Qubit dna br assay kit

All histology sections were reviewed, and the most representative was selected, marking the areas of cancer with at least 90% of tumor content for molecular analysis. Four 20 μm sections and two 10 μm sections were used for RNA and DNA extraction, respectively. Tumor material marked from each section was manually dissected. DNA was extracted using a QuickExtract FFPE DNA year.Extraction Kit (Epicentre, Madison, WI, USA). Total RNA enriched with miRNA was extracted using a RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Qubit DNA BR Assay Kit and Qubit RNA HS Assay Kit were used to evaluating DNA and RNA quantity (Thermo Fisher Scientific, Waltham, MA, USA), respectively.

A fecal suspension (10% w/v) prepared in Tris/HCl/Ca²⁺ buffer was used for nucleic acid extraction by a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s guidelines or using the silica method (Hernandez et al., 2018 (link)). The extracted RNA was quantified by a Qubit 2.0 fluorometer using the Qubit RNA BR assay kit (Thermo Fisher). Then, the samples were subjected to reverse transcription and purified using a cDNA synthesis system kit (Roche, Branford, CT, United States) to obtain pure double-stranded DNA. The DNA was quantified using the Qubit DNA BR assay kit (Thermo Fisher Scientific) and analyzed for fragmentation and quality profile using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States) as recommended by the manufacturer. Subsequently, libraries for sequencing were prepared using the Illumina Nextera XT DNA Library Prep kit and sequenced on an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA, United States) with the high-output V4 2 × 100-bp sequencing kit.

The genomes of C. sonorensis males and females were sequenced separately. Adults were separated by sex under a stereomicroscope and stored in 95% ethanol at room temperature prior to use. In order to obtain sufficient amount of DNA for genomic sequencing, DNA was extracted from 375 males in three pools of 100 and one pool of 75 individuals, and from 150 females in one pool of 100 and one pool of 50 individuals. Pooled samples were homogenised twice for 30 s in 75 μl of phosphate buffered saline using a Tissuelyser™ (Qiagen, UK) at a frequency of 25 Hz/s and a 3 mm stainless-steel ball bearing (DeJay Distribution Ltd., UK) within 2 ml screw-topped tubes. Genomic DNA extraction was conducted using gravity-flow anion-exchange tips (Qiagen’s Genomics Tip 20/G) with extraction of DNA up to 150 kb in size and a maximum of 20 μg of product. A user-developed protocol for mosquitoes and other insects was followed (see Additional file 1: Supplementary Methods). The DNA obtained from each extraction was pooled by sex and the concentration of the resulting samples was evaluated using a Qubit® 2.0 Fluorometer (ThermoFisher Scientific, UK) with the Qubit® DNA BR Assay Kit (ThermoFisher Scientific, UK). The integrity of the genomic DNA was visualized using a 5 μl sample on a 1% (weight/volume w/v) agarose gel.

RNA extraction from C. sonorensis was performed following the TRIzol® (ThermoFisher Scientific, UK) protocol (see Additional file 1: Supplementary Methods). In brief, samples were homogenised in 100 μl of Schneider’s Drosophila media (Gibco™, Thermofisher Scientific, UK) and RNA was extracted using TRIzol® and chloroform, followed by a precipitation with isopropanol. The integrity of the RNA was analysed using a Bioanalyser 2100 and the concentration was estimated on a Qubit® 2.0 Fluorometer (ThermoFisher Scientific, UK) with the Qubit® DNA BR Assay Kit (ThermoFisher Scientific, UK).
RNA was extracted from individual heads of C. sonorensis fed with blood:BTV-1 as described for the bodies (Additional file 1: Supplementary Methods). The corresponding bodies (abdomen and thorax) of the BTV-1-positive and BTV-1-negative heads were pooled separately for RNA extraction. In addition, the RNA from 100 C. sonorensis fed on horse blood 3 days post-emergence and left without access to sucrose for 8 days, and from 100 C. sonorensis fed on a 10% (w/v) sucrose solution from 3 to 8 days following emergence, were also extracted and their transcriptomes sequenced as controls.

cfTNA was extracted from 4 to 8 mL of plasma using the MagMAX Cell-Free Total Nucleic Acid Isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) or NextPrep-Mag cfDNA Automated Isolation Kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s protocol. Genomic DNA from buffy coat was extracted using the FlexiGene DNA Kit (Qiagen, Venlo, The Netherlands) or chemagic DNA Blood 400 Kit H96 (PerkinElmer, Waltham, MA, USA).
Genomic DNA of tumor tissue (both tumor and normal, if normal buffy coat DNA was absent) was extracted from ten 5 μm slices of formalin-fixed paraffin-embedded (FFPE) slides, which were macrodissected to leave only the tumor tissue, using GeneRead DNA FFPE Kit (Qiagen, Venlo, The Netherlands), according to the manufacturer’s protocol. The extracted cfTNA and genomic DNA were quantified using the Qubit DNA HS Assay Kit and Qubit DNA BR assay kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. The quality and size of extracted cfTNA were evaluated using the High Sensitivity D5000 ScreenTape (Agilent, Santa Clara, CA, USA), whereas the quality of genomic DNA was evaluated using the Genomic DNA ScreenTape (Agilent, Santa Clara, CA, USA) with TapeStation System (Agilent, Santa Clara, CA, USA).

Two milliliters of a fully grown pre-culture containing A. muciniphila in mBM was inoculated into 10 mL mBM supplemented with 0.1% ox-bile. Cell cultures were grown in triplicate under control and ox-bile conditions. After incubation at 37 °C, 7 mL of cell culture (OD₆₀₀ = ~ 1.0) was mixed with 14 mL of RNAprotect Bacteria Reagent (Qiagen GmbH, Hilden, Germany). After centrifugation at 8000×g for 10 min, the cell pellets were dissolved in 200 μL of TE buffer containing lysozyme (15 mg/mL), proteinase K (0.1 mg/mL), and mutanolysin (10 U/mL). After incubation for 40 min at room temperature, RTL buffer was added and the RNA extraction with DNase treatment was performed using a RNeasy mini kit and RNase-Free DNase Set according to the manufacturer’s instructions. RNA and DNA concentrations were measured using the Qubit RNA BR assay kit and the Qubit DNA BR assay kit, respectively (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The quality of the isolated RNA was assessed using a Qsep100 (BiOptic, La Canada Flintridge, CA, USA).