Nanodrop nd 1000 spectophotometer

Total RNA from the thigh muscles was extracted using TRIzol reagent (Invitrogen, San Diego, CA, USA). Briefly, after being mixed with TRIzol, each thigh muscle sample was transferred into a 1.5-mL microtube containing chloroform and was incubated for 5 min. Samples were centrifuged at 10,000× g (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 10 min. The pellet was precipitated using isopropanol and was washed with 75% ethanol. Each pellet was dried at 25 °C for 5 min. Then, the RNA pellet was re-suspended in 20 µL nuclease-free water.
The quality of the total RNA was assessed using a NanoDrop ND-1000 spectophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The 260:280 and 260:230 ratios of all of the samples were greater than 2.0. RNA integrity numbers were assessed using RNA 6000 Nano chips (Agilent Technologies), and all of the values were greater than 8.0.

Illumina sequencing was performed on a single DNA extract of A. solanicola that tested positive using the liberibacter generic, LG774F‐LG1463R, primer pair. The quality and quantity of the A. solanicola DNA was estimated using the Nanodrop ND‐1000 Spectophotometer (Thermo Scientific) and double‐stranded DNA (dsDNA) HS assay for the Qubit 2.0 fluorometer (Invitrogen). DNA was diluted to 0.2 ng μl⁻¹, and a library was prepared using the Nextera XT DNA Library Preparation kit (Illumina) following the manufacturer's instructions. Library quality was checked using the Qubit 2.0 fluorometer (Invitrogen, Netherlands), KAPA Library Quantification Kit (KAPA Biosystems, United States of America), and the 2200 TapeStation system (Agilent, United States of America) according to the High Sensitivity D1000 protocol. Initial paired‐end sequencing (2 × 250 bp) was performed on an Illumina MiSeq (Reagent Kit v3) to establish library quality, optimal library concentration and estimate the required sequencing depth. A final paired‐end sequencing run (2 x 150 bp) was performed on an Illumina NextSeq (Reagent Kit v2).

All samples were washed with phosphate-buffered saline solution containing 100 μg/ml tetrahydrouridine (Abcam, Cambridge, UK) and frozen at −80 °C. DNA and RNA were purified from frozen cell pellets using the All-In-One DNA/RNA Miniprep Kit (Astral Scientific, Sydney, NSW, Australia) following standard manufacturer recommendations, including RNase and DNaseI treatments to remove contaminating RNA or DNA as appropriate. Nucleic acids were quantified using the NanoDrop ND-1000 Spectophotometer (NanoDrop Technologies Inc., Thermo Fisher Scientific). Purified DNA and RNA samples were stored at −80 °C until further processing.

Quantitative PCR was performed, as we previously published [20 (link)]. Briefly, RNA was extracted from the mesothelium and mesothelioma samples using the High Pure FFPET RNA Isolation Kit (Roche). Concentration of the extracted RNA was measured with NanoDrop ND-1000 Spectophotometer (Nano DropTechnologies, Thermo Fisher Scientific, Waltham, MA). Reverse transcription was performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific) in the ProFlex PCR System Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Gene expression was analyzed using the Cobas z 480 instrument (Roche) according to the manufacturer’s instructions. Relative expression of selected genes was normalized against the endogenous control, RPLP0. The following gene expression assays were obtained from the Thermo Fisher Scientific: POU5F1 (Hs00999632_g1), NANOG (Hs04260366_g1), SOX2 (Hs01053049_s1), KRT5 (Hs00361185_m1), WT1 (Hs01103751_m1), PI3KCA (Hs00907957_m1), PIK3CD (Hs00192399_m1), AKT1 (Hs00178289_m1), AKT2 (Hs01086102_m1), AKT3 (Hs00987350_m1), BCL2 (Hs00608023_m1) and RPLP0 (Hs00420895_gH). The fold change in gene expression was calculated with the 2^−ΔΔCt method and the presented data were normalized to the mesothelium values.

Total RNA was isolated from ASCs in silica-membrane columns with the Qiagen RNeasy Mini Kit (Qiagen, Barcelona, Spain) according to the manufacturer’s instructions.
MirVana miRNA isolation kit (Life Technologies, Madrid, Spain) was used to extract miRNA from the cells, and miRNeasy Serum/Plasma Kit for the miRNA isolation from MVs, according to the manufacturer’s instructions.
RNA and miRNA quantity was determined with Nanodrop ND-1000 spectophotometer (Nanodrop Technologies, Wilmington, DE, USA). Isolated total RNA was reverse-transcribed into cDNA using a high capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA), and microRNA with the TaqMAn advanced miRNA assay (Life Technologies, Madrid, Spain). Gene expression analysis was carried out by quantitative PCR using TaqMan^® Gene Expression assays (Applied Biosystems, Madrid, Spain), and the Applied Biosystems Prism 7900HT Sequence Detection System (Applied Biosystems, Madrid, Spain) according to manufacturer’s instructions. Gene expression data are expressed as target gene mRNA expression relative to the correspondent housekeeping gene expression.