Total RNA was isolated from Pb-exposed and non-exposed cerebral cortex tissue according to the TRIzol method (Invitrogen, Carlsbad, CA). Purity and concentration were confirmed using a Nanodrop UV/vis Spectrophotometer (Thermo Scientific, Wilmington, DE). First strand complementary DNA (cDNA) was synthesized from 1 (μg total RNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) for mRNA and NCode™ VILO™ miRNA cDNA Synthesis Kit (Invitrogen, CA) for miRNA. cDNA was amplified using real-time polymerase chain reaction (PCR). The SYBR Green quantitative real-time PCR assay was performed in 12.5 μl (20 μl for miRNA) reactions using 1 μl cDNA template, 1 X SYBR Green master mix (Applied Biosystems, Foster City, CA), 0.5 μM forward and reverse primers, and deionized H2O. The primers utilized can be found in Table 2 . Amplification was performed on ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA) following the standard protocol: 50°C for 2min followed by 95 °C for 10min, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Results were analyzed with with ViiA 7, and expression was reported relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA with the 2−ΔΔCT method for mRNA and 2−ΔCT for miRNA which was normalized to small nucleolar RNA 202 (sno202).
Nanodrop uv vis spectrophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The NanoDrop UV-Vis spectrophotometer is a compact and versatile lab instrument designed for the measurement of absorbance and concentration of various samples. It utilizes a unique sample-retention technology that allows for direct measurement of nanoliter volumes without the need for cuvettes or other sample containers. The NanoDrop spectrophotometer provides accurate and reproducible results for a wide range of applications, including DNA, RNA, and protein quantification.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Rneasy mini kit, by Qiagen (7 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (5 mentions)
Quant it rna assay kit, by Thermo Fisher Scientific (4 mentions)
Rnase a, by Qiagen (4 mentions)
Mircury rna isolation kit cell and plant, by Qiagen (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Mirneasy mini kit, by Qiagen (3 mentions)
Mirneasy micro kit, by Qiagen (3 mentions)
Fastdna spin kit for soil, by MP Biomedicals (2 mentions)
Miseq platform, by Illumina (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Small rna kit, by Agilent Technologies (2 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
0.22 μm sterile filtration systems, by Merck Group (2 mentions)
Dneasy powersoil kit, by Qiagen (2 mentions)
121 protocols using nanodrop uv vis spectrophotometer
1
Quantification of mRNA and miRNA Levels in Pb-Exposed Cerebral Cortex
2
Metagenomics DNA Extraction and Sequencing
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Total DNA extraction was performed using FastDNA® Spin Kit for Soil (MP Biomedicals). DNA concentration and purity were measured by a NanoDrop UV–Vis spectrophotometer (Thermo Fisher Scientific), and structural integrity was determined by 1% agarose gel electrophoresis. Extracted DNA was shipped to Macrogen for library preparation and sequencing. Paired-end fragment libraries with a length of 450 nt from the 16S rDNA V3-V4 region were constructed using the primers 338F ACTCCTACGGGAGGCAGCA and 806R GGACTACHVGGGTWTCTAAT. 300 nt reads of each end were sequenced from fragments (Illumina MiSeq platform). Entire genomic DNA libraries were produced and sequenced (Illumina HiSeq) in 150 nt paired ends reads for WGS.
3
Quantitative Gene Expression Analysis
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After inducing differentiation, cells were harvested at different time points. Total RNA from (both treated and control wells) the cultured cells were isolated using RNAiso Plus (Takara). RNA quality and quantity were assessed by Nanodrop UV-VIS spectrophotometer (Thermo Fisher Scientific). Two micrograms of total RNA from each sample was reverse transcribed to cDNA using Prime Script RT reagent kit (Takara). Using SYBR Premix Ex Taq (Tli RNase H Plus, Takara), RT-qPCR was performed on Applied Biosystems Step one plus PCR machine. The RNA 18S gene was amplified as an internal standard reference gene (invariant control). Fold changes in the target gene expression were normalized to 18S ribosomal RNA gene expression using comparative CT method (2−ΔΔCT method) (Schmittgen and Livak, 2008 (link)).
4
Protein Purification via Ultrafiltration and Affinity Chromatography
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The harvested media containing the secreted proteins were concentrated 10- to 20-fold on a Prep/Scale Spiral Wound 10-KDa MWCO Ultrafiltration module (Millipore, cat. # SK1P003W4). Recombinant proteins were purified from the concentrated media by immobilized-metal affinity chromatography on Ni-NTA columns (Bio-Rad, cat. # 156-0135). Purified proteins were quantified using absorbance at A280 nm (NanoDrop UV-Vis spectrophotometer; Thermo Scientific) and confirmed by Coomassie blue staining after SDS–PAGE.
