Qiaamp 96 dna blood kit

Manufactured by Qiagen

Sourced in Germany, United States, Spain, Australia

The QIAamp 96 DNA Blood Kit is a nucleic acid purification system designed for the automated extraction of DNA from whole blood samples. The kit utilizes silica-based membrane technology to efficiently capture and purify DNA, which can then be used in various downstream applications.

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75 protocols using qiaamp 96 dna blood kit

Genomic DNA Extraction from Dried Blood Spots

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Genomic DNA was isolated from two DBS from each individual using a QIAamp 96 DNA Blood Kit (QIAGEN, Valencia, CA). DBS were punched out using a hole puncher and the genomic DNA was extracted simultaneously from each of the two DBS (such that the extracted genomic DNA from the two DBS was combined) following the supplementary protocol “Isolation of genomic DNA from dried blood spots using the QIAamp ®96 DNA Blood Kit–(EN)” from QIAGEN. Purified DNA was eluted using 150 μL DEPC-treated, nuclease-free water (Quality Biological, Inc., Gaithersburg, MD) and concentrated using a ZR-96 DNA Clean-up Kit^TM (Zymo Research, Irvine, CA). The resulting concentrated and purified genomic DNA was eluted in 12 μL DEPC-treated, nuclease-free water.

Figueroa D.B., Tillotson J., Li M., Piwowar-Manning E., Hendrix C.W., Holtz T.H., Bokoch K., Bekker L.G., van Griensven F., Mannheimer S., Hughes J.P., Grant R.M, & Bumpus N.N. (2018). Discovery of genetic variants of the kinases that activate tenofovir among individuals in the United States, Thailand, and South Africa: HPTN067. PLoS ONE, 13(4), e0195764.

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Quantifying P. falciparum Infection via qPCR

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Parasite and human genomic DNA were extracted from three punches (5 mm in diameter) of dried blood spots on filter papers with QIAamp 96 DNA blood kit (Qiagen, Germany) and eluted in 150µL of water, following the manufacturer’s recommendations. Five microliters of DNA were used as template for qPCR analysis targeting P. falciparum var genes acidic terminal sequence (varATS, ≈59 copies per genome) in StepOne Plus thermocycler (Applied Biosystems), as previously described [46 (link)]. The limit of detection of the varATS-qPCR was 0.1 parasite/μL as described elsewhere [47 (link)]. Samples with Ct value > 39.7 were considered negative.

Natama H.M., Rovira-Vallbona E., Krit M., Guetens P., Sorgho H., Somé M.A., Traoré-Coulibaly M., Valéa I., Mens P.F., Schallig H.D., Berkvens D., Kestens L., Tinto H, & Rosanas-Urgell A. (2021). Genetic variation in the immune system and malaria susceptibility in infants: a nested case–control study in Nanoro, Burkina Faso. Malaria Journal, 20, 94.

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Genetic Variation Analysis in GIP and GIPR

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Total genomic DNA was isolated from blood using the QIAamp 96 DNA blood kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. In this study, we analyzed rs2291725 (G/A, S103G) located in exon 4 of GIP and rs10423928 (T/A) in intron 12 of GIPR (Table 1). The GIP SNP rs2291725 was chosen since it is a high frequency missense variation in the GIP gene changing amino acid number 103 in the GIP protein (Ser to Gly). The rationale for selection of the GIPR SNP lies in the fact that in a combined analysis of several GWAS studies the risk genotype of rs10423928 showed impaired insulin secretion. This GIPR SNP is in strong linkage disequilibrium (r2 = 0.99) with the non-synonymous SNP rs1800437 (E354Q) analyzed in the study by Torekov et al. (2014) . Consequently the two SNPs reflect the same genetic variation in the gene.
From those who agreed to provide whole blood for DNA analyses, a total of 990 women from OPRA and 992 women from PEAK-25 were genotyped successfully using TaqMan (ABI, Foster City, USA). Approximately 3% of the samples from each cohort were genotyped in duplicate with 100% concordance. Both polymorphisms conformed to Hardy–Weinberg equilibrium and the minor allele frequencies did not differ from other European populations. Genotype and allele frequencies did not differ between cohorts.

Garg G., McGuigan F.E., Kumar J., Luthman H., Lyssenko V, & Akesson K. (2015). Glucose-dependent insulinotropic polypeptide (GIP) and GIP receptor (GIPR) genes: An association analysis of polymorphisms and bone in young and elderly women. Bone Reports, 4, 23-27.

