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Quant it ribogreen reagent

Manufactured by Thermo Fisher Scientific
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The Quant-iT™ RiboGreen® reagent is a fluorescent nucleic acid stain used for the quantitation of RNA. It provides a simple and sensitive method to measure the concentration of RNA samples.

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Lab products found in correlation

Dynabeads oligo dt 25, by Thermo Fisher Scientific (2 mentions) Zetasizer nano zsp, by Malvern Panalytical (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Cfx96 real time pcr detection system, by Bio-Rad (1 mentions) Taqman primer probe sets, by Integrated DNA Technologies (1 mentions) Lysotracker green, by Thermo Fisher Scientific (1 mentions) Cleancap mcherry mrna, by TriLink (1 mentions) Hek293t cell, by American Type Culture Collection (1 mentions) Hela cell, by American Type Culture Collection (1 mentions) Cleancap fluc mrna, by TriLink (1 mentions) Cleancap cyanine 5 fluc mrna, by TriLink (1 mentions) Eukaryote total rna nano series 2 chip, by Agilent Technologies (1 mentions) Stranded mrna seq kit, by Illumina (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Rneasy plant mini kit, by Qiagen (1 mentions) Nextseq 500, by Illumina (1 mentions) Rnase free dnase, by Qiagen (1 mentions)

9 protocols using quant it ribogreen reagent

1

Isolation of Poly(A)+ RNA Using Dynabeads

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Dynabeads Oligo(dT)25 (Life Technologies) were equilibrated with 50 µL Lysis/Binding Buffer, and total RNA was heat denatured (70°C for 2 minutes) prior to binding poly(A)+ RNA to Dynabeads. Isolation of mRNA was performed according to manufacturer’s instructions. Poly(A)+ RNA concentrations were measured with a fluorescence plate reader (Molecular Devices) using Quant-iT RiboGreen reagent (Life Technologies).
Hsu T.Y., Simon L.M., Neill N., Marcotte R., Sayad A., Bland C.S., Echeverria G.V., Sun T., Kurley S.J., Tyagi S., Karlin K.L., Dominguez-Vidaña R., Hartman J.D., Renwick A., Scorsone K., Bernardi R.J., Skinner S.O., Jain A., Orellana M., Lagisetti C., Golding I., Jung S.Y., Neilson J.R., Zhang X.H., Cooper T.A., Webb T.R., Neel B.G., Shaw C.A, & Westbrook T.F. (2015). The spliceosome is a therapeutic vulnerability in MYC-driven cancer. Nature, 525(7569), 384-388.
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2

Isolation of Poly(A)+ RNA Using Dynabeads

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Dynabeads Oligo(dT)25 (Life Technologies) were equilibrated with 50 µL Lysis/Binding Buffer, and total RNA was heat denatured (70°C for 2 minutes) prior to binding poly(A)+ RNA to Dynabeads. Isolation of mRNA was performed according to manufacturer’s instructions. Poly(A)+ RNA concentrations were measured with a fluorescence plate reader (Molecular Devices) using Quant-iT RiboGreen reagent (Life Technologies).
Hsu T.Y., Simon L.M., Neill N., Marcotte R., Sayad A., Bland C.S., Echeverria G.V., Sun T., Kurley S.J., Tyagi S., Karlin K.L., Dominguez-Vidaña R., Hartman J.D., Renwick A., Scorsone K., Bernardi R.J., Skinner S.O., Jain A., Orellana M., Lagisetti C., Golding I., Jung S.Y., Neilson J.R., Zhang X.H., Cooper T.A., Webb T.R., Neel B.G., Shaw C.A, & Westbrook T.F. (2015). The spliceosome is a therapeutic vulnerability in MYC-driven cancer. Nature, 525(7569), 384-388.
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3

