Genomic DNA of all participants was isolated from peripheral whole blood using a MagCore Genomic DNA Whole Blood Kit and HF-16 extractor (RBC Bioscience, Taiwan), as previously published.9 (link) Genotyping of rs75932628-T (p.R47H) was conducted by molecular beacon real-time PCR using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, California, USA) and then confirmed by Sanger sequencing using an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, California, USA). Sequences of primers and molecular beacons are given in table 2 . The amplification procedure consisted of 95°C for 20 s followed by 40 cycles of 95°C for 3 s, 54°C for 30 s and 72°C for 10 s. The fluorescence spectra of the molecular beacons were measured during the annealing step of the PCR cycle. The reference controls were two custom-made plasmids (Sangon Biotech, Shanghai, China).
Magcore genomic dna whole blood kit
Manufactured by RBC Bioscience
Sourced in Italy, United States
The MagCore Genomic DNA Whole Blood Kit is a laboratory product designed for the extraction and purification of genomic DNA from whole blood samples. The kit utilizes magnetic bead-based technology to efficiently isolate high-quality DNA, which can then be used in various downstream applications such as PCR, sequencing, and genetic analysis.
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Lab products found in correlation
Miniseq platform, by Illumina (3 mentions)
Trusight cardio sequencing kit, by Illumina (2 mentions)
Miseq platform, by Illumina (2 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Abi 3730xl automatic sequencer, by Thermo Fisher Scientific (1 mentions)
Qfx fluorometer, by DeNovix (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
Nimblegen seqcap ez exome v3, by Roche (1 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Truseq, by Illumina (1 mentions)
Leucosep, by Greiner (1 mentions)
Automacs running buffer, by Miltenyi Biotec (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Ibright cl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
Magcore nucleicacid extractor, by RBC Bioscience (1 mentions)
Nanophotometer p class, by Implen (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
19 protocols using magcore genomic dna whole blood kit
1
Genotyping of TREM2 rs75932628-T Variant
2
Genetic Testing Protocol for HCM
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Patients in the HCM group underwent genetic testing following a methodology that was described in detail elsewhere [14 (link),15 (link)]. In summary, blood samples were collected at enrollment, and DNA was isolated using the MagCore Genomic DNA Whole Blood Kit (RBC Bioscience, Taipei, Taiwan). Targeted sequencing was performed on an Illumina MiSeq platform using TruSight Cardio Sequencing Kit (Illumina, San Diego, California, SDSU, USA) following the manufacturer’s instructions.
The procedure targeted 47 core and emerging genes associated with HCM, including genes encoding for sarcomere and sarcomere-associated proteins. Variants were classified according to currently available standards and guidelines, taking into account evidence such as allele frequency in control populations, genotype segregation, amino-acid conservation, and in silico prediction [16 (link)].
The procedure targeted 47 core and emerging genes associated with HCM, including genes encoding for sarcomere and sarcomere-associated proteins. Variants were classified according to currently available standards and guidelines, taking into account evidence such as allele frequency in control populations, genotype segregation, amino-acid conservation, and in silico prediction [16 (link)].
3
Clinical DNA Extraction via MagCore
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DNA isolation from clinical samples was carried out by the use of the MagCore® Genomic DNA Whole Blood Kit (MagCore HF 16 nucleic acid extraction system, RBC Bioscience, New Taipei City, Taiwan).
For pneumococcal isolates, DNA extraction was carried out from a 24 h blood agar culture, as described previously [20 (link)].
For pneumococcal isolates, DNA extraction was carried out from a 24 h blood agar culture, as described previously [20 (link)].
4
Genetic Analysis of CYP24A1 Gene
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After obtaining a written informed consent from patient’s parents, molecular analysis of CYP24A1 gene was performed. Genomic DNA was extracted from peripheral blood leukocytes by an automatic DNA device (MagCore HF16 Plus, Diatech Lab Line, Jesi, Italy) with MagCore Genomic DNA Whole Blood Kit (RBC Bioscience Corp. TAIWAN). The DNA concentration and purity were tested using QFX Fluorometer (DeNovix Inc., Wilmington, USA) according to the manufacturer’s instructions.
5
Targeted Sequencing of Genetic Variants
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Genomic DNA was isolated from whole blood using the MagCore® Extractor System and MagCore® Genomic DNA Whole Blood Kit (RBC Bioscience, New Taipei City, Taiwan), following the manufacturer’s protocol. Genotyping was performed on a MiSeq sequencer (Illumina, San Diego, CA, USA) using a custom-made hotspot sequencing kit for 55 SNPs within 14 genes selected as previously being associated with increased lipids, non-alcoholic fatty liver, or cardiovascular disease [15 (link)].
Amplicon sequencing libraries were prepared from 20 ng of DNA per sample according to the AmpliSeq protocol (Illumina Inc, San Diego, CA, USA). Libraries were generated with dual indices (19 PCR cycles) followed by normalization and pooling. The pooled libraries were paired-end (2 × 150) sequenced on a micro flow cell with V2 chemistry on a MiSeq instrument (Illumina Inc, San Diego, CA, USA).
Amplicon sequencing libraries were prepared from 20 ng of DNA per sample according to the AmpliSeq protocol (Illumina Inc, San Diego, CA, USA). Libraries were generated with dual indices (19 PCR cycles) followed by normalization and pooling. The pooled libraries were paired-end (2 × 150) sequenced on a micro flow cell with V2 chemistry on a MiSeq instrument (Illumina Inc, San Diego, CA, USA).
