DNA was extracted from whole blood using the Qiagen QIAmp Mini Kit following manufacturer’s protocol. Total RNA was extracted from blood cells and liver tissue using a column method (RNeasy Kit; Qiagen, Hilden, Germany). RNA quality concentration was determined by a fluorometric technique (Qubit, Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.) and quality was verified by a small fragment analyzer (Bioanalyzer 2100, Agilent, Santa Clara, California, U.S.A.). The globin mRNA content of blood RNA samples (only) was first reduced using a commercial kit (Globin-Zero, Illumina, San Diego, California, U.S.A).
Qiamp mini kit
Manufactured by Qiagen
Sourced in Germany, United States, France
The QIAamp Mini Kit is a nucleic acid extraction kit designed for the purification of DNA from various sample types. It utilizes a silica-based membrane technology to efficiently capture and purify DNA, which can then be used in downstream applications such as PCR, sequencing, or other analyses.
Automatically generated - may contain errors
Lab products found in correlation
Humanmethylation450 beadchip, by Illumina (6 mentions)
Infinium humanmethylation450 beadchip kit, by Illumina (2 mentions)
Mic test strip method, by Liofilchem (2 mentions)
Abi 3100 avant automated dna sequencer, by Thermo Fisher Scientific (2 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (2 mentions)
Qubit dsdna hs assay, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Abi 3130xl, by Thermo Fisher Scientific (1 mentions)
Maxwell 16 instrument, by Promega (1 mentions)
Cobas dna sample preparation kit, by Roche (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Infinium human methylationepic beadchip, by Illumina (1 mentions)
Ez dna methylation, by Zymo Research (1 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
Isovitalex 2, by Thermo Fisher Scientific (1 mentions)
Humanomni1 quad beadchip, by Illumina (1 mentions)
Surescan microarray c scanner, by Agilent Technologies (1 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
Epitect fast bisulfite kit, by Qiagen (1 mentions)
42 protocols using qiamp mini kit
1
Whole Blood and Tissue RNA Extraction
2
Genetic Analysis of MEN1 and CDKN1B
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DNA was extracted from index patient's peripheral leukocytes with a Maxwell 16 Instrument according to the manufacturer's instructions (Promega Corp.). FFPE tissues were manually microdissected from two sections and samples were submitted to xylene deparaffination and then lysed and digested with proteinase K. DNA extraction was performed using the spin column procedure (QIAmp minikit; Qiagen). As no mutations in MEN1 were identified, the genetic analysis of the coding region and intron/exon boundaries of the CDKN1B (NM_004064.3) gene was carried out using the BigDye Sequencing Reaction Kit v.1.1. The reaction products were separated on an ABI 3130XL automatic sequencer (Applied Biosystems). To assess for loss-of-heterozigosity (LOH), the fragment of interest in the tumoral DNA was sequenced.
3
BRAF Codon 600 Mutations Sensitivity Analysis
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The cell lines used for the sensitivity studies contained four different mutations in BRAF codon 600 (V600E, V600K, V600D and V600R). The sensitivity tests were conducted using cell line DNA extracted from HT-29 (ATCC®; catalogue number: HTB-38TM), IGR-1 (DSMZ®; catalogue number: ACC 236TM), WM-115 (Rockland Immunochemicals®; catalogue number: WM115-01-0001TM) and HCT116 BRAF (V600R/+) (Horizon Discovery Group®; catalogue number: HD 104-037TM) (Table 1 ). Subcultures were made in appropriate media according to the manufacturer’s instructions and genomic DNA was isolated using the QIAmp Mini kit (Qiagen, Chatsworth, CA, USA).
4
FFPE DNA Extraction and Tumor Enrichment
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Quality and tumour cell content of the FFPE tissue block sections were evaluated by an experienced pathologist. Genomic DNA was extracted from representative FFPE tissue blocks, containing at least 50–70% neoplastic cells. In cases of low tumour tissue content, a macrodissection was performed to enrich the amount of tumour cells. Genomic DNA was extracted from a 5 μm thick section of MM tissue, using the Cobas® DNA Sample Preparation Kit (Roche) according to the manufacturer’s instructions. In CRC samples, genomic DNA was extracted from six 7 μm thick serial FFPE sections. DNA extraction was performed using the QIAmp Mini kit (Qiagen) according to the manufacturer’s instructions.
