Nanodrop one onec microvolume uv spectrophotometer

In this prospective and consecutive study, we used 78 PA patients recruited from 2018 to 2019 with confirmation by histopathology and/or surgery provided from Academic Biobank of Research on Cancer from the University of São Paulo, located in Centro de Investigação Translacional em Oncologia, Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo, Brazil. The Biobank protocol was approved by the Local Ethics Committee (CEP no. 031/12 and National Ethics Committee (CONEP no.023/2014). As control we used 256 subjects with non-pancreatic cancer, healthy blood donors or orthopedic patients provided from Hospital do Trabalhador, Curitiba PR, Brazil, with Local Ethics Committee (CEP CAAE no. 77979417.8.0000.5248 and 77979417.8.3001.5225) and National Ethics Committee (CONEP 77979417.8.0000.5248) approval. All approvals contemplated demographic and epidemiological data collection for both groups, while for PA cases clinical data were also collected. For all participants, the project was described and informed consent form was obtained in writing format. All the participants had 4 mL of peripheral blood collected and buffy-coat DNA was extracted with QIAmp DNA Blood Mini Kit (QIAGEN) as indicated. A quantification and purity of DNA were performed using NanoDrop One/OneC Microvolume UV Spectrophotometer® (Thermo Scientific).

mRNA and small RNA were extracted from tentacle and trunk tissues of two individuals using TRIzol (Ambion) and the miRVana Isolation kit, respectively. The concentrations were measured using a NanoDrop One/One C Microvolume UV Spectrophotometer (Thermo Fisher Scientific, Fitchburg, WI, USA). Agarose gel electrophoresis was used to assess the quality. Samples were stored at −80°C and sent to Novogene (Hong Kong) for transcriptome (Novaseq, PE150 platform for strand-specific library construction) and small RNA library (Novaseq, 50SE platform for strand-specific library construction) sequencing (electronic supplementary material, S2).

After cells were transferred to a lysis solution, RNA was isolated with a mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and purified with DNAse I. The initial RNA concentrations were measured with NanoDrop One/OneC Microvolume UV Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). At least 60 ng of each RNA was used for further experiments. Total miRNA libraries were prepared with an miRNA Library kit and miRNA NGS 12 Index IL kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s instruction. Quality assessment of the miRNA libraries was performed with the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and High Sensitivity DNA chips (Agilent, Santa Clara, CA, USA). Final library concentrations were measured with a Qubit dsDNA High Sensitivity Kit (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced on the MiSeqDx Instrument (Illumina, San Diego, CA, USA).