5
BALF Cell DNA Extraction Procedure
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The BALF specimens were centrifuged at 350 g for 15 min, followed by washing the cell pellets with saline solution three times. DNA was extracted from washed cell pellets using the conventional phenol-chloroform extraction method. DNA extracts were quantified using a NanoDrop-UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) and stored at −80°C until use.
6
Exosomal RNA Isolation and Quantification
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Total RNA was isolated as previously described24 (link). Briefly, with the purpose of degrading the residual RNA outside the vesicles, the exosome suspension was treated with RNAse A (Qiagen NV; Germany) (100 μg/ml final reaction concentration; 15 min at 37 °C). Total RNA was obtained from exosomes using the miRCURY RNA Isolation Kit-Cell and Plant (Exiqon; Denmark). RNA concentration was calculated by using the QUBIT fluorometer and the Quant-iT RNA Assay kit (Invitrogen; California, USA). All RNA samples presented an OD 260/280 nm ratio ≥ 1,7 when using a Nanodrop UV-Vis spectrophotometer (Thermo Fisher Scientific; Massachusetts, USA).
7
RNA Quality Evaluation via Spectrophotometry and Capillary Electrophoresis
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miRNAs concentration, A260/230, and A260/280 ratios were evaluated by NanoDrop UV/Vis spectrophotometer (2000, Thermo Fisher Scientific, Waltham, MA, USA). Later, the quality and the related size of total and small RNA was measured by capillary electrophoresis with 2100 Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) using two different chips: Agilent RNA 6000 Nano Kit for total RNA and Agilent Small RNA kit for low molecular weight RNA. Electropherograms were visualized with the Agilent 2100 Expert software.
8
Radiolabeling of ICOS Antibody with Zirconium-89
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89Zr radiolabeled ICOS antibody (rat clone 7E.17G9, Bio X cell) was prepared as previously described.12 (link) Briefly, 1 mg of antibody was diluted to 1 mg/mL in PBS, and the pH was adjusted to 8.8 to 9.0 prior to addition of deferoxamine-isothiocyanate (DFO-SCN) chelate (Macrocyclics) dissolved in DMSO (Thermofisher). The bioconjugation reaction was allowed to proceed for 1 hour at 37°C, after which the DFO-modified mAb was washed using a 2 mL Vivaspin filter with a 50 kDa cutoff (Sartorius) to remove unbound chelate. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was conducted on the bioconjugates to determine final chelate:mAb ratios (1.5-2.5 chelates per antibody), and final protein concentrations were determined using a Thermo Scientific NanoDrop UV-Vis spectrophotometer. For radiolabeling, ∼1 mCi 89Zr oxalate (3D Imaging), adjusted to pH 7.1 to 7.8 using 1 M Na2CO3, was incubated with the DFO-ICOS mAb for 1 hour at 37°C with gentle agitation. Free 89Zr oxalate was removed, and 89Zr-DFO-ICOS mAb was purified using 7K MW cutoff zeba spin desalting column (Thermofisher) and centrifuged for 1 minute at 1000g. Final radiochemical purity of >99% of 89Zr-DFO-ICOS mAb was determined using instant thin-layer chromatography using chromatography strips (Biodex).
9
Quantitative Gene Expression Analysis
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RNA was isolated from each group by TRIzol method according to the manufacturer’s instructions. The yield was measured using NanoDrop UV-Vis spectrophotometer (Thermo Scientific, USA). cDNA synthesis was performed using RevertAid kit according to manufacturer’s protocol. Gene expression levels of neuronal, glial, and germ layer markers were analyzed in each group by qPCR (Mastercycler, ep realplex, Eppendorf, Germany) using qPCR Master Mix. Primer sequences corresponding to these genes are presented inTable 1 . Reaction was performed with initial denaturation at 95°C for 10 minutes followed by 40 cycles (denaturation at 95°C for 15 seconds and annealing at 58°C for 1 minute). The Ct values obtained after completing the whole reaction were used to calculate the fold change.
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RNA was isolated from each group by TRIzol method according to the manufacturer’s instructions. The yield was measured using NanoDrop UV-Vis spectrophotometer (Thermo Scientific, USA). cDNA synthesis was performed using RevertAid kit according to manufacturer’s protocol. Gene expression levels of neuronal, glial, and germ layer markers were analyzed in each group by qPCR (Mastercycler, ep realplex, Eppendorf, Germany) using qPCR Master Mix. Primer sequences corresponding to these genes are presented in
10
Quantitative Gene Expression Analysis
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Total RNA from parental hESCs and clones was isolated using TRIzol reagent (Invitrogen). The RNA quality and quantity were assessed by Nanodrop UV-VIS spectrophotometer (Thermo Fisher Scientific). One microgram of total RNA from each sample was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). Using the QuantiNova SYBR Green PCR Kit (Qiagen), RT-qPCR was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The actin gene was amplified as an endogenous reference gene. Expression of the target gene was normalized to actin gene expression and represented as fold change using comparative CT method (2-ΔΔCT method) [48 (link)].
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