Magcore genomic dna ffpe one step kit

Two to four 3 μm-thick sections were cut from FFPE tissue samples and placed in Eppendorf Tube^® 1.5 mL. Subsequently, deparaffined using the MagCore^® Genomic DNA FFPE One-Step Kit (RBC Bioscience Corp. New Taipei City, Taiwan), following the manufacturer’s instructions (cartridge code: 405; execution time: 16 h; elution volume: 60 µL) using an extractor automated MagCore^® HF16 Plus nucleic acid (RBC Bioscience Corp.). Genotyping, viral load and co-infections were detected by Anyplex II HPV28 kit (Seegene, Seoul, Republic of Korea), which simultaneously identifies 28 genotypes: 19 high-risk HPV types (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73 and 82) and 9 low-risk HPV types (6, 11, 40, 42, 43, 44, 54, 61 and 70) by performing a multiplex PCR with CFX96 thermal cycler (Bio-Rad, Hercules, CA, USA). Data analysis and interpretation were automated with Seegene viewer software, according to the manufacturer’s instructions.

HPV DNA from the paraffin-embedded tissue was extracted with the MagCore Genomic DNA FFPE One-Step Kit (RBC Bioscience) according to the manufacturer’s protocol.
HPV DNA detection and genotyping were performed by qualitative real-time PCR with the AmoyDx Human Papillomavirus Genotyping Detection Kit (Amoy Diagnostics). The test is designed for the specific amplification of the L1 gene in HPV DNA to detect and genotype 19 high-risk HPVs and 2 low-risk HPVs (HPV 6 and 11). The sensitivity of the test is 100 copies of HPV DNA per reaction. An internal control is provided in the assay to test for sample quality and the presence of inhibiting factors.
HPV DNA⁺/p16⁺ samples were considered HPV-positive.

DNA isolation: cancer genomic DNA was extracted from formalin-fixed paraffin-embedded tissue using the MagCore^® Genomic DNA FFPE One-Step Kit (RBC Bioscience, Taiwan, China). The quality was quantified using DeNovix DS-11 Spectrophotometer (DeNovix, Wilmington, DE, USA) and QuantiFluo^® ONE dsDNA System (Promega, WI, USA).
The assay generated a library of 207 gene-specific amplicons and targeted ~2800 clinically relevant mutations.
Sequencing: the products were analyzed by next-generation sequencing (NGS) using the Illumina platform, MiSeq Dx.
Data analysis: an analysis of the NGS data was performed using the GALAXY platform (usegalaxy.org). Sequencing reads (FASTQ files) were aligned to the human reference genome hg19 using the Bowtie2 tool. Variant calling was performed using the Varscan2 tool. Parameters used for the analysis were minimum allele frequency—0.05, minimum quality—20, and minimum coverage ×80. All variants were annotated with ANNOVAR (Available online: https://wannovar.wglab.org, accessed on 2 May 2022). The results were visualized using the R Bioconductor package Maftools (Available online: http://bioconductor.org/, accessed on 2 May 2022) [19 (link)].

DNA was extracted from cell suspension using the MagCore Viral Nucleic Acid Extraction Kit (RBC Bioscience, Taiwan), according to the manufacturer´s protocol.
DNA was extracted from the paraffin-embedded placental and fetal membrane tissue with the MagCore Genomic DNA FFPE One-Step Kit (RBC Bioscience, Taiwan), according to the manufacturer´s protocol.
HPV DNA detection and genotyping was performed using qualitative real-time PCR with the AmoyDx Human Papillomavirus Genotyping Detection Kit (Amoy Diagnostics, China). The test has been designed for specific amplification of L1 gene in HPV DNA to detect and genotype 19 high-risk HPVs (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82) and 2 low-risk HPVs (HPV 6 and 11). The test was sensitive to a level of 100 copies of HPV DNA per reaction. An internal control in the assay was provided to test for sample quality and the presence of inhibiting factors.

All hematoxylin and eosin (HE) staining data related to the diagnosis of leiomyosarcoma were collected and evaluated by a dedicated gyneco-pathologist to determine the percentages of tumor cells (%TCs) and tumor necrosis (%TN) in the macrodissected areas [28 (link)]. FFPE tissue blocks, representative of the tumor lesions, were collected and cut into 10 μm thick slides. DNA was extracted from the 10 μm thick unstained FFPE slide using the MagCore^® Genomic DNA FFPE One-Step kit (RBC Bioscience, New Taipei City, Taiwan) on the automated platform MagCore^® HF16Plus (Diatech Lab Line, Jesi, Italy), following the manufacturer’s instructions. The quantitation of the extracted DNA was performed using Qubit dsDNA HS fluorimetric assays (Life Technologies, Gaithersburg, MD, USA). Finally, the qualitative evaluation of the DNA integrity was performed using the DNA Fragmentation Quantification Assay (EntroGen Inc., Los Angeles, CA, USA) on the LightCycler^® 480 Real-time system (Roche Diagnostics, Basel, Switzerland). Only tissue samples with at least 50% of amplifiable DNA ≥ 150 bp were analyzed.

DNA isolation: Cancer genomic DNA was extracted from formalin-fixed paraffin-embedded tissue using a MagCore^® Genomic DNA FFPE One-Step Kit (RBC Bioscience, Taiwan). The quality was quantified using a DeNovix DS-11 Spectrophotometer (DeNovix, Wilmington, DE, USA) and a QuantiFluo^® ONE dsDNA System (Promega, Madison, WI, USA).
The assay generates a library of 207 gene-specific amplicons and targets ~2800 clinically relevant mutations.
Sequencing: The products were analyzed via next-generation sequencing (NGS) using an Illumina platform, MiSeq Dx.
Data analysis: the analysis of NGS data was performed using the GALAXY platform (usegal-axy.org, accessed on). Sequencing reads (FASTQ files) were aligned to the human reference genome hg19 using the Bowtie2 tool. Variant calling was performed using the Varscan2 tool. The parameters used for the analysis were minimum allele frequency—0.05, minimum quality—20, and minimum coverage ×80. All variants were annotated with ANNOVAR (https://wannovar.wglab.org, accessed on 2 May 2022). The results were visualized using the R Bioconductor package by Maftools (http://bioconductor.org/, accessed on 2 May 2022). More details have been described in previous study [18 (link)].