Blackprep ffpe dna kit

FFPE tissue specimens and matched blood samples were collected from 117 patients who were diagnosed with gynecologic cancer. Tumor DNA was isolated from FFPE specimens with the blackPREP FFPE DNA Kit (Analytik Jena AG, Jena, Germany) according to the manufacturer’s instructions. Each DNA sample was obtained from four 2-μm tissue specimens and quantified with the Qubit dsDNA HS Assay kit (Life Technologies, USA) according to the manufacturer’s recommended protocol. Blood lymphocytes were isolated by centrifugation of whole blood at 1600 g for 10 min at room temperature. Tiangen whole blood DNA kits (Tiangen, Beijing, PRC) were used to extract DNA from peripheral blood lymphocytes according to the manufacturer’s instruction. Tumor DNA and matched genomic DNA were sheared into 150–200-bp fragments by a Covaris M220 Focused-Ultrasonicator (Covaris, Massachusetts, USA). Fragmented DNA libraries were constructed with a KAPA HTP Library Preparation Kit (Illumina Platform) (KAPA Biosystems, Massachusetts, USA) according the manufacturer’s instruction. DNA libraries were captured with a designed 1086-gene panel, NimbleGen SeqCap EZ Library (Roche, Wisconsin, USA), that includes major tumor-related genes. The captured samples were then subjected to Illumina HiSeq X-Ten for paired-end sequencing.

DNA extraction from FFPE tissue was performed with the blackPREP FFPE DNA Kit (Analytik Jena, Jena, Germany) and quantified with a Qubit 3.0 System using the Qubit™ dsDNA HS Assay Kit (Invitrogen, Dreieich, Germany).

Tumor cells were enriched from formalin-fixed paraffin-embedded (FFPE) tissue slides from biopsy or surgical specimens by microdissection. For the VP and ulcWGS assays, DNA was extracted with the Maxwell RSC 48 (Promega, Walldorf, Germany), using the Maxwell RSC FFPE Plus DNA Kit or the blackPREP FFPE DNA Kit (Analytik Jena, Jena, Germany), respectively. RNA for the FP assay was isolated by applying the innuPREP FFPE total RNA Kit (Analytik Jena). Nucleic acids were quantified by using a Quantus Fluorometer with the QuantiFluor ONE dsDNA System (Promega) or a Qubit device (Thermo Fisher Scientific, Waltham, MA, USA) with dsDNA HS or RNA HS assays, as appropriate.

From EDTA blood and tumor samples (primary tumor, metastasis), genomic DNA was isolated according to the manufacturers’ instructions using QIAamp DNA Blood Maxi Kit on a QiaSymphony instrument (Qiagen, Hilden, Germany) or blackPREP FFPE DNA Kit (Analytik Jena, Jena, Germany), respectively. DNA isolation of tumor DNA was performed after macro dissection by pathologist or neuropathologist. DNA quantity and quality were determined using Qubit® Fluorometer (Thermo Fisher Scientific, Dreieich, Germany) and Fragment Analyzer (AATI, Heidelberg, Germany), respectively. All coding regions and flanking intronic regions of 710 genes were enriched using Agilent in-solution bait-hybridization technology. For sequencing we used either Illumina HiSeq2500, HiSeq4000, or NovaSeq6000 systems (Illumina, San Diego, USA).