Qubit fluorometric system

Our study included the whole-genome sequencing of 63 samples from 29 patients diagnosed with breast carcinoma (ethical approval: 14383-2017 ETT-TUKEB).
The study consisted of two patient cohorts, stratified based on treatment protocol. The adjuvant cohort included 16 primary tumor surgical samples of 15 patients (one with samples from two different localizations) and their 15 matched adjacent normal breast tissue samples. These patients received no chemotherapy prior to surgery. The neoadjuvant cohort consisted of 14 tumor core biopsy samples of 14 patients obtained prior to neoadjuvant chemotherapy, 14 corresponding surgical samples of adjacent normal breast tissue, and 4 additional surgical tumor samples obtained after neoadjuvant treatment.
Samples were collected between 2004 and 2017 in the adjuvant cohort and between 2012 and 2014 in the neoadjuvant cohort, at the Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, Budapest, Hungary and at the Department of Pathology, University of Szeged, Hungary, respectively. Tissue was frozen at −80 °C until DNA extraction. Genomic DNA was extracted with the QIAamp DNA Micro Kit (50), according to the manufacturer’s protocol. DNA quantification was performed with a Qubit fluorometric system (Life Technologies, Waltham, MA, USA).

Surface-sterilized seeds were ground using sterilized mortars and liquid nitrogen. Per DNA extraction, 50 seeds were used, equaling 97.5 mg of seeds. The number of seeds was estimated based on the weight of 1000 P. lanceolata seeds, which is 1.95 g for plants from “The Jena Experiment” field site [16 (link)]. For blossoms with a lower amount of seeds, all material was used. Seed DNA was extracted following a phenol/chloroform/isoamyl alcohol-based method [17 (link)]. Sample lysis was performed using Lysing Matrix E tubes (MP Biomedicals™, Germany). The beat beating was done using a TissueLyser II bead beater (QIAGEN®, Germany) at a frequency of 15 Hz for 2 min. The resulting DNA was quantified by Qubit fluorometric system (Thermo Fisher Scientific, Germany) using the broad range assay kit. The quality of the DNA was checked using the Nanodrop photometric system (Thermo Fisher Scientific, Germany) and by agarose gel electrophoresis. To exclude contamination during DNA extraction, a blank control without seed material was processed.

A label free quantitative (LFQ) MS approach was applied in order to identify differential protein levels between serum samples of asymptomatic donors (Neg n:29; PCR + n:8; IgG + n:27). Also, the protein changes in CACs after the incubation with the different sets of serum samples (CACs + Neg, n:8; CACs + PCR, n:8; CACs + IgG, n:8) were analyzed following the same LFQ approach.
Serum samples (10 μl) were supplemented with protease inhibitors (04693132001; Roche) and precipitated with acetone, over-night, centrifuged at 14,000 rpm, 25 min and the pellet resuspended in 8 M urea. Similarly, the cell pellets were resuspended in 50 μl of 8 M urea containing protease inhibitors (04693132001; Roche) for protein extraction and further proteomic analysis. For all samples, protein amount was quantified with the Qubit Fluorometric system (ThermoFisher Scientific) following manufacturer´s guidelines, and 50 µg of proteins in 8 M urea per sample were reduced (10 mM Dithiothreitol) and alkylated (50 mM Iodoacetamide). Samples were diluted four times with 50 mM ammonium bicarbonate and digested with Trypsin/LysC (V5073; Promega) (enzyme/substrate ratio 1:50) at 37 °C overnight. Finally, digestion was quenched with 0.1% TFA before peptide purification with C18 micro-columns, as described (Palmisano et al. 2010 (link)), and eluates were dried with a speed-vac system.

Total RNA was extracted using QIAGEN RNeasy (Qiagen, Mississauga, ON, Canada) according to the manufacturer’s instructions and quantified via Qubit Fluorometric system (Thermo Scientific, Waltham, MA, USA). cDNA was synthesized from 1 μg of total RNA using High Capacity cDNA Reverse Transcription kit (Thermo Scientific, Waltham, MA, USA). Quantitative reverse transcription PCR (qRT-PCR) was performed using TaqMan QuantiTect Probe kit (Qiagen, Mississauga, ON, Canada) with primers specific for CAP1 (Hs02860542_g1 ThermoFisher Scientific, Waltham, MA, USA). GAPDH (Hs99999905_m1 ThermoFisher Scientific, Waltham, MA, USA) was used as reference gene. All transcripts were measured in minimum duplicates and normalized to GAPDH. Relative CAP1 mRNA expression levels in CAP1 silenced cells compared with control were determined by 2−ΔΔCt method in three independent experiments (Additional file 1A).

Faecal sub-samples (0.25 g) were thawed and then genomic DNA extracted using the PowerSoil^® DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA) as per manufacturer instructions, with the following modifications. Prior to extraction, sub-samples were re-weighed to ensure accurate quantification. DNA was eluted in a final volume of 50 μL sterile RNA/DNA free water. DNA yields in final eluates were quantified using a Qubit fluorometric system (Thermo Fisher Scientific, Canada). All extracts were stored at -20°C prior to 16S amplicon sequencing.

Samples were processed for analysis on the NanoString nCounter Flex system using the 770 gene nCounter^® PanCancer Pathways Panel (NanoString Technologies, Inc., Seattle, WA, USA), as previously reported (17 (link)). This panel assesses 13 cancer-associated canonical pathways related to basic cancer biology (Notch, Wnt, Hedgehog, Chromatin modification, Transcriptional regulation, DNA damage control, TGF-β, MAPK, STAT, PI3K, RAS, Cell cycle, Apoptosis). Briefly, 100 ng of total RNA, quantified by a Qubit Fluorometric System (Thermo Fisher Scientific, USA), from each sample was hybridized for 21 hours at 65°C, followed by purification and RNA/probe complex immobilization in nCounter PrepStation (NanoString Technologies, Inc., Seattle, WA, USA) and cartridge scanning in a digital analyzer (NanoString Technologies, Inc., Seattle, WA, USA), according to the manufacturer’s protocol. Reading with 280 field-of-views (FOVs) was used in the study samples.