A 10mL peripheral blood from patients were collected in 5-mL EDTA tubes (BD Diagnostics) which would be processed as soon as possible within 12 hours to ensure the reliability of results. After that, the blood was layered slowly over equal volume of Ficoll medium (Solarbio, Beijing) in a 50-mL centrifuge tube (Corning, USA) and then density gradient centrifugated at 500 g for 25 minutes at 4°Cwithout brake. Peripheral blood mononuclear cells (PBMCs) and CTCs were harvested by pipetting the mid-layer, transferred into a 15-mL clean tube, and washed twice with phosphate buffered saline (PBS). Finally, cells were resuspended with PBS for further experiments.
5 ml edta tubes
Manufactured by BD
Sourced in United Kingdom
The 5-mL EDTA tubes are primary blood collection tubes designed for the collection, transportation, and storage of whole blood samples. They contain an anticoagulant, ethylenediaminetetraacetic acid (EDTA), which prevents the blood from clotting. These tubes are commonly used for hematology and other blood-based laboratory analyses.
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2 protocols using 5 ml edta tubes
1
Isolation of PBMCs and CTCs from Peripheral Blood
2
Genomic DNA Extraction and Genotyping
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Biological samples were collected as either a venous blood or saliva sample. Briefly, 5 mL of blood was collected from a superficial forearm vein by a trained phlebotomist into 5 mL EDTA tubes (BD Vacutainer Systems, Plymouth, UK). Samples were stored at −20 °C before further processing. Saliva samples were collected using Oragene DNA OG-500 collection tubes (DNA Genotek Inc., ON, Canada) following the manufacturer’s instructions and stored at room temperature until DNA extraction. Genomic DNA was extracted from the collected samples using a QIAcube, QIAamp DNA Blood Mini kit and standard spin column protocol (Qiagen, Crawley, UK). For genotyping, two techniques were used; EP1 Fluidigm (Fluidigm, Cambridge, UK) and StepOnePlus (Applied Biosystems®, Paisley, Scotland, UK). A brief description of genotyping procedures using both techniques is presented in our previous papers [36 (link),37 (link)] and the genotypes for the selected SNPs were called based on end-point fluorescence (https://www.thermofisher.com/np/en/home.html ) (attached in Table S2 ). All samples were analysed in duplicate [38 (link)].
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