACTTGGGTGTTTGAGCATTGCCT-3’ (reverse); TGIF, 5’-TAGAGGAGACCCCATTTCATTCC-3’ (forward) and 5’-GGGGATGACGGCTTAGGAGA-3’ (reverse); β2-microglobulin, 5′-TGTCTGGGTTTCATCCATCCGACA-3′ (forward) and 5′-TCACACGGCAGGCATACTCATCTT-3′ (reverse).

Liu X., Hubchak S.C., Browne J.A, & Schnaper H.W. (2014). Epidermal Growth Factor Inhibits Transforming Growth Factor-β-Induced Fibrogenic Differentiation Marker Expression through ERK Activation. Cellular signalling, 26(10), 2276-2283.

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Characterization of siRNA-Loaded Lipid Nanoparticles

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The LNPs were diluted to an siRNA concentration of 0.5–1.25 µg/mL in PBS for the characterization studies. The siRNA entrapment efficiency was determined using Quant-iT Ribogreen assay (Invitrogen) following the manufacturer’s protocol. The siRNA entrapment efficiency is calculated using Equation 1:

E_{siRNA} (%) = \frac{(F_{total} - F_{unencapsulated})}{F_{total}} \times 100

where E_siRNA is the siRNA entrapment percentage, F_total is the total siRNA fluorescence, and F_{unencapsulated} is the fluorescence of the siRNA outside of the nanoparticles. LNP particle size was measured using a Malvern Zetasizer Nano (Malvern Instruments, UK). Each LNP sample was measured three times, and size value was reported as the z-average hydrodynamic diameter.

Ball R.L., Bajaj P, & Whitehead K.A. (2016). Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization. International Journal of Nanomedicine, 12, 305-315.

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Neutrophil RNA Extraction and Sequencing

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Neutrophil RNA was extracted as described previously [22 (link)] using TRIzol, according to the manufacturer’s instructions. Trace DNA was removed by DNase I (Life Technologies, Carlsbad, CA) treatment. RNA was purified using the RNeasy MinElute Cleanup Kit (Qiagen, Venlo, Limburg, Netherlands) and suspended in RNase-free water. The purity and concentration of RNA in each sample were measured by absorbance at 260 nm and 280 nm on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA), and 200 ng to 400 ng of RNA was reserved for sequencing. Precise RNA concentration was measured via the Quant-iT RiboGreen Assay (Invitrogen, Carlsbad, CA) with a TBS-380 Fluorometer (Promega, Madison, WI), and the quality of the RNA samples was measured with an Agilent 2100 BioAnalyzer RNA 6000 Pico Chip (Agilent, Las Vegas, NV). RNA samples to be sequenced had acceptable purity (260/280 ratio of ~ 1.8 or greater, range: 1.76–2.12) and integrity (RIN of ~ 5 or greater, range: 4.5–9.1) prior to RNA sequencing.

Poppenberg K.E., Tutino V.M., Li L., Waqas M., June A., Chaves L., Jiang K., Jarvis J.N., Sun Y., Snyder K.V., Levy E.I., Siddiqui A.H., Kolega J, & Meng H. (2020). Classification models using circulating neutrophil transcripts can detect unruptured intracranial aneurysm. Journal of Translational Medicine, 18, 392.

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RNA Encapsulation Efficiency Quantification

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The RNA encapsulation efficiency was determined by the Quant‐iT RiboGreen Assay (R11491, Invitrogen). Quantification of RNA in DP7‐C was conducted using a standard curve generated from a dilution series of the corresponding RNA stock. Fluorescence was measured using a fluorescence microplate reader (BioTek) set at 480‐nm excitation and 520‐nm emission.

Liu M., Xie D., Hu D., Zhang R., Wang Y., Tang L., Zhou B., Zhao B, & Yang L. (2023). In Situ Cocktail Nanovaccine for Cancer Immunotherapy. Advanced Science, 10(31), 2207697.

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Femoral Blood RNA Extraction and Sequencing

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For this work, 2.5 mL of blood was drawn off the femoral access sheath during DSA and transferred into a PAXgene blood RNA tube (PreAnalytiX, Hombrechtikon, Switzerland). The PAXgene Blood RNA kit was used to extract RNA from blood following manufacturer’s instructions. We removed globin mRNA from samples using magnetic bead capture and the GLOBINclear Kit (Ambion, Austin, TX, USA) according to manufacturer’s instructions. Concentration and purity of isolated RNA was analyzed by absorbance at 260 nm using NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Prior to sequencing, RNA concentration was accurately measured by the Agilent 2100 BioAnalyzer RNA 6000 Pico Chip (Agilent, Las Vegas, NV, USA), while purity was precisely assessed by the Quant-iT RiboGreen Assay (Invitrogen, Carlsbad, CA, USA). Samples of sufficient quality (260/280 ratio ~2, RNA integrity number ≥ 6.0) were selected for RNA-sequencing (RNAseq).

Poppenberg K.E., Chien A., Santo B.A., Baig A.A., Monteiro A., Dmytriw A.A., Burkhardt J.K., Mokin M., Snyder K.V., Siddiqui A.H, & Tutino V.M. (2023). RNA Expression Signatures of Intracranial Aneurysm Growth Trajectory Identified in Circulating Whole Blood. Journal of Personalized Medicine, 13(2), 266.

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