Quantifying tRNA Thermal Stability

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In vitro transcribed full-length unlabeled tRNA (1 µg) was mixed with 400-fold diluted Quant-iT™ Ribogreen® reagent (Life technologies, Carlsbad, CA, USA) and the fluorescence intensities and associated melting temperatures (Tm) were measured on a CFX96 Real-Time PCR Detection system (Bio-Rad) using the in-system melting curve protocol [21 (link)].
Krishnamohan A., Dodbele S, & Jackman J.E. (2019). Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from Thermococcus kodakarensis. Genes, 10(2), 100.
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4

Quantitative RT-PCR for mRNA Profiling

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RNA was extracted and purified with a glass fiber filter plate (Pall # 5072) and chiotropic salts. Messenger RNA expression levels were quantitated with quantitative reverse transcription PCR on the QuantStudio7 PCR Instrument (Applied Biosystems). Briefly, 10 μl RT-qPCR reactions containing 4 μl of RNA were run with Agpath-ID reagents (Applied Biosystems) and gene specific TaqMan primer probe sets (Integrated DNA Technologies) following the manufacturer's instructions. Total RNA levels, measured with Quant-iT Ribogreen Reagent (Thermo Fisher), were used to normalize the RT-qPCR data.
Migawa M.T., Shen W., Wan W.B., Vasquez G., Oestergaard M.E., Low A., De Hoyos C.L., Gupta R., Murray S., Tanowitz M., Bell M., Nichols J.G., Gaus H., Liang X.H., Swayze E.E., Crooke S.T, & Seth P.P. (2019). Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins. Nucleic Acids Research, 47(11), 5465-5479.
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5

Quantification of siRNA Loading in Nanoparticles

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siRNA actual loading was
evaluated indirectly by quantitation of non-encapsulated siRNA. Briefly,
just after production, hNPs were collected by centrifugation (7000
rcf for 20 min at 4 °C) and the supernatant was analyzed for
siRNA content using Quant-IT RiboGreen reagent (Thermo Fisher Scientific,
Massachusetts, USA) according to the manufacturer’s instructions.
Quantitative analysis was performed by spectrofluorimetry at λex/λem 480 nm/520 nm (ProMega GloMax Plate
reader, USA). Results are reported as actual loading (nmol of encapsulated
siRNA per mg of hNPs) and encapsulation efficiency (actual loading/theoretical
loading × 100) ± SD of values collected from three different
batches.
Conte G., Costabile G., Baldassi D., Rondelli V., Bassi R., Colombo D., Linardos G., Fiscarelli E.V., Sorrentino R., Miro A., Quaglia F., Brocca P., d’Angelo I., Merkel O.M, & Ungaro F. (2022). Hybrid Lipid/Polymer Nanoparticles to Tackle the Cystic Fibrosis Mucus Barrier in siRNA Delivery to the Lungs: Does PEGylation Make the Difference?. ACS Applied Materials & Interfaces, 14(6), 7565-7578.
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6

Nanoparticle Size and DNA Loading

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Particle sizes were measured and calculated by intensity using Zetasizer Nano ZSP (Malvern Panalytical, Malvern, UK) according to the manufacturer’s protocol [26 ]. The loading efficiency of pDNA was analyzed by using the Quant-iT™ RiboGreen® reagent (Thermo Fisher Scientific R11491, Waltham, MA, USA) as described earlier [27 ]. First, a calibration curve was obtained using standard pDNA in the concentration range 0.03–0.2 µg/mL. After that, DNA concentration was measured in obtained nanoparticles before and after disruption with the Triton X-100 buffer. The difference in these values shows the portion of loaded pDNA.
Shashkovskaya V.S., Vetosheva P.I., Shokhina A.G., Aparin I.O., Prikazchikova T.A., Mikaelyan A.S., Kotelevtsev Y.V., Belousov V.V., Zatsepin T.S, & Abakumova T.O. (2023). Delivery of Lipid Nanoparticles with ROS Probes for Improved Visualization of Hepatocellular Carcinoma. Biomedicines, 11(7), 1783.
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7

