Quibit fluorometer

Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the FlexiGene DNA kit (Qiagen) according to the manufacturer’s instructions. The DNA was quantified by fluorescence using the Quibit Fluorometer (Invitrogen) and the quality assessed by running <500 ng on a 1% TAE agarose gel for 1 h at 70 V. Whole genome sequencing was performed on 3.5–7.5 ng DNA on either the Illumina HiSeq2000 or the HiSeq2500 run in standard mode using v2.5 or v3 sequencing chemistry (Core Genomics, WTCHG). Briefly, the genomic DNA was fragmented, end-paired, A-tailed and adapter-ligated before size selection and amplification for a multiplexed library preparation. The libraries were then paired end sequenced and sequenced to an average coverage between 27 and 40 quality reads per base (100 base pair reads).

After human fibroblasts were incubated with predetermined concentrations of BPE-CO₂A for 48 hours, total RNA was extracted using TriReagent Solution (Applied Biosystems) and quantified using a Quant-iT RNA Assay Kit (Invitrogen) and a Quibit Fluorometer (Invitrogen). The tests were conducted in a StepOnePlus sequence detection system (Applied Biosystems).
The gene expression of RAR, RXR, and EGFR was evaluated using a commercially available kit (TaqMan RNA-to-CT 1-Step, Applied Biosystems) and probes (TaqMan Gene Expression Assays: RAR: Hs0023097_m1; RXR: Hs01067635_m1; EGFR: Hs01075087_m1; B2M: Hs00984230_m1, Applied Biosystems). The B2M (beta-2-microglobulin) gene was used as a reference (endogenous control). The RT-PCR conditions were 48°C for 15 min for reverse transcription and 95°C for 10 minutes for the activation of the Ultra-Pure AmpliTaq Gold DNA Polymerase, followed by 40 cycles of 94°C for 15 seconds and 60°C for 1 minute for denaturation and annealing, respectively.
The relative amount of mRNA was calculated with the 2-ΔΔCT method. Gene expression was considered significant when the expression values were greater than 1.5 times compared to the control. For expression inhibition, values less than 0.5-fold were considered relevant.

Total RNA was isolated from homogenized placental tissue using the RNeasy Mini Kit (Qiagen, Valencia, CA) and stored in RNAse-free water at −80 °C. The yield was quantified using a Quibit Fluorometer (Thermo Scientific, Waltham, MA) and the integrity was assessed using an Agilent Bioanalyzer (Agilent, Santa Clara, CA). Ribosomal RNA was removed using a Ribo-Zero Kit [57 ]. RNA was converted to cDNA using random hexamers (Thermo Scientific, Waltham, MA). Transcriptome-wide 50 bp single-end RNA sequencing was conducted using the HiSeq 2500 platform (Illumina, San Diego, CA) [58 ]. Samples were run in three sequencing batches, with 10% of the samples run in triplicate within each batch.

Three biological replicates, corresponding to a pool of leaves from four plants for the distal sampling, and to individual stems (−1 cm) for the local sampling, were used for RNA extraction. Total RNA was isolated using a CTAB-based extraction protocol followed by a DNase treatment as described in Blanco-Ulate et al. (2013b (link)). RNA concentration and purity were measured using a Quibit fluorometer (Thermo Scientific) and a NanoDrop 2000c Spectrophotometer (Thermo Scientific), respectively. Libraries were prepared using the Illumina TruSeq RNA sample preparation kit v.2 (Illumina, CA, USA). Final libraries were evaluated for quantity and quality using the High Sensitivity DNA kit on a Bioanalyzer 2100 (Agilent Technologies, CA).

The samples collected in replicate, yeast cells C0 and P0 (0 h), and C8 and P8 (8 h) were centrifuged at 3g at 4 °C for 10 min, and washed three times with PBS buffer. Total RNA from these two samples was isolated using RNeasy mini kit (QIAGEN—Hilden, Germany), according to the manufacturer’s protocol. The amount of RNA in each sample was determined using a Quibit fluorometer (Thermo Fisher Scientific—Waltham, Massachusetts, EUA), with the quality determined based on the RIN value and by visualization using a BioAnalyzer (Agilent Genomic—Santa Clara, CA, EUA). Purified total RNA was fragmented (fragments of approximately 76–200 bp) in a fragmentation buffer Illumina (San Diego, California, EUA). First-strand cDNA was synthesized using random hexamer primers. Double-stranded cDNA was purified using QuiaQuick PCR extraction kit (QIAGEN), which was followed by end-polishing. Sequencing adapters Illumina were added to the ends of RNA fragments, and the fragments were enriched by PCR amplification. Finally, the DNA library products were sequenced using an Illumina MiSeq platform.