Total RNA was isolated from human BBB brain microvascular endothelial cells (BMECs) using a QIAGEN RNeasy mini kit (Qiagen, Inc., Valencia, CA, USA). RNA purity and concentration were measured using Nanodrop (NanoDrop 1000 UV-VIS Spectrophotometer Software Ver.3.8.1, Thermofisher Scientific). A ratio of 260/280 of 2.0 was considered adequate for analysis. Reverse transcription to cDNA was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Thermofisher Scientific, Waltham MA, USA) in a total reaction of 20 μL. Quantitative real-time PCR was performed using Taqman fast advanced master mix (Applied Biosystem, Thermofisher Scientific, Waltham MA, USA) and TaqMan Gene Expression assay (Applied Biosystem, Foster City, CA, USA) specific for: Claudin 4 (Hs00976831_s1), ZO-1 (Hs01551871_m1), and internal control beta actin (Actb) (Hs01060665_g1). RT-PCR was run using the StepOnePlus Real-Time PCR System. Calculation of relative mRNA expression was performed using delta–delta CT and presented as relative fold-change to the control group.
Nanodrop 1000 uv vis spectrophotometer software ver 3
Manufactured by Thermo Fisher Scientific
The NanoDrop 1000 UV-VIS Spectrophotometer Software Ver.3.8.1 is a compact and efficient instrument used for the quantification and analysis of nucleic acids and proteins. It measures the absorbance of a small sample volume (1-2 μL) across a wide range of the UV-Vis spectrum. The software version 3.8.1 provides the necessary functionality to operate the instrument and analyze the obtained data.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
2 protocols using nanodrop 1000 uv vis spectrophotometer software ver 3
1
Quantification of Tight Junction Proteins
2
Quantifying Immune Markers in Dendritic Cells
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Total RNA was isolated from DCs in vitro and from gingival tissue of the experimental groups used for in vivo studies using QIAGEN RNeasy mini kit (Qiagen, Inc., Valencia, CA, and USA). RNA concentration and purity were assessed with Nanodrop (NanoDrop 1000 UV-VIS Spectrophotometer Software Ver.3.8.1, Thermofisher Scientific). Ratio of 260/280 of 2.0 was deemed acceptable for further analysis, and was reverse transcribed to cDNA. Amplification by PCR was performed using the High-Capacity cDNA Reverse Transcription Kit and PCR in total reaction of 20μL. Quantitative real-time PCR was performed using TaqMan gene expression primers specific for IL6 (Mm00446190_m1), IL12 (Mm01288989_m1), IL23 (Mm00518984_m1), TGFB1 (Mm01178820_m1) and TNF (Mm00443258_m1) IL10(Mm01288386_m1), FOXP3 (Mm00475162_m1), CTLA4 (Mm00486849_m1), Rankl (Mm00441906_m1) and Beta Actin (Mm02619580_g1), all from Thermofisher Scientific. RT-PCR was run in StepOnePlus Real-Time PCR System. Relative gene expression was calculated using delta-delta CT and plotted as relative fold change.
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