Dna blood midi mini kit

gDNA was extracted from the umbilical cord and parental blood using a QIAGEN DNA Blood Midi/Mini Kit. DNA libraries were prepared using an Agilent liquid capture system (Agilent SureSelect Human All Exon V6) according to the manufacturer’s protocol. The size distribution and concentration of the libraries were determined using an Agilent 2100 Bioanalyzer and quantified using real-time polymerase chain reaction. The DNA library was sequenced on an Illumina Hiseq 4000 or Illumina Novaseq for paired-end 150 bp-long reads according to the manufacturer’s protocol. The mean sequencing coverage on target regions of whole-exome sequencing was 103-fold. Raw image files were processed using bcl2fastq (Illumina) for base calling and generating raw data. Low-quality sequencing reads were filtered using a quality score of 20 (Q20). The average read depths were 103X for each case. The reads were aligned with the NCBI human reference genome (hg19/GRCh37) using the Burrows–Wheeler Aligner. The BAM files were subjected to SNP analysis, duplication marking, indel realignment, and recalibration using GATK and Picard.

Genomic DNA was extracted from umbilical cord blood of the proband and peripheral blood of the family members separately, using a Qiagen DNA Blood Midi/Mini kit (Qiagen GmbH, Hilden, Germany, 69,506). NanoDrop spectrophotometer and agarose gel electrophoresis were employed in determining the purity and yield of DNA products.

Genomic DNA (gDNA) was extracted from the FFPE samples using the GeneRead DNA FFPE Kit (Qiagen, United States), from the fresh tumor tissue samples using AllPrep DNA/RNA Mini (Qiagen, United States), from bulk WBC samples using the DNA Blood Midi/Mini kit (Qiagen, United States), or large DNA fragments (>500 bp) from the hydrothorax samples using the MagMAX^TM Cell-Free DNA Isolation Kit (Life Technology, United States) according to the manufacturer’s instructions, respectively. The quality of purified DNA was assayed by gel electrophoresis and quantified by a Qubit^® 4.0 Fluorometer (Life Technologies, United States).

A total of 96 patients histologically confirmed with mucinous pulmonary adenocarcinoma (MPA) were recruited from Shandong Cancer Hospital and Institute (SCH). They were diagnosed with MPA from 14, April 2015 to 19, August 2019, except for one patient who was pathologically confirmed with MPA on 26, December 2013. The last follow-up date was 22, July 2021. All the diagnoses were independently confirmed by two experienced pathologists. All patients in this study provided written informed consent and this study was approved by the Ethics Committee of SCH. It also conforms to the provisions of the Declaration of Helsinki. MPA specimens and attached non-tumor samples were obtained by biopsies. A strict quality inspection was carried out to remove contaminated and insufficient DNA samples. The overall survival (OS) time was defined as the interval between diagnosis and death, or between diagnosis and the last observation point. Clinical pathological data were retrieved from patients’ medical records. Biopsied tumor tissues were fixed with formalin, then embedded in paraffin (FFPE). Corresponding non-tumor samples were set as controls. Genomic DNA was extracted from each FFPE sample using the GeneRead DNA FFPE Kit (Qiagen, #180134, USA) and from the blood sample using the DNA Blood Midi/Mini kit (Qiagen, #51185, USA).

The genomic DNA (gDNA) was extracted from amniocytes or peripheral blood using a Qiagen DNA Blood Midi/Mini kit (Qiagen), following the manufacturer's protocol. Variant screening of the TYR gene in 12 families (family 1‐10, 19, and 20) was performed with direct Sanger sequencing. Polymerase chain reaction (PCR) primers were designed by Primer Premier version 5.0 and contained the entire coding regions and the flanking introns of the TYR gene (Table S1). The 20 μL PCR reaction mixture contained 10‐50 ng template DNA, 10 μL Premix EX Taq HS (Takara), and 1 μL of each primer. Touchdown PCR was performed as follows: 95°C for 15 minutes; 11 cycles of 95°C for 45 seconds, 60°C‐0.5°C for 45 seconds, 72°C for 45 seconds; 24 cycles of 95°C for 45 s, 54°C for 45 seconds, 72°C for 45 seconds; and 72°C for 7 minutes. The PCR products were sequenced using an ABI 3130 automated DNA sequencer (Applied Biosystems). DNASTAR Lasergene SeqMan software was used for DNA sequence assembly, and sequences were compared with a wild‐type reference sequence.

Genomic DNA was extracted from the peripheral blood (for adults) and cord blood (for foetuses) samples (200 µL each) by using the DNA Blood Midi/Minikit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's protocol.