Paxgene tissue dna kit

Patients undergoing coronary artery bypass graft surgery at Beth Israel Deaconess Medical Center were recruited to participate in a study of DNA methylation and atrial fibrillation. PBL, atrium and artery tissue were collected from 18 participants using PaxGene tubes for blood and PaxGene (Qiagen, Valencia, CA, USA) tissue containers. After surgery, six participants developed atrial fibrillation. Blood DNA was extracted using the PaxGene Blood DNA Kit (Qiagen, Valencia, CA, USA); atrial and artery tissue DNA was extracted using the PaxGene Tissue DNA Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Among the four samples with duplicates, correlation R² between duplicates was >99%. For 18 participants, one or more tissue types were available. There were 14 individuals with methylation measures in all three tissues that met quality control standards and were included in downstream analysis.

Genomic DNA was extracted from pre-clonal and clonal cell lines using PAXgene Tissue DNA Kit (Qiagen, USA). 200 ng of good quality DNA from each sample was submitted to Sandor Proteomics (Hyderabad, India) for sample preparation and genome wide SNP array using Illumina Infinium assay (Human660W-quad BeadArray chip) following manufacturer’s standard protocol. Array data was pre-processed using GenomeStudio (Illumina Inc., USA) for quality control check. To retain only good quality genotyping calls, a threshold GenCall score of 0.25 was used across all samples. A total of 396, 266 SNPs were retained after this filtering. These SNPs were then used for copy number analysis using Genome Studio plug-in cnvPartition 3.2 and an R package Genome Alteration Print (GAP) [20 (link)]. Inferred copy numbers were then annotated with genomic features using BedTools (v. 2.17.0) [21 (link)]. Copy number segments of more than 10 Mb in size were classified as arm-level amplifications and were identified as non-significant alterations. Focal amplifications (less than 10 Mb) were used for further analysis.

Tumor samples containing >60% tumor cellularity were selected for DNA extraction using PAXgene Tissue DNA kit (Qiagen). Gentra Puregene Blood Kit (Qiagen) was used to extract genomic DNA from blood. Extracted DNA was quality controlled for its purity, quantity, and integrity. Identity of each extracted DNA sample was tested using AmpFlSTR Identifiler PCR Amplification Kit (Thermo Fisher Scientific). Samples that match >80% of the short tandem repeat profiles between tumor and germline DNA were considered authentic. RNA was extracted from PAXgene fixed tissues using the PAXgene Tissue RNA kit (Qiagen). RNA integrity (RIN) was determined for all samples by the RIN score given by the TapeStation (Agilent) read out. RNA samples that had RIN scores of 4 and above were included in downstream sequencing analysis.

In the germline ADO cohort of 290 LFS/LFL cases, the genomic DNA was extracted from peripheral blood lymphocytes by Qiagen columns (QIAamp DNA Blood Mini Kit; Cataloque number 51106) according to manufacturers protocol or in some cases by conventional phenol chloroform method. The entire coding region of the TP53 gene was sequenced by Sanger sequencing. If no germline TP53 mutation was identified, Large Genomic Rearrangement analysis was done by the Multiplex Ligation dependent Probe Amplification (MLPA) kit (MRC Holland) as per manufacturer’s instructions. In selected LFS/LFL cases without an identified TP53 mutation on Sanger Sequencing and MLPA, either germline exome sequencing was done (n = 3) or targeted re-sequencing with multigene NGS panel in commercial laboratories (n = 8). In the Oral Cancer somatic ADO cohort, DNA was extracted from peripheral blood and tumour tissue with at least 60% viable tumour, using Qiagen columns (PAXgene Tissue DNA Kit (50). Exome sequencing was done on paired DNA from tumour and blood as described earlier^{13 (link)}. In addition, Sanger sequencing for the entire TP53 gene was done on tumour DNA for validation.

The BRAFV600E mutation was also included in the statistical analyses since its presence increases the recurrence risk according to the current ATA guidelines [4 (link)]. However, since molecular analyses of thyroid cancers started to be performed in our center in 2011, BRAF mutation analysis could be performed on only 455 of the 706 patients constituting the study sample. The records of the molecular analysis of the BRAF gene were obtained from the Department of Medical Genetics and the Laboratory of Molecular Pathology of the Department of Pathology. In these 455 patients, the tumor tissue containing at least 10–30% of tumor cells was isolated from the sections of the largest tumor foci. Subsequently, DNA purification was performed using the nucleic acid isolation kit for paraffin-embedded tissue (QIAamp^® DNA FFPE Tissue Kit (50), QIAGEN (Hilden, Germany) Catalog No. 56404, EZ1^® DNA Tissue Kit (48), QIAGEN (Hilden, Germany) 953034, PAXgene^® Tissue Containers (10), QIAGEN (Hilden, Germany) Catalog No. 765112, PAXgene Tissue DNA Kit (50), QIAGEN (Hilden, Germany) Catalogue No. 767134). Per the polymerase chain reaction (PCR) procedures, pyrosequencing analysis was performed on PyroMarkQ24, using sequencing primers including the Seq Primer BRAF 600E or Seq Primer BRAF 600E 464–469 (QIAGEN Catalogue No. 970470).

Breast tumors were selected for sequencing following the TCGA guidelines^{6 (link)}. Tumor samples containing > 60% tumor cellularity were selected for DNA extraction using PAXgene Tissue DNA kit (Qiagen). Gentra Puregene Blood Kit (Qiagen) was used to extract genomic DNA from blood. Extracted DNA were quality controlled for its purity, quantity, and integrity. Identity of the extracted DNA were tested using AmpFlSTR Identifiler PCR Amplification Kit (Thermo Fisher Scientific). Samples that match > 80% of the short tandem repeat profiles between tumor and germline DNA were considered authentic. RNA was extracted from PAXgene fixed tissues using the PAXgene Tissue RNA kit (Qiagen). RNA integrity (RIN) was determined for all samples by the RIN score given by the TapeStation (Agilent) read out. RNA samples that had RIN scores of 4 and above were included in downstream sequencing analysis.