After getting written informed consent, ten milliliters of whole blood were drawn from each patient in EDTA tubes and sent to central laboratory at National Institute of Health, Maputo, Mozambique, for analysis. TCD4+ counting was performed using150ul of blood using Becton Dickinson (BD) FACSCalibur (Becton-Dickison, Fraklin Lakes, NJ), and these were performed at HIV Reference Laboratory in Mozambique. Buffy coat and plasma were separated from remnant blood and stored at -80°C and latter shipped to Laboratório de Virologia Molecular, Universidade Federal do Rio de Janeiro, Brazil, were genotyping was performed. HIV-1 viral load was quantified using COBAS TaqMan48 (Roche Diagnostics, USA), at the HIV Reference laboratory in Mozambique. Genomic DNA was extracted using QIAamp blood DNA extraction kit (QIAGEN, Germany), following manufacturers manual. Nested PCR was used to amplify a 1000bp fragment of the pol gene spanning the complete PR (297bp) and the polymerase domain of the RT(703bp). Both PCR and sequencing conditions have been described elsewhere [20 (link),21 (link)]
Qiaamp dna blood extraction kit
Manufactured by Qiagen
Sourced in Germany
The QIAamp DNA Blood Mini Kit is a product designed for the rapid and efficient purification of DNA from whole blood, buffy coat, and plasma or serum samples. The kit utilizes a silica-based membrane technology to selectively bind DNA, allowing for the removal of impurities and the subsequent elution of high-quality genomic DNA.
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Lab products found in correlation
Oxiselect oxidative dna damage elisa kit, by Cell Biolabs (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Cobas taqman 48, by Roche (1 mentions)
Genomestudio software, by Illumina (1 mentions)
Infinium oncoarray 500k beadchip, by Illumina (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time system, by Thermo Fisher Scientific (1 mentions)
Copycaller v2, by Thermo Fisher Scientific (1 mentions)
Experion bioanalyzer, by Bio-Rad (1 mentions)
Nextera rapid capture custom kit, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Thermoscript, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Taqman genotyping master mix, by Thermo Fisher Scientific (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
21 protocols using qiaamp dna blood extraction kit
1
HIV-1 Viral Load and Genotyping
2
DNA Extraction and Genotyping Protocol
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DNA was isolated from EDTA-blood using a QiaAmp blood DNA Extraction kit (Qiagen, Hilden, Germany). Briefly, 200 lL EDTA-blood was treated with protease for 15 min at 56°C followed by addition of AL lysis buffer and ethanol. The mixture was passed through a spin column and washed according to the manufacturer's instructions. DNA was eluted with 100 l-LAE buffer and quantified on agarose gel using lambda DNA as the standard. The DNA fragment spanning C/T-13910 variants were amplified using the forward primer [5#-GGA TGC ACT GCT GTG ATG AG-3#] and reverse primer [5#-CCC ACT GAC CTA TCC TCG TG-3#] to also include positions.
Both sequencing and RFLP were carried out by using this PCR product, which was done without knowledge of the clinical data and the results of the LTT and LBT.
Both sequencing and RFLP were carried out by using this PCR product, which was done without knowledge of the clinical data and the results of the LTT and LBT.
3
Quantitative Analysis of HBV Viral Load
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For evaluating the HBV viral titers, HBV genomes from cell culture supernatants and pellets were purified using the QIAamp Blood DNA extraction kit (QIAGEN, Hilden, Germany) and quantitated by qPCR using a primer pair specific to the small S gene (SF: 5′-TTG ACA AGA ATC CTC ACA ATA CC-3′) and antisense primer SR (positions 309–328, 5′-GGA GGT TGG GGA CTG CGA AT-3′). The HBV DNA plasma standard containing 1 × 106 HBV genomic DNA copies/ml (HBV DNA Quantiplex, Chiron) was used to standardize the viral titers. The following primer sets were used to investigate the mRNA expression levels with RT-qPCR: 18S-F: 5′-AGTCCCTGCCCTTTGTACACA-3′ and 18S-R: 5′-CGATCCGAGGGCCTCACTA-3′, HO-1-F: 5′-TTG CCAGTGCCACCAAGTTC-3′, and HO-1-R: 5′-TCAGCAG CTCCTGCAACTCC-3′, IFNb-F: 5′-TTGTGCTTCTCCACTA CAGC-3′ and hIFNb-R: 5′-CTGTAAGTCTGTTAATGAAG-3′, mtDNA1-F: 5′CATGCCCATCGTCCTAGAAT-3′ and mtDNA1-R: 5′-ACGGGCCCTATTTCAAAGAT-3′, mtDNA2-F: 5′-CCCTAACACCAGCCTAACCA-3′ and mtDNA2-R: 5′-AA AGTGCATACCGCC7AAAAG-3′, mtDNA3-F: 5′-TCCAACT CATGAGACCCACA-3′ and mtDNA3-R: 5′-TGAGGCT TGGATTAGCGTTT-3′).
