Nanodrop nd 1000 uv vis spectrophotometer

L. pentosus L33 was originally isolated from fermented sausages (Pavli et al., 2016 (link)), and was acquired by the Institute of Technology of Agricultural Products, Hellenic Agricultural Organization DIMITRA (Athens, Greece). It was maintained in de Man, Rogosa, and Sharpe (MRS) broth (Condalab, Madrid, Spain) at 37°C for 16–18 h under anaerobic conditions, prior to DNA extraction. Bacterial cells were collected by centrifugation at 8,000 g for 4 min. Total genomic DNA was extracted from the cell pellets using the NucleoSpin^® Tissue kit (Macherey-Nagel, Düren, Germany), according to manufacturer’s instructions. DNA purity and quantity were confirmed spectrophotometrically at 260 nm using NanoDrop^® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States).

RNA was prepared from whole blood collected and stored in PAXgene Blood RNA Tubes (BD, Heidelberg, Germany) using the PAXgene Blood miRNA Kit (Qiagen, Hilden, Germany). Isolation of RNA was performed using a QIAcube according to protocols provided by the manufacturer Qiagen. Purity and concentration of RNA were determined using a NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Scientific, Hennigsdorf, Germany). To ensure a consistently high RNA quality, all preparations were analyzed using RNA 6000 Nano LabChips on a 2100 Bioanalyzer (both from Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. Samples exhibiting a RNA integrity number (RIN) less than seven were excluded from subsequent analyses. The Illumina TotalPrep-96 RNA Amplification Kit (Ambion, Darmstadt, Germany) was used for reverse transcription of 500 ng RNA into double-stranded (ds) cDNA and subsequent synthesis of biotin-UTP-labeled antisense-cRNA using this cDNA as the template. Finally, in total 3,000 ng of cRNA were hybridized with a single array on the Illumina HumanHT-12 v3 BeadChips, followed by washing and detection steps in accordance with the Illumina protocol. BeadChips were scanned using the Illumina Bead Array Reader. Further details on expression data transformation and quality control are available elsewhere [11 (link)].

RNA was extracted from cell pellets using the Total RNA Purification Kit (Norgen, Canada), according to the manufacturer’s protocol. Extracted RNA was quantified using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, USA) and 300 ng of RNA were retrotranscribed to cDNA using the PrimeScriptTM RT reagent kit (Takara Bio Inc., Japan). cDNA was diluted 1/10 in ultrapure water and qPCR mix was prepared by adding cDNA (12 ng), SYBR green supermix (Bio Rad, USA), forward and reverse primers (0.2μM). The primers and their respective sequences are listed in Supplementary Table 1. qPCR protocol included one step at 95° C for 30 s, 40 cycles at 95° C for 10s, and 60° C for 1min. The results were analyzed with Bio-Rad CFX Manager software. BACT was used as a housekeeping gene to normalize gene expression.

Placentas (stored at +4 °C) were sampled within 1 h after vaginal delivery or caesarean section. Full-thickness blocks of 2–3 cm were taken from the middle region of the placenta. Collected tissue samples were washed with 1 × PBS to remove contamination of maternal blood, placed immediately into RNAlater solution (Ambion Inc, Life Technologies) and kept at −80 °C until RNA isolation. All samples were collected by the same medical personnel and using identical protocol. Total RNA was extracted from 200–300 mg of homogenized placental tissue using TRIzol reagent (Invitrogen, Life Technologies) and purified with RNeasy MinElute columns (Qiagen, Netherlands) for RNA-Seq or NucleoSpin^® II Isolation Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) for Taqman RT-qPCR according to the manufacturers’ protocols. Purity level and concentration of isolated total RNA was measured using NanoDrop^® ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific Inc., USA) and RIN (RNA integrity number) was estimated using Agilent 2100 Bioanalyzer (Agilent Technologies, USA).

Total RNA was purified from the whole cells using TRIzol reagent as per manufacturer's instructions and quantified via NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). Reverse transcription to initiate cDNA synthesis for qRT-PCR analysis was carried out using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA (5–10 ng) was mixed with 1× TaqMan Universal PCR Master Mix (Applied Biosystems) and universal TaqMan-based probes (Applied Biosystems, Supplementary Table S3). The qRT-PCR was performed on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). The recommendations of the manufacturer were followed when performing the reverse transcription and real time-PCR.

An RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) was used to isolate total RNA from the melanoma cell lines. The concentration and purity of the RNA were determined using a NanoDrop ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Bioscience, Budapest, Hungary), and only samples with a ratio greater than 1.8 (260/280 nm) were included in the analysis. The reverse transcription of total RNA (600 ng) was performed using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. qRT-PCR measurements were carried out in triplicate using the SYBR^® Premix Ex Taq™ master mix (Takara Holding Inc., Kyoto, Japan). The primer sequences of the candidate genes are listed in Supplementary Table S1. qRT-PCR was performed with a LightCycler 480 System (Roche Diagnostics GmbH, Mannheim, Germany). The PCR data were analyzed using the Livak method (2^−ΔΔCT), and glyceraldehyde-3-phosphate dehydrogenase (Hs9999 9905_m1) served as the reference gene.

DNA was extracted from 0.4 g soil taken from each layer of the soil cores obtained at −99 h, −3 h, 3 h, 24 h, and 69 h of incubation (n = 3) using a FastDNA spin kit for soil (MP Biomedical, Santa Ana, CA, USA). To remove humic substances capable of potentially inhibiting enzymatic reactions in downstream analyses^{65 (link)}, additional purification was performed using a MicroSpin S-400 HR column (GE Healthcare, Little Chalfont, UK) and a DNA clean & concentrator-5 kit (Zymo Research, Irvine, CA, USA). The concentrations of extracted DNA were measured using a NanoDrop ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).