Nucleospin plasma xs kit

In the adolescent cohort, from 75 subjects enrolled in the 10-year follow-up, genomic DNA was isolated from the blood with QiAmp DNA Blood Mini Kit 250 (Qiagen, Germany) [18] (link). From original study in 1997, we randomly selected 280 subjects whose sera were enough for DNA isolation. The DNA isolation was made by using NucleoSpin Plasma XS kit (Macherey-Nagel GmbH&Co, Germany). In infant cohort, genomic DNA was isolated from 213 subjects as described earlier [26] (link). Genotyping of the three MBL2 polymorphism sites on exon1; codons 52, 54 and 57, were done with single pyrosequencing reaction. Before pyrosequencing, PCR reaction and the cycles were performed as described by Roos et al. (2006) [27] (link). The PCR product was verified with agarose electrophoresis, the specific band for MBL was 240 bp. Pyrosequencing of MBL2 was performed as described previously [27] (link) with minor modifications described earlier [18] (link). The MBL genotypes were categorized as A/A as the wild type, heterozygotes variants as A/O and homozygotes variants as O/O. O stands for any variant alleles B, C or D.

Preoperative blood samples were usually collected on the day prior to RC at a median of 39 days [interquartile range (IQR): 27; 61] after the preceding TURB. Serum and plasma were prepared from 6 ml whole blood. cfDNA was extracted from serum and plasma using diverse DNA extraction kits (i.e., QiAmp DNA Blood Mini kit, Qiagen, Hilden, Germany; QiAmp Circulating Nucleic Acid kit, Qiagen; NucleoSpin Plasma XS kit, Macherey Nagel, Düren, Germany; PME free-circulating DNA Extraction kit, Analytik Jena, Germany). cfDNA was extracted from 2 ml serum or plasma as well as leukocytes (reference) from 6 ml EDTA blood, and performed according to the manufacturer´s instructions. Quantification and quality of the extracted cfDNA were determined spectrophotometrically using the NanoDrop Spectrometer ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA).

Blood samples were immediately centrifuged at 1200 g
for 15 minutes and the top plasma layer was centrifuged
at 16000 g for 10 minutes. To check plasma hemolysis,
the absorbance of plasma samples at 414 and 375 nm
was measured and A414/A375 <2 was considered as
hemolysis free plasma. Plasma cfDNA was isolated
using the NucleoSpin® Plasma XS kit (Macherey-Nagel,
Germany) based on the manufacturer’
s procedures with
several modifications (28 ).

cfDNA was extracted using the NucleoSpin Plasma XS kit (Macherey Nagel) with optimized manufacturer’s protocols. Fresh tissue DNA and whole blood DNA were extracted using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s protocols. FFPE samples were de-paraffinized with xylene and DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s protocols. Purified DNA was qualified by Nanodrop2000 (Thermo Fisher Scientific) and quantified by Qubit 2.0 using the dsDNA HS Assay Kit (Life Technologies) according to the manufacturer’s recommendations.
The size distribution of cfDNA was analyzed by the Agilent Technologies 2100 Bioanalyzer using the Agilent High Sensitivity DNA kit (Agilent Technologies) according to the manufacturer’s instructions. For cfDNA samples contaminated with genomic DNA, size selection was performed using Agencourt AMPure XP beads (Beckman Coulter) according to the manufacturer’s recommendations.

After collection all CSF samples were centrifuged at 1200 rpm for 20 min at 4 °C, aliquoted and kept frozen at − 80 °C until analysis. Each 225 µl of CSF supernatant/standard was filtrated using Millex-HV Syringe Filter Unit (Merck KgaA, Germany) with a pore size of 0.45 μm and digested with 2U of TURBO DNase (Thermo Fisher Scientific, USA) for 30 min.
Next, 250 μl of filtrated and digested CSF/standard were subjected to RNA extraction with TRIzol LS (Thermo Fisher Scientific, USA) or DNA extraction using NucleoSpin Plasma XS kit (Macherey–Nagel, Germany), following manufacturers' protocols. RNA and DNA were eluted in 5 μl and 12 μl of water, respectively.

ccfDNA from hemolymph (5 ml) was carried out using the NucleoSpin Plasma XS kit (Macherey Nagel) with optimized manufacturer’s protocols. Purified DNA was quantified by the PicoGreen assay according to the manufacturer’s recommendations [21 ].

This study was approved by the Ethics Review committee of Peking Union Medical College. HCC patients were eligible if they agreed to undergo a circulating cfDNA assay on plasma obtained before surgery and provided signed informed consent. No more restriction was existed. The peripheral blood was drawn into EDTA tubes and within 1 hour subjected to centrifugation at 800g for 10 min. The plasma was separated from blood cells and subjected to an additional centrifugation step at a high speed of 16,000g for 10 min to remove any remaining cellular debris [26 (link)]. The plasma supernatant and matched blood cells were stored separately at −80°C. Corresponding tumor samples, about 1 cm*1 cm, were taken from the central part of the tumor tissue and frozen in liquid nitrogen immediately after surgery, and then were transferred to −80°C for storage. The necrotic tissue was also avoided when tissue samples were obtained. The pathologic diagnosis of tumor was based on histologic criteria [27 (link)].
DNA was extracted from frozen tissue and matched blood cells using the QIAamp DNA mini kit (Qiagen Co. Ltd, DE). Circulating cfDNA was extracted from 720μl of plasma per sample using the NucleoSpin Plasma XS kit (Macherey & Nagel GmbH & Co. KG, DE) strictly following the manufacturer's instructions.