4
Oxidative DNA Damage Assay in HepG2 Cells
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After the HepG2-2.15 cells were seeded into six-well plates for 12 h, they were treated with PBS (0.5%) or GV1001 (5 or 10 μM) for 12 h. From the pellets, genomic DNA was extracted using a QIAamp Blood DNA extraction kit (QIAGEN, Hilden, Germany). For the detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) activity, competitive ELISAs from an 8-OHdG analysis kit (OxiSelect Oxidative DNA Damage ELISA kit, Cell Biolabs, San Diego, CA, United States) was used according to the manufacturer’s protocol.
5
Genotyping and Imputation for Telomere Length
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Genomic DNA was isolated from peripheral blood using the QIAamp blood DNA extraction kit (Qiagen, Valencia, CA, USA). All the genotyping was done in the Genotyping Core of MD Anderson Cancer Center using Illumina’s Infinium OncoArray-500K Beadchip. Genome Studio software (Illumina, San Diego, CA, USA) was utilized to analyze the genotyping data. The mean concordance rate of 2% replicated samples was 99.2%. We removed nonconcordant SNPs for analyses. All the samples had an overall SNP call rate >95%. Individual SNPs with minor allele frequency (MAF) <1% and call rate <90% were excluded for analysis. Imputation was performed using the Michigan Imputation Server (https://imputationserver.sph.umich.edu/ ), an online server that generates phased and imputed genotypes using the Haplotype Reference Consortium (HRC Version r1.1) reference panels [64 (link)]. Eleven independent SNPs were associated with LTL by large scale GWAS [40 (link),41 (link),42 (link)] and were used to construct a genetic risk score (GRS). Among these SNPs, four SNPs (rs10936599, rs2736100, rs9420907, and rs755017) were directly genotyped on OncoArray-500K, and the other seven were imputed with a high imputation accuracy (mean R2) of 0.96.
6
Quantifying Oxidative DNA Damage
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Genomic DNA was extracted from the transfected cells using QIAamp Blood DNA extraction kit (QIAGEN, Hilden, Germany). For the detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) activity, a competitive ELISA for 8-OHdG analysis kit (OxiSelect Oxidative DNA Damage ELISA kit, Cell Biolabs, San Diego, CA, USA) was used according to the manufacture's protocol.
7
DNA Extraction from Blood Samples
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The QIAamp blood DNA extraction kit (Qiagen, Hilden, Germany) was used to extract DNA from the blood samples. The DNA was dissolved with 1X AE solution (Qiagen), and stored at −20°C for the long-term.
8
Relative Leukocyte Telomere Length Measurement
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Genomic DNA was isolated from peripheral blood leukocytes using the QIAamp Blood DNA Extraction Kit (Qiagen). Relative LTL was measured using a modified real-time qPCR method (48) . Detailed assay procedures and quality control steps were described previously (49) (50) (51) (52) (53) . Briefly, two separate PCR reactions (telomere amplification and globulin amplification) were performed to determine the ratio of the telomere repeats copy number (T) to the single gene (human globulin) copy number (S; T/S), which was used as a parameter to represent relative LTL. The PCR for telomere amplification consisted of 1x SYBR Green Master Mix (Applied Biosystems), 200 nmol/L Tel-1 primer, 200 nmol/L Tel-2 primer, and 5 ng of genomic DNA. The PCR for human globulin (Hgb) amplification consisted of 1x SYBR Green Master Mix, 200 nmol/L Hgb-1, 200 nmol/L Hgb-2 primer, and 5 ng of genomic DNA. Each sample was done in duplicates. The intraassay and interassay coefficient of variation was <3% and <5%, respectively; the intraclass correlation coefficient was 0.959 [95% confidence interval (CI), 0.954-0.962] for telomere assay and 0.986 (95% CI, 0.985-0.988) for Hgb assay.
9
Comparison of DNA Extraction Methods
10
Determination of FUT2 Genotype
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The FUT2 genotype was determined in the individuals from whom we had obtained blood samples (n = 98). DNA was extracted from a 50-μL sample of whole blood with the QIAamp Blood DNA extraction kit (Qiagen, Les Ulis, France), according to the manufacturer's recommendations. The FUT2 genotypes were determined by partial sequencing of the FUT2 gene as described previously [28] .
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