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Quantitative HHV-6 and CMV Detection

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DNA was extracted from plasma samples utilizing the QIAamp 96 DNA Blood Kit (Qiagen, Inc., Santa Clarita, CA). Detection of HHV-6 DNA was performed using a real-time quantitative fluorescent probe polymerase chain reaction (PCR) assay as previously described (25 (link)). Detection of 1 copy of HHV-6 DNA/reaction (25 copies/mL of plasma) was the lower limit of detection of our assay and considered a positive test. A highly conserved region of the U94 gene in HHV-6A and HHV-6B was amplified and used to distinguish species. Testing for chromosomally integrated HHV-6 was considered for patients with suggestive test results (viral load >3.5 log₁₀ copies/mL or persistent levels >100 copies/mL in >80% of plasma samples) (26 (link)). CMV PCR test results from our original publication were used for this study, and testing was performed as previously described (27 (link)).

Roa P.L., Hill J.A., Kirby K.A., Leisenring W.M., Huang M.L., Santo T.K., Jerome K.R., Boeckh M, & Limaye A.P. (2015). Co-Reactivation of HHV-6 and CMV is Associated with Worse Clinical Outcome in Critically Ill Adults. Critical care medicine, 43(7), 1415-1422.

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Molecular Diagnosis of Malaria by qPCR

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For the molecular diagnosis by qPCR, filter papers with dried blood samples from maternal peripheral, placental and cord blood were punched and three circles of 5 mm in diameter was used for DNA extraction with QIAamp 96 DNA blood kit (Qiagen, Germany). Extracted DNA was eluted in 150μL of water. Five microliters of DNA were used for qPCR analysis targeting P. falciparum var gene acidic terminal sequence (varATS, ≈59 copies per genome) as previously described²². The limit of detection in our laboratory was 0.1 parasite/μL using DNA extracted from blood spot on filter paper. Parasite densities were obtained by extrapolating cycle thresholds (Ct) from a standard curve prepared with titrated samples containing known numbers of infected erythrocytes diluted in whole blood (100,000 to 0.1 parasites/μL). Samples with Ct value ≤39.7 were considered positive.

Natama H.M., Ouedraogo D.F., Sorgho H., Rovira-Vallbona E., Serra-Casas E., Somé M.A., Coulibaly-Traoré M., Mens P.F., Kestens L., Tinto H, & Rosanas-Urgell A. (2017). Diagnosing congenital malaria in a high-transmission setting: clinical relevance and usefulness of P. falciparum HRP2-based testing. Scientific Reports, 7, 2080.

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Idiopathic Scoliosis Family Study

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A three-generation family (Fig 1) with a high burden of idiopathic scoliosis participated in the study. All participating family members (n = 15) were blood-sampled and radiographed. All but one (I:II) were examined by a spine surgeon; however this individual had no scoliosis on radiographs. Individuals with a curve angle of 10 degrees or more, as measured by the Cobb method [14 ], were diagnosed with scoliosis. No one had any signs of a non-idiopathic scoliosis and all had an onset in adolescence, i.e. after the age of ten years (S1 Table). DNA was extracted from blood either by a salt precipitation method on the Autopure LS system (Qiagen, Hilden, Germany) or the QIAamp 96 DNA blood kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

Einarsdottir E., Grauers A., Wang J., Jiao H., Escher S.A., Danielsson A., Simony A., Andersen M., Christensen S.B., Åkesson K., Kou I., Khanshour A.M., Ohlin A., Wise C., Ikegawa S., Kere J, & Gerdhem P. (2017). CELSR2 is a candidate susceptibility gene in idiopathic scoliosis. PLoS ONE, 12(12), e0189591.

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DNA Extraction and APOE Genotyping of Iwaki Residents

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The DNA of Iwaki residents was purified from peripheral whole blood using the QIAamp® 96 DNA Blood Kit (QIAGEN, Hilden, Germany), and the APOE genotype was identified by Toshiba Corporation using the Japonica Array consisting of population‐specific SNP markers designed from the 1070 whole genome reference panel. The primers used were previously reported.¹⁵, ¹⁸

Nakamura T., Kawarabayashi T., Nakahata N., Itoh K., Ihara K., Nakaji S., Ikeda Y., Takatama M, & Shoji M. (2023). Annual stability of the plasma Aß40/42 ratio and associated factors. Annals of Clinical and Translational Neurology, 10(6), 879-891.

