Quant it ribogreen rna kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Quant-iT RiboGreen RNA kit is a fluorescent nucleic acid stain used for the quantitation of RNA. It provides a sensitive and accurate method for measuring RNA concentrations in purified samples or crude cellular extracts.

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Lab products found in correlation

Labchip gx touch ht genomic dna reagent kit, by PerkinElmer (3 mentions) Labchip rna high ht pico sensitivity reagent kit, by PerkinElmer (3 mentions) Rna 6000 pico kit, by Agilent Technologies (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Quant it picogreen dsdna reagent kit, by Thermo Fisher Scientific (2 mentions) Hiseq 4000 system, by Illumina (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Mint cdna synthesis kit, by Evrogen (1 mentions) 454 gs flx titanium, by Roche (1 mentions) Trimmer cdna normalization kit, by Evrogen (1 mentions) Slide a lyzer mini dialysis unit, by Thermo Fisher Scientific (1 mentions) Malvern nano zs, by Malvern Panalytical (1 mentions) Ffpe rna extraction kit, by Qiagen (1 mentions) Bioanalyzer 2100 system, by Agilent Technologies (1 mentions) Multi mode microplate readers, by Agilent Technologies (1 mentions) Uv transilluminator, by Bio-Rad (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Chitosan powder, by Merck Group (1 mentions) Sybr safe, by Thermo Fisher Scientific (1 mentions) Endo free plasmid dna extraction mini kit, by Favorgen Biotech (1 mentions)

19 protocols using quant it ribogreen rna kit

Normalized cDNA Preparation for 454 Sequencing

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Total RNA from each tissue/condition was used as the source of starting material for cDNA synthesis and production of normalized cDNA libraries intended for 454 sequencing. Briefly, the total RNA quality was verified on Agilent 2100 Bioanalyzer with the RNA 6000 Pico kit (Agilent Technologies, Waldbronn, Germany) and the quantity assessed by fluorimetry with the Quant-iT RiboGreen RNA kit (Invitrogen, CA, USA). A fraction of 1–2 μg of total RNA was used for cDNA synthesis with the MINT cDNA synthesis kit (Evrogen, Moscow, Russia), a strategy based on the SMART double-stranded cDNA synthesis methodology using a modified template-switching approach that allows the introduction of known adapter sequences to both ends of the first-strand cDNA. Amplified cDNA was then normalized with TRIMMER cDNA Normalization kit (Evrogen, Moscow, Russia) using the Duplex-Specific Nuclease-technology [20 (link), 29 ].
Normalized cDNA was quantified by fluorescence and sequenced in 454 GS FLX Titanium according to the standard manufacturer’s instructions (Roche-454 Life Sciences, Brandford, CT, USA) at Biocant (Cantanhede, Portugal).

Pereira-Leal J.B., Abreu I.A., Alabaça C.S., Almeida M.H., Almeida P., Almeida T., Amorim M.I., Araújo S., Azevedo H., Badia A., Batista D., Bohn A., Capote T., Carrasquinho I., Chaves I., Coelho A.C., Costa M.M., Costa R., Cravador A., Egas C., Faro C., Fortes A.M., Fortunato A.S., Gaspar M.J., Gonçalves S., Graça J., Horta M., Inácio V., Leitão J.M., Lino-Neto T., Marum L., Matos J., Mendonça D., Miguel A., Miguel C.M., Morais-Cecílio L., Neves I., Nóbrega F., Oliveira M.M., Oliveira R., Pais M.S., Paiva J.A., Paulo O.S., Pinheiro M., Raimundo J.A., Ramalho J.C., Ribeiro A.I., Ribeiro T., Rocheta M., Rodrigues A.I., Rodrigues J.C., Saibo N.J., Santo T.E., Santos A.M., Sá-Pereira P., Sebastiana M., Simões F., Sobral R.S., Tavares R., Teixeira R., Varela C., Veloso M.M, & Ricardo C.P. (2014). A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing. BMC Genomics, 15(1), 371.

