Polymeric DNA structures were prepared by the addition of EDTA (50 mM), a strong Mg2+ scavenger, because of the disruption of polymeric DNA/inorganic composite structures of ODN-MS. The concentration of polymeric ssDNA and short ssDNA (ODN control) were measured according to manufacturer’s instructions for Quant-iT OliGreen ssDNA Assay Kit (Life Technologies, Carlsbad, CA). Briefly polymeric DNA or standard solution (10 μL) were mixed well with Quant-iT OliGreen ssDNA reagent (190 μL) by pipetting. Then the mixtures were placed into a Corning Clear Flat Bottom 96-wells plate and incubated for 5 min at 24 °C. Fluorescence (λex = 480 nm, λem = 520 nm) was measured using a fluorescence microplate reader (Tecan Infinite 200 PRO). The concentration of polymeric DNAs was determined by comparing with an ODN standard curve of fluorescence intensity versus concentration.
Quant it oligreen ssdna assay kit
Manufactured by Thermo Fisher Scientific
Sourced in Australia, United States
The Quant-iT OliGreen ssDNA Assay Kit is a fluorescent nucleic acid stain designed to quantify single-stranded DNA (ssDNA) in solution. It provides a sensitive and accurate method for measuring ssDNA concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Quantitect reverse transcription kit, by Qiagen (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Rnase h, by Thermo Fisher Scientific (3 mentions)
Dnase 1 amplification grade, by Thermo Fisher Scientific (2 mentions)
Quantifast sybr green pcr master mix, by Qiagen (2 mentions)
Quantitech primer assay, by Qiagen (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Zeta pals particle size analyzer, by Brookhaven Instruments (1 mentions)
Quant it oligreen ssdna reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (1 mentions)
Axis 165, by Shimadzu (1 mentions)
Ultima 4 xrd, by Rigaku (1 mentions)
Cpg odn 1826, by Integrated DNA Technologies (1 mentions)
23 protocols using quant it oligreen ssdna assay kit
1
Quantification of Polymeric DNA Structures
2
cDNA Quantification and Normalization
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RNA was treated with DNase I Amplification Grade (Life Technologies) and first-strand cDNA was generated as described above. cDNA was then treated with RNase H (Life Technologies) according to the manufacturer’s protocol. Single-strand DNA was quantified using the Quant it OliGreen ssDNA Assay Kit (Life Technologies) according to the manufacturer’s instructions and used for mRNA PCR normalization.
3
Characterizing Lipid Nanoparticles and ASO Loading
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Hydrodynamic size and zeta potential for LNPs were recorded on a Zeta-PALS particle size analyzer (Brookhaven Instruments). The ASO loading efficiency was determined by the Quant-iT OliGreen ssDNA Assay Kit (Life Technologies). TEM images were captured by an FEI Technai Spirit Transmission Electron Microscope.
4
Quantitative mRNA Expression Analysis
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Total mRNA was isolated from the prefrontal cortex and hippocampus using the miRNeasy mini kit (Qiagen Inc.) according to the manufacturer’s recommendations. Total RNA concentration and purity were measured by a Nanodrop (Thermo Scientific) at 235, 260 and 280 nm. cDNA was prepared from 1ug RNA in a 20-μL reaction using QuantiTect Reverse Transcription Kit (Qiagen). Each PCR (20 μL), containing 2 μL cDNA (12 ng), 10 μL Quanti Fast SYBR Green PCR Master Mix (Qiagen) and 2 μL PCR primer (QuantiTech Primer Assay, Qiagen), was run on a LightCycler 480 (Roche, Sweden). The following primers were used: Efna3 QuantiTech Primer Assay (QT00320026), Epha4 QuantiTech Primer Assay (QT00093576), Dlg4 QuantiTech Primer Assay (QT00121695) and Syp QuantiTech Primer Assay (QT01042314), all from Qiagen. Melting curve analysis was performed to ensure that only one PCR product was obtained. For quantification and for estimation amplification efficiency, a standard curve was generated using increasing concentrations of cDNA. The amplified transcripts were quantified with the relative standard curve and normalized by the cDNA concentration using the Quant-IT OliGreen ssDNA Assay kit (Fisher Scientific).
5
Hippocampal Gene Expression Analysis
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At P12 and P45 (n = 10 per treatment group per sex), mice were deeply anaesthetized via intraperitoneal administration of pentobarbital (Pentacour), and the right and left hippocampi were rapidly dissected and stored at −80°C until analysis. Total mRNA was isolated from the hippocampus using the miRNeasy mini kit (Qiagen Inc.) according to the manufacturer's recommendations. Total RNA concentration and purity were measured by a Nanodrop (Thermo Scientific) at 235, 260, and 280 nm. cDNA was prepared from 1ug RNA in a 20‐μl reaction using QuantiTect Reverse Transcription Kit (Qiagen). Each PCR (20 μl), contained 2‐μl cDNA (12 ng), 10‐μl Quanti Fast SYBR Green PCR Master Mix (Qiagen), and 2‐μl PCR primer (QuantiTech Primer Assay, Qiagen), were run on a LightCycler 480 (Roche, Sweden). The following primers were used Nlgn1 QuantiTech Primer Assay (QT00167580), Nrxn3 QuantiTech Primer Assay (QT00166621), and Pvrl1 (nectin‐1) QuantiTech Primer Assay (QT00171703) all from Qiagen. Melting curve analysis was performed to ensure that only one PCR product was obtained. For quantification and for estimation amplification efficiency, a standard curve was generated using increasing concentrations of cDNA. The amplified transcripts were quantified with the relative standard curve and normalized by the cDNA concentration using the Quant‐IT OliGreen ssDNA Assay kit (Fisher Scientific).