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Genotyping malaria parasites using HRM

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Genotyping was done by locus-specific high-resolution melting (HRM) assays with parasite DNA extracted from filter-paper-dried blood spots (DBS). To recover parasite DNA, DBS were punched onto plates containing 96 deep wells, using punchers and forceps that were rinsed in 1% bleach and alpha-Q water after each sampling step to limit cross-contamination. For each plate, 4 negative and 4 positive controls were included. Genomic DNA (gDNA) was manually extracted using a QIAamp 96 DNA blood kit (Qiagen, Hilden, Germany) and the manufacturer’s instructions. The DNA concentrations of the eluates were quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific) and stored at –20°C until use. One micromolar volume of gDNA of approximately 10 pg (1 ng/μl) was used for genotyping assays. HRM genotyping reactions were performed for alleles pfcrt C72/M74/N75/K76, pfmdr1 N86, pfmdr1 Y184, pfdhps S436/A437, pfdhfr N51/C59, and pfk13 C580 on a LightCycler 96 real-time PCR system (Roche). The primers and probes used for PCRs with a 2.5× LightScanner master mix (Biofire) were as previously described (38 (link)). Each reaction mixture had final forward and reverse primer concentrations of 0.05 μM and 0.2 μM, respectively (asymmetric PCR) and a 0.2 μM concentration for allele-specific probes. The PCR conditions and analysis method used were as previously described (38 (link)).

Mbye H., Bojang F., Jawara A.S., Njie B., Mohammed N.I., Okebe J., D’Alessandro U, & Amambua-Ngwa A. (2020). Tolerance of Gambian Plasmodium falciparum to Dihydroartemisinin and Lumefantrine Detected by Ex Vivo Parasite Survival Rate Assay. Antimicrobial Agents and Chemotherapy, 65(1), e00720-20.

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Genotyping from Whole Blood DNA

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DNA extraction for genotyping was carried out from the whole blood using the QIAamp 96 DNA Blood Kit (cat # 51161) from Qiagen, Valencia, USA. Concentration and quality of all extracted DNA were assessed using Nanodrop 1000. Samples were processed on Illumina HumanCytoSNP-12 v2.1 chips with 299,140 markers and read on the BeadArray Reader. Image data was processed in BeadStudio software to generate genotype calls.
Quality control was conducted as described previously for 5,499 individuals typed for 299,140 SNPs (23 (link), 29 (link)). We removed DNA samples with call rates <97% (n = 13), gender mismatches (n = 79), as well as technical duplicates (n = 53). We removed SNPs that were poorly called (<90%) or monomorphic (n = 38,753), and then removed SNPs with call rates <95% (n = 1,045) or HWE p-values<10⁻¹⁰ (n = 634, which produces no HWE p-values <10⁻⁷ in a subset of 1,842 unrelated participants). This QC resulted in 5,354 individuals with high-quality genotype data for 257,747 SNPs. The MaCH software (30 (link)) was used to conduct genotype imputation using 1,000 genomes reference haplotypes (1KG phase3 v5, which includes South Asian populations). Only high-quality autosomal imputed SNPs (imputation r²>0.5) with MAF>0.01 were included in this analysis, yielding 8,512,165 imputed SNPs.

Argos M., Tong L., Roy S., Sabarinathan M., Ahmed A., Islam M.T., Islam T., Rakibuz-Zaman M., Sarwar G., Shahriar H., Rahman M., Yunus M., Graziano J.H., Jasmine F., Kibriya M.G., Zhou X., Ahsan H, & Pierce B.L. (2018). Screening for gene-environment (GxE) interaction using omics data from exposed individuals: an application to gene-arsenic interaction. Mammalian genome : official journal of the International Mammalian Genome Society, 29(1-2), 101-111.

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Malaria Infection Detection Protocols

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Genomic DNA was extracted from whole blood using the QIAamp 96 DNA blood kit (Qiagen, Valencia, CA) or the 96-well genomic DNA extraction kit (Favorgen, Taiwan) according to the manufacturer instructions. The presence of P. falciparum and P. vivax infections was determined by a post-PCR, ligation detection reaction—fluorescent microsphere assay (LDR-FMA: 2006 surveys) [30 (link)] and a qPCR assay (2010 survey) [31 (link), 32 (link)].

Barnadas C., Timinao L., Javati S., Iga J., Malau E., Koepfli C., Robinson L.J., Senn N., Kiniboro B., Rare L., Reeder J.C., Siba P.M., Zimmerman P.A., Karunajeewa H., Davis T.M, & Mueller I. (2015). Significant geographical differences in prevalence of mutations associated with Plasmodium falciparum and Plasmodiumvivax drug resistance in two regions from Papua New Guinea. Malaria Journal, 14, 399.

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