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Release Kinetics of CpG-Encapsulated Nanoparticles

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The release kinetics was determined in 10 mM sodium phosphate buffer (pH 7.0) and 10 mM acetate buffer (pH 5.0). The Slide-A-Lyzer MINI Dialysis units (10 kDa MWCO, Thermo Fisher Scientific) were loaded with 100 μL of CpG-encapsulated nanoparticles and placed in tanks containing aforementioned buffers at 37°C. After 15 min, 30 min, 1, 2, 4, 8, 24, 48, 72 and 96 hr, the nanoparticles remaining in dialysis units were collected and disrupted by 100% acetone. After acetone was evaporated, remaining CpG was analyzed by the reagent in Quant-iT™ RiboGreen^® RNA Kit (Invitrogen).

Lin S.Y., Yao B.Y., Hu C.M, & Chen H.W. (2020). Induction of Robust Immune Responses by CpG-ODN-Loaded Hollow Polymeric Nanoparticles for Antiviral and Vaccine Applications in Chickens. International Journal of Nanomedicine, 15, 3303-3318.

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mRNA Polyplexes Formulation and Characterization

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Unless specified, polymer:mRNA polyplexes were prepared at a 100:1
polymer:mRNA weight ratio in 25 mM sodium acetate buffer (pH 5.8). For
in vitro experiment, a solution at 10 µg mRNA/mL
was prepared: 1 µL of polymer solution (100 mg/mL in DMSO) was first
diluted in 50 µL sodium acetate buffer. After brief vortexing, the
polymer solution was mixed with 1 µg mRNA diluted in 50 µL
sodium acetate buffer, and vortexed again. The polymer:mRNA mixture was
incubated at room temperature for 10 min before use. For in
vivo experiments, a solution at 100 µg mRNA/mL in sodium
acetate buffer was prepared by the same method.
The hydrodynamic diameter of the polyplexes was measured by Dynamic
Light Scattering (DLS) using a Malvern Nano-ZS (Malvern Instruments, UK), after
dilution of polyplexes in DI water at a concentration of 2 µg/mL of
mRNA. To measure zeta potential, the same solution was loaded into a disposable
capillary cell and analyzed on a Malvern Nano-ZS.
Encapsulation efficiency (EE) of mRNA in the polyplexes was measured
using the Quant-IT RiboGreen RNA kit (Invitrogen, #R11491) according to
manufacturer instructions. As the RiboGreen assay measures the amount of free
mRNA in solution, this amount was substracted to the initial amount added to
form the polyplexes, to obtain the amount of mRNA complexed within the
polyplexes.

Jiang Y., Gaudin A., Zhang J., Agarwal T., Song E., Kauffman A.C., Tietjen G.T., Wang Y., Jiang Z., Cheng C.J, & Saltzman W.M. (2018). A "top-down" approach to actuate poly(amine-co-ester) terpolymers for potent and safe mRNA delivery. Biomaterials, 176, 122-130.

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CpG Release Kinetics from Polymer Nanoparticles

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The release kinetics was determined in 10 mM sodium phosphate buffer (pH 7.0) and 10 mM acetate buffer (pH 5.0). The Slide-A-Laser MINI Dialysis units (10 kDa MWCO, Thermo Fisher Scientific) were loaded with 100 μL of CpG encapsulated CSNPs and placed in tanks containing aforementioned buffers at 37 °C. After 20, 40, 60, 80, and 100 h, the nanoparticles remaining in dialysis units were collected and disrupted by 100% acetone. After acetone was evaporated, remaining CpG was analyzed by the reagent in Quant-iT^™ RiboGreen^® RNA Kit (Invitrogen).

Piri-Gharaghie T., Doosti A, & Mirzaei S.A. (2023). Novel adjuvant nano-vaccine induced immune response against Acinetobacter baumannii. AMB Express, 13, 31.

