Mirneasy serum plasma kit

Total RNA was extracted using the miRNeasy Serum/Plasma Kit (Qiagen, catalogue number 217184, Milan, Italy). Serum was thawed on ice and centrifuged at 3000 × g for 5 min at 4 °C. An aliquot of 200 μl per sample was transferred to a new tube, and RNA was extracted using miRNeasy Serum/Plasma Kits (Qiagen, catalogue number 217184, Milano, Italy) in accordance with the manufacturer’s instructions. Animals were divided into six pools of 5 by the presence or absence of Brucella infection, and the pools were sequenced.
Libraries were prepared using TruSeq SmallRNA Sample Prep kits (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. Both RNA samples and final libraries were quantified using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and quality tested by the Agilent 2100 Bioanalyzer RNA Nano assay (Agilent Technologies, Santa Clara, CA, USA). Libraries were then processed with Illumina cBot for cluster generation on flowcells, following the manufacturer’s instructions and sequenced on single-end mode at the multiplexing level requested on HiSeq. 2500 (Illumina, San Diego, CA). The CASAVA 1.8.2 version of the Illumina pipeline was used to process raw data for both format conversion and demultiplexing^{58 (link)}. The reads obtained were mapped to the Bos taurus database because the complete Bubalus bubalis genome is lacking.

Total RNA was extracted from 200 μL plasma samples of OC patients and healthy controls using the miRNeasy Serum/Plasma Kit for purification of total RNA, including miRNA (Qiagen, Germany) and QIAzol Lysis Reagent (Qiagen, Germany) according to the manufacturer's instructions. The proposed method for the extraction of total RNA from plasma is as follows. A five times volume of QIAzol Lysis Reagent and an equal volume of chloroform were added to the plasma sample, then centrifuged for 15 min at 12,000 g at 4 °C. One and a half times the volume of 97–100% ethanol was added to the resulting double supernatant and applied to mini-columns from the miRNeasy Serum/Plasma Kit (QIAGEN), washing the membrane from unbound biopolymers and chemical agents. The sorbent was washed to remove impurities of non-nucleotide biopolymers and chemical agents RWT and RPE with buffer solutions according to the manufacturer's instructions. After that, the total RNA were eluted with 14 μm RNase-free water from the sorbent, with incubation for 10 min on ice between the elution steps. RNA purity and concentration were determined using a Nano-Drop 2000 (Thermo Scientific) and consistently yielded A260:A280 and A260:A230 ratios close to 2.0. All isolated total RNA was stored at a −80 °C freezer until use.

Serum RNA was extracted using miRNeasy Serum/Plasma Kit (Qiagen, Germany). Since our commen use housekeping genes may change its expression in tumor serum, thus we introduced an external reference, pGL3 [5 (link)]. The pGL3 (1 ng, approximately 2 × 10⁸ copies) was added to serum samples according to the manufacturer’s protocol using an miRNeasy Serum/Plasma Kit (Qiagen, Germany). The extracted serum RNA was reverse transcribed using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). Forward (F) and reverse (R) primers were synthesized by TSINGKE Biological Technology Company (China), as follows: LOC284454-F, 5′-ATTACAGGTGGCTCAGGTGT-3′, LOC284454-R, 5′-CTTCAGTGTGCCTCCTCAGT-3′; and pGL3-F, 5′-TCCATCTTGCTCCAACACCC-3′, pGL3-R, 5′-TCGTCTTTCCGTGCTCCAAA-3′. The probe sequences were as follows: LOC284454-P, 5′-FAM-CGTGCCTGGCTTTTCTCCACTATCTTG-BHQ1–3′ and pGL3-P, 5′-HEX-ACGCAGGTGTCGCAGGTCTTCC-BHQ1–3′. Conventional SYBR-qPCR was performed using iTaq universal SYBR Green Supermix (Bio-Rad, USA). TaqMan-qPCR was performed using iTaq Universal Probes Supermix (Bio-Rad,USA). All RT-qPCR procedures were performed using a Bio-Rad CFX96 Multicolor Real-time PCR Detection System. TaqMan-qPCR allowed the simultaneous detection of two probes in the same tube (Bio-Rad, USA).

Small RNA was extracted from 200 μL plasma or serum samples using miRNeasy Serum/Plasma Kit (Qiagen, Germany), according to the manufacturer’s instructions, and the RNA was resuspended in 12 μL of RNase-free water and then stored at −80 °C until library preparation.
Small RNA libraries were prepared using the QIAseq miRNA Library Kit (Qiagen, Germany) according to the manufacturer’s protocol. Briefly, RNA samples were ligated with 3′ and 5′ adapters. After ligation, reverse transcription was done. The cDNA was purified using magnetic beads, then eluted with 17 µL nuclease-free water. The cDNA samples were used as templates for subsequent PCR. The samples were barcoded by unique indexes during the PCR amplification to allow pooling of libraries before sequencing. The PCR products were cleaned up using the magnetic beads and eluted with 25 µL nuclease-free water. For assessment of the yield, size distribution, and molar concentration of the amplified DNA libraries, the samples were run on 2200 Tapestation Instrument with High Sensitivity D1K ScreenTape and High Sensitivity D1K Reagents (all purchased from Agilent Technologies, Santa Clara, CA, USA). The quantity of libraries required for sequencing was determined according to the manufacturer’s protocol using the miRNeasy Serum/Plasma Kit (Qiagen, Germany).