DNA was extracted from frozen granulocytes or buffy coats with the use of QIAamp-96 spin blood kits (QIAGEN, Stockholm, Sweden) at the DNA extraction facility supported by SWEGENE. We successfully genotyped the plasma cystatin C associated SNP rs13038305 at the cystatin C locus on chromosome 20 in 27 618 subjects of the MDC. Primers and probes were custom synthesized by Applied Biosystems (Foster City, CA) according to standard recommendations for the AB Prism 7900HT analysis system, and genotyped with polymerase chain reaction-based TaqMan method[29 (link)].
Qiaamp 96 spin blood kit
Manufactured by Qiagen
Sourced in Sweden, United States
The QIAamp-96 spin blood kits are a collection of laboratory equipment designed for the rapid and efficient extraction of DNA from whole blood samples. The kits provide a standardized protocol and necessary components to isolate high-quality genomic DNA from multiple samples simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Veracode technology, by Illumina (2 mentions)
Goldengate genotyping assay system, by Illumina (2 mentions)
Beadxpress reader, by Illumina (2 mentions)
Genomestudio data analysis software v2011, by Illumina (2 mentions)
Ab prism 7900ht, by Thermo Fisher Scientific (1 mentions)
Humanomniexpress beadchip, by Illumina (1 mentions)
Viia7, by Thermo Fisher Scientific (1 mentions)
Iscan system, by Illumina (1 mentions)
Omni swabs, by Cytiva (1 mentions)
6 protocols using qiaamp 96 spin blood kit
1
Genotyping of Cystatin C SNP
2
Genotyping Cystatin C SNP in 3,105 Subjects
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DNA was extracted from frozen granulocytes or buffy coats with the use of QIAamp-96 spin blood kits (QIAGEN, Stockholm, Sweden) at the DNA extraction facility supported by SWEGENE. We successfully genotyped the plasma cystatin C associated SNP rs13038305 at the cystatin C locus on chromosome 20 in 3,105 subjects of the MDC-CC-re-exam using TaqMan® Assay by Design primers and probes, with a real-time PCR assay using the ViiA7 equipment (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions [19 (link)]. As this cohort has been analyzed with a GWAS (HumanOmniExpressBeadChip and iScan system (Illumina, San Diego, CA, USA)) we were able to check for concordance between the TaqMan based and GWAS-based method, which showed more than 99.5% concordance.
3
Genotyping AVPR1B Polymorphisms in DNA
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DNA was extracted from frozen granulocyte or buffy coat with the use of QIAamp-96 spin blood kits (Qiagen) at the DNA extraction facility supported by SWEGENE. To analyse the AVPR1B polymorphism and capture the genetic variance of AVPR1B, data from the online catalogue HapMap were used (http://www.hapmap.org ) to select four tag single nucleotide polymorphisms (SNPs): rs35810727, rs28373064, rs35439639 and rs35608965. Tag SNPs are SNPs that correlate with all the other SNPs in the same chromosome segment and they are selected to capture the maximum amount of information on the genetic variation. Primers and probes were custom synthesized by Applied Biosystems according to standard recommendations for the AB Prism 7900HT analysis system and genotyped with polymerase chain reaction-based TaqMan method (33) (link).
4
Genotyping HIV+ and HIV- Blood Samples
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DNA was extracted from 200 μl of packed blood pellets from HIV+ patients and whole-blood samples from HIV− donors using the QIAamp 96 spin blood kit (QIAGEN, Valencia, CA). DNA concentrations were measured using Qubit® Fluorometer (Invitrogen, Carlsbad, CA). SNPs were genotyped using Illumina’s GoldenGate® genotyping assay system combined with VeraCode® Technology (Illumina Inc., San Diego, CA). Allelic discrimination was performed using a BeadXpress® Reader (Illumina) according to the manufacturer’s instructions.
The genotype data were uploaded and filtered using the GenomeStudio data analysis software v2011.1 (Illumina Inc., San Diego, CA). SNPs were filtered by genotype call frequency (<0.9, n = 1) and replicate errors (n = 2). Samples with genotype call frequency <0.9 were excluded (n = 4). Subsequently, SNPs were excluded from analysis if genotypic distribution among HIV− donors, stratified by race, deviated from the Hardy-Weinberg equilibrium (HWE) with a significant cutoff value of P ≤ 0.001 (n = 1). Thus, in the final analysis, 41 SNPs, as listed inSupplementary Table A , were examined in a total of 276 subjects (HIV+, n = 180; HIV−, n = 96).
The genotype data were uploaded and filtered using the GenomeStudio data analysis software v2011.1 (Illumina Inc., San Diego, CA). SNPs were filtered by genotype call frequency (<0.9, n = 1) and replicate errors (n = 2). Samples with genotype call frequency <0.9 were excluded (n = 4). Subsequently, SNPs were excluded from analysis if genotypic distribution among HIV− donors, stratified by race, deviated from the Hardy-Weinberg equilibrium (HWE) with a significant cutoff value of P ≤ 0.001 (n = 1). Thus, in the final analysis, 41 SNPs, as listed in
5
Measuring HPA-1a Antibody Levels and Typing
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HPA-1a antibody (IgG) levels were measured using a modified MAIPA assay.[12 (link)]
DNA from the immunized women’s mothers was obtained from buccal swabs (Omni swabs, Whatman®, GE Healthcare UK Limited Buckinghamshire, UK). Purification of DNA was performed using a DNA isolation kit (QIAamp 96 Spin Blood kit, QIAGEN Inc., Valencia, CA, USA).
HPA-1 typing was performed using fluorogenic probes and a modified FAST 5´ Nuclease assay (NA)[13 (link)] or by flow cytometry.[14 (link)]
The HLA DRB3 typing was performed by sequencing the HLA DRB3 gene when present. For the PCR, we used intron-located amplification primers previously described by Kotsch et al.[15 (link)]
DNA from the immunized women’s mothers was obtained from buccal swabs (Omni swabs, Whatman®, GE Healthcare UK Limited Buckinghamshire, UK). Purification of DNA was performed using a DNA isolation kit (QIAamp 96 Spin Blood kit, QIAGEN Inc., Valencia, CA, USA).
HPA-1 typing was performed using fluorogenic probes and a modified FAST 5´ Nuclease assay (NA)[13 (link)] or by flow cytometry.[14 (link)]
The HLA DRB3 typing was performed by sequencing the HLA DRB3 gene when present. For the PCR, we used intron-located amplification primers previously described by Kotsch et al.[15 (link)]
6
Genotyping HIV+ and HIV- Blood Samples
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