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Total RNA Extraction from Preserved Samples

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Total RNA extractions were performed by slowly thawing samples on ice and removing preservation solution using a syringe (Frias-Lopez et al., 2008 (link)). Then, 350 μl of MirVana denaturing lysis buffer (Ambion, #AM854OG, Austin, TX) was added to the filter unit, capped, and vortexed for 1 min. Next, 350 μl of RNase-free water was added to the filter unit. Lysate was recovered from the filter unit and added to 600 μl of RNase-free water. Prior to purification, lysate was spilt into two aliquots of 675 μl each. Purification and DNase treatment were performed on a Chemagic MSM robotic lab instrument (Perkin Elmer, Waltham, MA) using an RNA Tissue kit (Perkin Elmer, CMG-1212A, Waltham, MA). Total RNA quality was analyzed using a fragment analyzer with the high sensitivity RNA kit from Agilent (DNF-472-0500, Santa Clara, CA), and quantification performed using the Quant-IT Ribogreen RNA kit (Invitrogen, R11490, Carlsbad, CA).

Vislova A., Sosa O.A., Eppley J.M., Romano A.E, & DeLong E.F. (2019). Diel Oscillation of Microbial Gene Transcripts Declines With Depth in Oligotrophic Ocean Waters. Frontiers in Microbiology, 10, 2191.

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FACS-Sorted Dopamine Neuron RNA Extraction

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RNA from bulk collected FACS sorted dopamine neurons was extracted using an FFPE RNA extraction kit (QIAGEN) as per manufacturer’s instructions, with minor modifications. RNA integrity analysis was analyzed using a 2100 bioanalyzer system and a RNA 6000 pico kit (Agilent). Concentration was obtained and confirmed utilizing two methods; the 2100 bioanalyzer system and a Quant-iT RiboGreen RNA kit (Invitrogen), as per manufacturer’s instructions.

Lang C., Campbell K.R., Ryan B.J., Carling P., Attar M., Vowles J., Perestenko O.V., Bowden R., Baig F., Kasten M., Hu M.T., Cowley S.A., Webber C, & Wade-Martins R. (2019). Single-Cell Sequencing of iPSC-Dopamine Neurons Reconstructs Disease Progression and Identifies HDAC4 as a Regulator of Parkinson Cell Phenotypes. Cell Stem Cell, 24(1), 93-106.e6.

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Quantification of siRNA Encapsulation Efficiency

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Encapsulation efficiency was performed by the Quant-iT RiboGreen RNA Kit (Invitrogen, Grand Island, NY, USA) as described previously.^{44 (link)} The unencapsulated siRNA content and the total siRNA content that was obtained upon lysis of the TLNP_κ by 1% Triton were determined according to the manufacturer’s instruction using Multi-Mode Microplate Readers (Biotek, Winooski, VT, USA) at 480 nm λ_ex and 520 nm λ_em. The encapsulation efficiency (EE) of siRNA was calculated with the following equation:

EE = \frac{Total siRNA - unencapsulated siRNA}{Total siRNA} \times 100 %

The encapsulation efficiency of siRNA encapsulated keratinocyte targeted lipid nanoparticles (TLNP_k/si) was also re-verified using gel retardation assay via 1% agarose gel. Electrophoresis was performed at 100 V for 20 min and visualized under a UV transilluminator (Bio-Rad laboratories, CA, USA).

Zhou X., Brown B.A., Siegel A.P., El Masry M.S., Zeng X., Song W., Das A., Khandelwal P., Clark A., Singh K., Guda P.R., Gorain M., Timsina L., Xuan Y., Jacobson S.C., Novotny M.V., Roy S., Agarwal M., Lee R.J., Sen C.K., Clemmer D.E, & Ghatak S. (2020). Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing. ACS nano, 14(10), 12732-12748.

