For total lung RNA preparation, the left lung was flash frozen in liquid nitrogen immediately after dissection. Frozen lungs were homogenized in 2 mL precooled RLT buffer (Qiagen, Hilden, Germany) + 1% β-mercaptoethanol (Sigma-Aldrich) using the Peqlab Precellys 24 Dual Homogenizer and 7 mL-ceramic bead tubes (#91-PCS-CK28L, Peqlab, Erlangen). 150 µL homogenate were then mixed with 550 µL QIAzol Lysis Reagent (#79306, Qiagen). After addition of 140 µL chloroform (Sigma-Aldrich), the mixture was shaken vigorously for 15 s and centrifuged for 5 min at 12,000 xg and 4 °C. 350 µL of the upper aqueous phase (containing RNA) were then further purified using the miRNeasy 96 Kit (#217061, Qiagen) according to the manufacturer’s instructions. After purification, RNA concentration was determined using a Synergy HT multimode microplate reader and the Take3 module (BioTek Instruments, Winooski, VT, USA). RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technologies).
Take3 module
Manufactured by Agilent Technologies
Sourced in United States
The Take3 module is a compact and versatile piece of lab equipment designed for Agilent Technologies. It serves as a microvolume sample handling accessory, enabling efficient and precise measurements of small-volume samples.
Automatically generated - may contain errors
Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Synergy htx plate reader, by Agilent Technologies (2 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Chloroform, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Precellys 24 dual homogenizer, by Avantor (1 mentions)
Mirneasy 96 kit, by Qiagen (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Synergy mx, by Agilent Technologies (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Magna pure lc rna isolation kit high performance, by Roche (1 mentions)
Lysis binding buffer, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Superscript 3 first strand synthesis supermix for rt qpcr, by Thermo Fisher Scientific (1 mentions)
Cfx96 system, by Bio-Rad (1 mentions)
6 protocols using take3 module
1
Lung Total RNA Extraction Protocol
2
RNA Extraction and Sequencing Protocol
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RNA was extracted using Trizol® Reagent (Life Technologies) according to the manufacturer’s protocol. Dissected tissues and entire females were initially crushed with pestles in Trizol reagent before proceeding with the extraction protocol. RNA concentration and quality were evaluated using standard procedures: Take3 Module (BioTek SynergyHT) reading and gel electrophoresis. RNA samples were treated with DNAseI (Ambion). Amounts of total RNA used to synthesize mRNA TruSeq libraries were the following: 500 Antennae (A): 1.4 μg; 500 Antennae (B): 1.9 μg; 500 Palps (A): 500 ng; 500 Palps (B): 400 ng 10 Female Body (A): 4.5 μg; 10 Female Body (B): 4.2 μg; 15 Male Heads (A): 3.3 μg; 15 Male Heads (B): 3 μg. cDNA library preparation, including fragmentation and barcoding (ligation of specific adapters), was performed following the Illumina TruSeq RNA Library v2 protocol (Illumina, San Diego, CA, USA). After quality evaluation with an Agilent 2010 Bioanalyzer, as recommended for Illumina sequencing, the libraries were diluted with an elution buffer and loaded on an Illumina HiSeq2000 for sequencing (paired-end, PE, 2 × 100 bp), and each cDNA library was sequenced.
3
RNA Isolation and Quality Evaluation
4
RNA Isolation from Stimulated Cells
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Cells were harvested from three replicates for each condition at 8 and 24 h stimulation and centrifuged 5 min at 300×g. The pellet was suspended in 200 µl PBS and lysed in 550 µl Lysis/binding buffer (Roche) supplemented with 1% DTT (Sigma Aldrich). Cell lysates were stored at − 80 °C until RNA isolation with the MagNA Pure LC RNA isolation kit-High performance (Roche) on the MagNA Pure LC 2.0 instrument following the manufacturer’s protocol. Isolated RNA samples were quantified using UV spectroscopy (Take3 module, Synergy Mx, BioTek, Winooski, USA). RNA integrity was analysed by agarose gel electrophoresis using 2% E-Gel EX pre-stained gels (Thermo Fischer Scientific).
5
Gene Expression Analysis in Human Tissues
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RNA was isolated from tissues using the RNeasy Mini Kit (Qiagen, Hilden, Germany) per the manufacturer’s protocol and was quantified on a Synergy HTX plate reader equipped with a Take3 module (BioTek, Winooski, VT, USA). RNA quality was evaluated based on a 280/260 ratio. cDNA was synthesized using the SuperScript III First-Strand Synthesis Supermix for RT-qPCR (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) per the manufacturer’s protocol except half volumes of 2X RT Reaction Mix and RT Enzyme Mix were used for a final volume of 10 µL. RT-qPCR reactions were performed using a BioRad CFX96 system (BioRad Laboratories, Hercules, CA, USA). The thermocycler was programmed for 1 cycle 95 °C for 1 min initial denaturing, 40 cycles 95 °C for 15 s denaturing, 60 °C for 30 s annealing/elongation, and a melt curve 65–95 °C/0.5° per 5 s for validating single product amplification. Human CYP1A1, CYP1B1, ALDH3A1, GSTA, HMOX1, NQO1, GJA1, TJP2, and DDB2 levels were normalized to PPIA using the ΔΔCt comparative method with primers from Invitrogen (Table S3 , Invitrogen, Waltham, MA, USA).
6
Characterizing EETg Phenolics and Infrared Profile
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EETg was characterized in relation to its phenolic composition by Folin-Ciocalteu colorimetric method (Pessoa et al., 2016) (link) as well as infrared spectroscopy (Gonzaga et al., 2013) (link). The total phenol content was determined by reaction with Folin-Ciocalteu reagent followed by absorbance reading at 700 nm in a spectrophotometer (Epoch, Take 3 module, BioTek, Winooski, USA). The analysis was performed in triplicate and results expressed in micrograms of gallic acid equivalents per milligram of extract. Afterwards, the extract was prepared in KBr pellets. The spectrum profile in infrared region was obtained using a Shimadzu FT-IR spectrophotometer model 8300 (Shimadzu Corporation, Kyoto, Japan). The maximum absorption was reported in wavenumbers (cm -1 ) which was then used to determine organic groups indicative of secondary metabolites present in EETg.
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