Formulation and Characterization of siRNA-LNP

Cited 1 time
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We used one small interfering RNA (siRNA) described by Leuschner et al.51 (link) and in Supplementary Methods. siRNA was selected to avoid off-target activity based on several known criteria as described previously52 (link). siRNA modification with 2′-OMe pyrimidine nucleotides and 3′-internucleotide phosphorothioates should reduce immune response and increase stability in vivo. Potency of siRNA targeting CCR2 were studied by transfection in Hepa1–6 cells followed by qPCR analysis after 24 h. siRNA were formulated in lipid nanoparticles (LNPs), as previously described52 (link). Particle size measurements were performed using a Zetasizer Nano ZSP (Malvern Panalytical) according to the manufacturer’s protocol, siRNA entrapment efficiency was determined using the Quant-iT™ RiboGreen® reagent (R11491, Thermo Fisher Scientific) as described52 (link).
Flamini S., Sergeev P., Viana de Barros Z., Mello T., Biagioli M., Paglialunga M., Fiorucci C., Prikazchikova T., Pagano S., Gagliardi A., Riccardi C., Zatsepin T., Migliorati G., Bereshchenko O, & Bruscoli S. (2021). Glucocorticoid-induced leucine zipper regulates liver fibrosis by suppressing CCL2-mediated leukocyte recruitment. Cell Death & Disease, 12(5), 421.
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8

mRNA Transfection and Luminescence Assay

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CleanCap Fluc mRNA, CleanCap mCherry mRNA, and CleanCap Cyanine 5 Fluc mRNA were purchased from TriLink Biotechnologies. QUANT-iT Ribogreen reagent, LysoTracker Green, and Hoechst 33342 were purchased from ThermoFisher Scientific. One-Glo + Tox were purchased from Promega. HEK293T cells and HeLa cells were purchased from ATCC.
Álvarez-Benedicto E., Farbiak L., Ramírez M.M., Wang X., Johnson L.T., Mian O., Guerrero E.D, & Siegwart D.J. (2022). Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA). Biomaterials science, 10(2), 549-559.
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9

Midgut Tissue RNA Extraction and Sequencing

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Total RNA was extracted from midgut tissue using Qiagen RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) and treated with RNase Free DNase (Qiagen, Valencia, CA, USA) as per manufacturer’s protocol. RNA quality was assessed with an Agilent 2100 Bioanalyzer and a Eukaryote Total RNA Nano Series II chip (Agilent, Santa Clara, CA, USA). RNA ribosomal integrity numbers (RIN) from all samples except one, ranged between 8.1 and 9.1, where any RIN above eight is considered high quality (Fleige & Pfaffl, 2006 (link)). RNA was quantified with Quant-iT™ RiboGreen® reagent and a Labsystems Fluoroskan Ascent™ Microplate Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Samples were precipitated with 1/10th volume sodium acetate and 2.5x volume pure ethanol and stored at −80 °C prior to shipment. Eight samples with RIN greater than eight from each treatment were shipped to the Georgia Genomic Facility (GGF) for library preparation and sequencing. The quality of the total RNA was confirmed by GGF using an Agilent 2100 Bioanalyzer. GGF prepared barcoded cDNA libraries using a KAPA Stranded mRNA-Seq Kit (Wilmington, MA) and sequenced them on the Illumina NextSeq 500 using paired-end sequencing with a NextSeq 2×75 High Output Flow Cell.
Fisher K.E., Tillett R.L., Footohi M., Caldwell C., Petereit J., Schlauch K., Tittiger C., Blomquist G.J, & MacLean M. (2021). RNA-Seq used to identify Ipsdienone reductase (IDONER): A novel monoterpene carbon-carbon double bond reductase central to Ips confusus pheromone production. Insect biochemistry and molecular biology, 129, 103513.
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