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Plasmid DNA Extraction and TLR-9 Activation

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Chitosan powder (Sigma-Aldrich, USA), Endo-free Plasmid DNA Extraction mini kit (Favorgen, Taiwan), CpG ODN C274 and pcDNA3.1 ( +), by BGI Genomics Company (Shenzhen, China), SYBR Safe (Invitrogen), TLR-9 activation was performed as follows by abeomics, Inc (San Diego, CA 92121, United States). Quant-iT^™ RiboGreen^® RNA Kit (Invitrogen), karmania pars gene ELISA kits (KPG, IRAN), LB medium, fetal bovine serum (FBS) and antibiotic- ampicillin were all purchased from Invitrogen (Carlsbad, CA).

Piri-Gharaghie T., Doosti A, & Mirzaei S.A. (2023). Novel adjuvant nano-vaccine induced immune response against Acinetobacter baumannii. AMB Express, 13, 31.

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Formulation and Characterization of Lipid Nanoparticle Vaccines

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LNP formulations were prepared using a modified version of a procedure from Precision Nanosystems. Briefly, ionizable lipids were dissolved in ethanol at a volume ratio of 1:1 (25 mM). The lipid mixture was combined with the PNI Formulation Buffer containing RBD_beta, HLA-EPs, HLA-NC, or Luc mRNA at a concentration of 174 μg/mL using microfluidic cartridges. The formulations were concentrated using 10-kDa Amicon Ultra Centrifugal Filters, passed through a 0.22-μm filter, and stored at 4 °C until use. The concentrations of all formulations defined as LNP-RBD_beta, LNP-HLA-EPs, and LNP-NC were tested using the Quant-iT RiboGreen RNA Kit (Invitrogen, Cat. No# R11490). The particle size distribution of LNPs was analyzed by dynamic light scattering (Dynapro NanoStar).

Tai W., Feng S., Chai B., Lu S., Zhao G., Chen D., Yu W., Ren L., Shi H., Lu J., Cai Z., Pang M., Tan X., Wang P., Lin J., Sun Q., Peng X, & Cheng G. (2023). An mRNA-based T-cell-inducing antigen strengthens COVID-19 vaccine against SARS-CoV-2 variants. Nature Communications, 14, 2962.

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Cisplatin-Loaded NCP-1 Nanoparticles for siRNA Delivery

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DOPA-capped NCP-1 nanoparticles carrying a cisplatin prodrug were prepared according to our previous report. NCP-1/siRNAs was prepared by adding 10 μL of aqueous solution of DSPE-siRNA (DSPE-siRNA/DOPA-coated NCP = 1:16 in weight ratio) and a THF solution (80 μL) of cholesterol, DSPC (cholesterol/DOPC = 1:2 in molar ratio), 20 mol % DSPE-PEG2k, and DOPA-coated NCP to 500 μL of 30% (v/v) ethanol/water at 50 °C. The mixture was stirred at 1700 rpm for 1 min. THF and ethanol were completely evaporated, and the NCP-1/siRNAs solution was allowed to cool down to room temperature.
ICP-MS (Agilent 7700X, Agilent Technologies, USA) was utilized to analyze the Pt concentration of NCP to calculate cisplatin loadings. The amounts of siRNA loaded were quantified by Quant-iT RiboGreen RNA kit (Invitrogen, USA). The particle size and ζ potential of NCP-1/siRNAs in PBS were determined by Zetasizer (Nano ZS, Malvern, UK). Transmission electron microscopy (TEM, Tecnai Spirit, FEI, USA) was used to observe the morphology of NCP-1/siRNAs.

He C., Poon C., Chan C., Yamada S.D, & Lin W. (2016). Nanoscale Coordination Polymers Codeliver Chemotherapeutics and siRNAs to Eradicate Tumors of Cisplatin-Resistant Ovarian Cancer. Journal of the American Chemical Society, 138(18), 6010-6019.

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