Nanophotometer c40

DNA was extracted from breast tissue and blood sample using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, USA) and ReliaPrep™ Blood gDNA isolation kit (Promega, USA) respectively according to manufacturer protocols. The concentration and quality of DNA was measured using NanoPhotometer C40 (Implen, Germany).We have developed a panel comprised of four high impact genes in breast cancer using high throughput sequencing technology. We have designed (using Primer 3 plus software, IDT and UCSC Genome Browser) 52 sets of primers targeting all exons and splicing junctions of BRCA1, BRCA2, TP53 and ERBB2 genes (Additional file 1). In total, 13 sets of multiplex PCR were carried out to amplify all the 52 amplicons (Additional file 1: Table S1-S5) using GoTaq® Hot Start Colorless Master Mix and GoTaq Long PCR Master Mix (Promega, USA). The amplicons were visually confirmed by 0.8% agarose gel electrophoresis. After confirmation, amplicons were purified using the Agencourt AMPure XP PCR purification bead (Beckman Coulter, Pasadena, CA) and quantified using the QuantiFluor® ONE dsDNA System (Promega, USA). Then 1 ng target amplicons were used for library preparation. Nextera XT library preparation kit (Illumina, Inc., San Diego, CA), using the manufacturer’s recommended protocol, was used for library preparation.

DNA was extracted using ZymoBIOMICS™ DNA Miniprep kit (Zymo Research, USA). Briefly, 750 µL of fecal suspension were lysed in a ZR BashingBead™ lysis tube using TissueLyser LT (Qiagen, Germany) at 50 Hz for 3 min. The cell lysate was then extracted following the manufacturer’s instruction. The concentration of DNA was determined using A₂₆₀/_280 nm by NanoPhotometer® C40 (Implen, Germany).

RNA extraction was performed using the PureLink RNA Mini Kit (Thermo Fisher Scientific, Life Technologies, Waltham, MA, USA) and random hexamers according to the manufacturer’s instructions. The quantity and quality of the RNA samples were determined using a NanoPhotometer^® C40 (Implen, München, GermanyUSA). Total RNA (500 ng/20 µL) was transcribed into complementary DNA (cDNA) using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Life Technologies, Waltham, MA, USA) according to the manufacturer’s protocols. The reaction parameters were: 25 °C for 5 min, 60 °C for 60 min and 70 °C for 5 min. The cDNA samples were stored at −80 °C until real-time PCR analysis was performed.

The individual large roundworm was homogenized by high-speed shaking at 50 Hz for 5 min in plastic tubes with lysis buffer and small stainless beads using TissueLyser LT (Qiagen, Germany). The genomic DNA was extracted from human feces and intestinal tissue of large roundworm samples using the GenUP gDNA extraction kit (Biotechrabbit, Germany) following the manufacturer’s recommendations after removal of small amounts of debris by centrifugation at 13,000 rpm for 5 min. The concentrations and quality of DNA were measured using a NanoPhotometer C40 (Implen, Germany).

For the quantitative determination of G6PD activity, the Glucose-6-Phosphate Dehydrogenase Reagent Set was used (Pointe Scientific, catalog G7583180). Following the manufacturer’s protocol, 10 μL of whole unwashed RBCs were assayed using a heated cuvette spectrophotometer (Nanophotometer C40, Implen); hemoglobin (g/dL) was measured using approximately 70 μL whole, unwashed RBCs on an ABLX90 (Radiometer). Results are presented as G6PD activity (U/g hemoglobin).

The total RNA was extracted from samples (egg, cercaia, and adult parasites). All samples were lysed by lysis buffer and homogenized by TissueLyser LT (Qiagen, Germany) at 50 Hz for 5 min using RNeasy Mini Kit (Qiagen, Germany) following the manufacture’s instruction. RNA concentrations were measured using NanoPhotometer^® C40 (Implen, Germany) and Qubit RNA HS assay kit (Invitrogen, USA). To ensure the quality of RNA before proceeding with library preparation and sequencing, the integrity and concentration of RNA were assessed using a Bioanalyzer. Only RNA samples that were passed the quality criteria specified by Vishuo Biomedical Pte., Ltd. for small RNA sequencing were used.