6
Cardiac Gene Expression Analysis
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Total RNA was extracted from the heart (n=5-9/genotype/timepoint) using TRIzol/chloroform, followed by the RNeasy Fibrous Tissue Mini Kit (Qiagen) as per the manufacturer's instructions. The concentration and quality of RNA samples were determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Real-time reverse transcription PCR was performed as described previously (Murphy et al., 2019 (link); Swiderski et al., 2016 (link)) using the following forward and reverse primer sequences: F4/80, 5′-CATCAGCCATGTGGGTACAG-3′ and 5′-CATCACTGCCTCCACTAGCA-3′; TGFβ, 5′-TGAGTGGCTGTCTTTTGACG-3′ and 5′-TCTCTGTGGAGCTGAAGCAA-3′; Col1a1, 5′-CACCCTCAAGAGCCTGAGTC-3′ and 5′-GTTCGGGCTGATGTACCAGT-3′; Col3a1, 5′-ACCAAAAGGTGATGCTGGAC-3′ and 5′-GACCTCGTGCTCCAGTTAGC-3′; Col6a1, 5′-CCCCATTGGACCTAAAGGAT-3′ and 5′-TCTCCCACTTCACCCTCATC-3′; Runx2, 5′-GCCTTCAAGGTTGTAGCCCT-3′ and 5′-GTTCTCATCATTCCCGGCCA-3′; Ocn, 5′-TTCTGCTCACTCTGCTGACC-3′ and 5′-GGGACTGAGGCTCCAAGGTA-3′; and Alpl, 5′-CAGGCCGCCTTCATAAGCA-3′ and 5′-AATTGACGTTCCGATCCTGC-3′. Gene expression was quantified using a cycle threshold (CT) method. Relative gene expression was calculated using the expression 2−ΔCT, normalized to total cDNA content as determined using the Quant-iT OliGreen ssDNA assay kit and Quant-iT OliGreen ssDNA reagent (Thermo Fisher Scientific).
7
Characterization of Iron Oxide Nanoparticles
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To determine the density of DNA probes on the IONPs surface, the reaction was conducted as above, and the supernatant as well as three previous washes were collected right after the reaction was completed. DNA concentration was estimated using Quant-iT OliGreen ssDNA Assay Kit (Thermo Fisher Scientific).
IONP size and morphology were determined by field emission high- resolution scanning electron microscopy (SEM) with a Hitachi- S4800 HR instrument set at 30 kV and by transmission electron microscopy (TEM) imaging with a JEOL TEM-2200FS instrument. Samples were prepared by dropping the nanoparticle suspension on a 400-mesh carbon grid and drying it in a vacuum oven for 2 h. X-ray powder diffraction (XRD) patterns were collected using a Rigaku XRD Ultima IV instrument to study the structural properties of IONPs. X-ray photoelectron spectra (XPS) were taken on a Kratos AXIS 165 electron spectrometer with 150 W monochromatized A1 Kα radiation (1486.6 eV), whereby all peaks were referred to the signature C1s peak for adventitious carbon at 284.8 eV.
IONP size and morphology were determined by field emission high- resolution scanning electron microscopy (SEM) with a Hitachi- S4800 HR instrument set at 30 kV and by transmission electron microscopy (TEM) imaging with a JEOL TEM-2200FS instrument. Samples were prepared by dropping the nanoparticle suspension on a 400-mesh carbon grid and drying it in a vacuum oven for 2 h. X-ray powder diffraction (XRD) patterns were collected using a Rigaku XRD Ultima IV instrument to study the structural properties of IONPs. X-ray photoelectron spectra (XPS) were taken on a Kratos AXIS 165 electron spectrometer with 150 W monochromatized A1 Kα radiation (1486.6 eV), whereby all peaks were referred to the signature C1s peak for adventitious carbon at 284.8 eV.