5
H3F3A Mutation Amplification Protocol
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DNA was extracted using the Qiamp Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer´s instructions. The PCR reaction to amplify the mutation spanning H3F3A region consisted of 1U Hot Star Taq polymerase (Thermo Fisher Scientific, Dreieich, Germany), 0.8 µl dNTPs (10 mM each, Fermentas, St. Leon-Rot, Germany), 2 µl of each primer (10 µM, Sigma-Aldrich, Taufkirchen, Germany) and 100 ng genomic DNA as template in a total volume of 40 µl. Samples were incubated at 95 °C for 15 min followed by 10 cycles of denaturation at 94 °C for 45 s, annealing at 61–56 °C for 30 s (touchdown −0.5 °C/cycle) and extension at 72 °C for 60 s followed by 25 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 30 s and extension at 72 °C for 60 s and a final extension step at 72 °C for 10 min. Sequencing was performed by GATC Biotech AG, Konstanz, Germany. All primers are listed in Supplementary Data 5 .
6
Comprehensive DNA Methylation Analysis
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Genomic DNA was extracted in triplicate from early and Sen BMSCs and HUVECs and from BMSC-derived adipocytes and osteoblasts using Qiagen’s QiAmp mini kit following the manufacturer’s recommendations. Next, 1 μg DNA was bisulfite-converted using EZ DNA Methylation (Zymo Research, Irvine, CA, USA) and analyzed by the Infinium Human MethylationEPIC BeadChip (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Raw data were extracted using minfi R package [20 (link)] and normalized using the preprocessFunnorm function. Not-reliable probes according to Zhou and collaborators [21 (link)] and probes on X and Y chromosomes were removed. The remaining 748,241 probes were annotated according to IlluminaHumanMethylationEPICanno.ilm10b4.hg19 annotation data.
7
DNA Methylation Analysis from Blood Samples
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EDTA-treated peripheral blood samples were stored immediately until use at −80 °C. Genomic DNA was extracted using QIAmp Mini Kit (Qiagen, Germany). For each sample, 1 μg of genomic DNA was bisulfite-converted using Zymo EZ DNA-methylation Kit. The methylation level was measured using the Infinium HumanMethylation450 Beadchip Kit (Illumina, Inc.) at Aros Applied Biotechnology A/S.
8
DNA Methylation Profiling of Ovarian Cell Lines
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Genomic DNA was extracted from eight human ovarian cell lines (SK-OV-3, PA-1, Caov-3, TOV-21G, TOV-112D, OV-90, and OVCAR-3) using the QIAmp mini kit (Qiagen, Inc.), according to the manufacturer's instructions. For genome-wide screening of DNA methylation, the Illumina HumanMethylation450 BeadChip (Illumina, Inc., San Diego, CA, USA) was used, which targets 450,000 specific CpG sites. DNA methylation values were described by β-values, which were determined by subtracting the background obtained from negative controls on the array and calculating the ratio of the methylated signal intensity to the sum of the methylated and unmethylated signals. β-values range from 0 (completely unmethylated) to 1 (fully methylated) on a continuous scale for each CpG site. To identify differentially methylated CpG sites, we calculated the difference in mean β-value (Δβ; mean β-value in resistant group – mean β-value in sensitive group). If the absolute difference in mean β-values (|Δβ|) was >0.3, the sites were defined as differentially methylated CpG sites.
9
Genome-Wide DNA Methylation Analysis
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Genomic DNA was extracted from the cell line and tumor tissues using QIAmp mini kit (Qiagen), according to the manufacturer's instructions. For analysis of genome-wide screening of DNA methylation, the Illumina HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA) targeted 450000 specific CpG sites. DNA methylation values were described as β-values, which are calculated by subtracting background using negative controls on the array and taking the ratio of the methylated signal intensity against the sum of both methylated and unmethylated signals. β-values range from 0 (completely unmethylated) to 1 (fully methylated) on a continuous scale for each CpG site. To identify differentially methylated CpG sites, we applied the difference in mean β-value (Δβ; mean β-value in tumors-mean β-value in SK-OV-3). If the absolute difference in mean β-values (￨Δβ￨) >0.06 (0.06 means a standard deviation at identical CpG site), the sites were defined as differentially methylated CpG sites. We described hypermethylated CpG sites/gene if Δβ was greater than 0.06 and hypomethylated CpG sites/gene if Δβ was less than -0.06.
10
Serogroup Identification of Neisseria Isolates
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Isolates were cultured on Thayer-Martin agar plates with IsoVitaleX 2% (Oxoid, Ltd.) in 5% CO2 atmosphere at 37°C. The serogroup was identified by slide agglutination with commercial antisera (Thermo Scientific, Waltham, Massachusetts, USA) or by multiplex PCR on bacterial DNA extracted using the QiAmp mini kit (Qiagen, Hilden, Germany) [17 (link)], from an overnight culture or directly from clinical sample.
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