6
Magnetic Bead-Based DNA Extraction
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DNA was isolated using the MagCore® Genomic DNA Whole Blood Kit (RBC Bioscience Corp., New Taipei City, Taiwan) based on separation on magnetic beads and quantitated using an Epoch™ spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).
7
Whole-Exome Sequencing of IPF Families
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Blood samples from nine family members were collected and processed for genomic DNA isolation using the MagCore® Genomic DNA Whole Blood Kit (RBC Bioscience, USA). We performed whole-exome sequencing (WES) on samples from four family members (I-2, I-3, II-1, and III-1). Whole-exome libraries were prepared using the Kapa Hyper Prep Kit (Roche, USA) according to the protocol for NimbleGen SeqCap EZ Exome v3 (Roche, USA). Paired-end 2 × 75 bp sequencing was performed on an Illumina NextSeq 500 sequencer (Illumina Inc., USA). The raw sequencing reads were aligned to the GRCh37 human reference genome using the BWA mem algorithm, version 0.7.15. PCR duplicates were identified with the MarkDuplicates tool from Picard version 2.9.2. GATK HaplotypeCaller, version 3.7, was used to detect germline single nucleotide variants (SNV) and indels. Obtained variants/indels have been annotated using Annovar program version (2018Apr16).
On the basis of the current knowledge, we have chosen 30 candidate genes previously associated with IPF: TERC, TERT, SFTPC, SFTPA1, SFTPA2, MUC5B, MUC5C, RTEL1, PARN, ABCA3, DKC1, TINF2, IL1RN, IL8, FAM13A, TLR3, HLA- DRB1, HLA- DQB1, DSP, OBFC1, MUC2, TOLLIP, ATP11A, MDGA2, MAPT, SPPL2C, DPP9, TGFB1, NAF1, and OBFC17 (link)–17 (link). We then looked more deeply into the exonic variants of these genes.
On the basis of the current knowledge, we have chosen 30 candidate genes previously associated with IPF: TERC, TERT, SFTPC, SFTPA1, SFTPA2, MUC5B, MUC5C, RTEL1, PARN, ABCA3, DKC1, TINF2, IL1RN, IL8, FAM13A, TLR3, HLA- DRB1, HLA- DQB1, DSP, OBFC1, MUC2, TOLLIP, ATP11A, MDGA2, MAPT, SPPL2C, DPP9, TGFB1, NAF1, and OBFC17 (link)–17 (link). We then looked more deeply into the exonic variants of these genes.
8
Targeted NGS for Genetic Profiling
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The genetic testing methodology has been previously reported [14 (link)]. Briefly, blood samples were collected at enrolment and total DNA was isolated using MagCore Genomic DNA Whole Blood Kit (RBC Bioscience) following the manufacturer’s protocol, and subsequently being quantified using Qubit dsDNA HS assay kit (Life Technologies). Targeted next generation sequencing (NGS) was performed on an Illumina MiSeq platform using TruSight Cardio Sequencing Kit (Illumina) according to manufacturer’s instructions. An initial amount of 50 ng of genomic DNA was used for optimal gene enrichment.
9
Genetic Analysis of Osteogenesis Imperfecta
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Molecular testing using NGS technique was performed in the entire study group. Genomic DNA was automatically extracted from peripheral blood leukocytes using a MagCore Genomic DNA Whole Blood Kit (RBC Bioscience, Taiwan). The pathogenic variants were detected using a custom gene panel containing OI and OI-related genes (COL1A1, COL1A2, IFITM5, SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, SP7, BMP1, TMEM38B, WNT1, CREB3L1, SPARC, PLS3, SEC24D, P4HB, PLOD2, TNFSF11, LRP5, DKK1, RANKL, XYLT2, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, OPG, SOST, IL6, and IGF1) for resequencing the exons and flanking non-coding sequences (Illumina Design Studio software). The gene panel, composed of 710 amplicons with a mean length of 175 bp, reached 99% coverage. TruSeq Custom Amplicon libraries were prepared according to the manufacturer’s protocol and sequenced on the MiniSeq platform (Illumina, United States) using two types of cartridge (Mid Output and High Output Kit, 300 cycles; Illumina, United States).
10
Taiwanese Population Genome Profiling
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After informed consent was obtained from participants, a 3 mL venous blood sample was collected and preserved in an EDTA tube. Genomic DNA was extracted from 200 µL peripheral blood samples by using the MagCore Genomic DNA Whole Blood Kit (RBC Bioscience, New Taipei City, Taiwan) in accordance with the manufacturer’s instructions. Genetic information from individuals in the Taiwanese population was obtained using the Affymetrix Axiom genotyping platform, specifically the Axiom Taiwan Precision Medicine (TPM)-customised SNP array (Thermo Fisher Scientific, Santa Clara, California, USA). This array encompasses 714 457 SNPs across the entire human genome. PLINK V.1.9 was used for analysis, and samples and SNPs with missing rates were excluded. Variants failed to meet the Hardy-Weinberg equilibrium criteria (p<1e−6 and a minor allele frequency (MAF) of <1e−4) were also excluded. The TPM arrays were phased using SHAPEIT4 and imputation was performed using Beagle V.5.2, known for its efficacy and accuracy compared with other imputation tools. The imputed data were filtered on the basis of criteria such as an r2 alternate allele dosage of <0.3 and a genotype posterior probability of <0.9.28 (link)
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