8
HDM Vaccine Formulation and Characterization
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The HDM vaccine
contained 2 primary components: 50 μg of class B CpG ODN 1826
(Integrated DNA technologies, Coralville, IA)-loaded poly(lactide-co-glycolide) PLGA (Resomer RG 530, Evonik, Germany) NPs
and 100 μg of purified Der p1 and Der p 2 (80:20) (lot numbers:
02.01.71 and 02.01.72, respectively, CiteQ Biologics, Groningen, Netherlands)
dispersed in 150 μL of saline (Baxter, Deerfield, IL). CpG-loaded
PLGA NPs were fabricated using a double emulsion solvent evaporation
method as previously described by Joshi et al. with some modifications
(Figure 2 a, Method S1 ).37 (link) To characterize
CpG NPs, hydrodynamic diameter and zeta potential were measured using
a Zetasizer (Zetasizer Nano ZS, Malvern Instrument Ltd., Westborough,
MA). The primary particle size and surface morphology of the NPs were
determined by scanning electron microscopy (SEM). CpG loading was
measured using a Quant-iT Oligreen ssDNA assay kit (ThermoFisher Scientific,
Waltham, MA).
contained 2 primary components: 50 μg of class B CpG ODN 1826
(Integrated DNA technologies, Coralville, IA)-loaded poly(lactide-co-glycolide) PLGA (Resomer RG 530, Evonik, Germany) NPs
and 100 μg of purified Der p1 and Der p 2 (80:20) (lot numbers:
02.01.71 and 02.01.72, respectively, CiteQ Biologics, Groningen, Netherlands)
dispersed in 150 μL of saline (Baxter, Deerfield, IL). CpG-loaded
PLGA NPs were fabricated using a double emulsion solvent evaporation
method as previously described by Joshi et al. with some modifications
(
CpG NPs, hydrodynamic diameter and zeta potential were measured using
a Zetasizer (Zetasizer Nano ZS, Malvern Instrument Ltd., Westborough,
MA). The primary particle size and surface morphology of the NPs were
determined by scanning electron microscopy (SEM). CpG loading was
measured using a Quant-iT Oligreen ssDNA assay kit (ThermoFisher Scientific,
Waltham, MA).
9
Quantification of mRNA Expression
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Total RNA was extracted from ∼2 mg of powdered freeze-dried TA muscle tissue using Tri-Reagent (Ambion Inc, Austin, TX, USA). Samples were treated with DNAse I (Life Technologies, Mulgrave, Victoria, Australia) to remove any contaminating genomic DNA as recommended by the manufacturer. cDNA was synthesized using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) from half a microgram of RNA followed by treatment with RNase H to remove remaining RNA as per the manufacturer's instructions. Semi-quantitative polymerase chain reaction (qPCR) was performed on cDNA diluted 1:20 using the Mx3000 PCR system (Stratagene, La Jolla, CA, USA) with SYBR Green Master Mix (Applied Biosystems) and 300 nM primers. Primer sequences and gene accession numbers are listed in Supplementary Table 2 . The qPCR cycling conditions were: 95 °C for 10 min (1 cycle), 30 s at 95 °C and 60 °C for one min (40 cycles). The mRNA expression was calculated using the 2−ΔCt formula following normalization to cDNA input as determined by the Quant-iT™ OliGreen™ ssDNA Assay Kit (Thermo Fisher Scientific, Scoresby, Victoria, Australia). Data were then normalized to mean of the scrambled condition.
10
RNA Extraction and qRT-PCR Analysis
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Brain tissue was manually homogenized in RNase-free PBS, and total RNA was
extracted using an miRNeasy Mini kit (Cat. No. 205313, Qiagen) and quantified
using a spectrophotometer (NanoDrop 2000, Thermo scientific) at 260 nm
absorbance (n = 10/group, that was decided based on our previous experiments30
). A total of 1 μg RNA was used for complementary DNA (cDNA) generation
using a QuantiTect Reverse Transcription kit (Qiagen). The reverse transcription
protocol included 2 min at 42°C, 30 min at 42°C, and 3 min at 95°C. SYBR
Green-based primers (Qiagen) for Hmox1 (QT00175994),
Hspb1 (QT00179501), and Alox15(QT00181265) were used for gene amplification. The QuantiFast SYBR Green
(Qiagen) amplification protocol included 45 cycles of 10 s at 95°C and 30 s at
60°C. A Quant-iT OliGreen ssDNA Assay Kit (Thermo Fisher Scientific) was used to
determine the amount of single-stranded DNA in each cDNA sample in a fluorometer
with excitation at 480 nm and emission at 520 nm. Relative quantification of
gene expression was corrected using the cDNA concentration ratio between the
samples.
extracted using an miRNeasy Mini kit (Cat. No. 205313, Qiagen) and quantified
using a spectrophotometer (NanoDrop 2000, Thermo scientific) at 260 nm
absorbance (n = 10/group, that was decided based on our previous experiments30
). A total of 1 μg RNA was used for complementary DNA (cDNA) generation
using a QuantiTect Reverse Transcription kit (Qiagen). The reverse transcription
protocol included 2 min at 42°C, 30 min at 42°C, and 3 min at 95°C. SYBR
Green-based primers (Qiagen) for Hmox1 (QT00175994),
Hspb1 (QT00179501), and Alox15(QT00181265) were used for gene amplification. The QuantiFast SYBR Green
(Qiagen) amplification protocol included 45 cycles of 10 s at 95°C and 30 s at
60°C. A Quant-iT OliGreen ssDNA Assay Kit (Thermo Fisher Scientific) was used to
determine the amount of single-stranded DNA in each cDNA sample in a fluorometer
with excitation at 480 nm and emission at 520 nm. Relative quantification of
gene expression was corrected using the cDNA concentration ratio between the
samples.
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