Genomic DNA was extracted from whole blood (QIAamp: Qiagen, Tokyo, Japan). The RHD genotype was analyzed by polymerase chain reaction (PCR), with sequence-specific primers essentially as described previously [5 (link)]. Using PCR-sequence-specific primers, the RHD deletion (RHD*01N.01) of the hybrid Rhesus box and the c.1227G > A mutation of the RHD*01EL.01 were determined. Multiplex PCR was used to amplify all exons 1 through 10 of the RHD gene, with exon 8 as a positive control as it has no nucleotide difference between the RHD and RHCE genes. When alleles were encountered in DEL samples that could not be explained by our screening methods, RHD exons 1–10 were sequenced as previously described [5 (link)].
Qiaamp
Manufactured by Qiagen
Sourced in Germany, United States, Japan, Australia
The QIAamp is a laboratory equipment product designed for nucleic acid purification and extraction. It utilizes a silica-based membrane technology to isolate DNA, RNA, or viral nucleic acids from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Purelink, by Thermo Fisher Scientific (1 mentions)
16 capillary 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Hotstartaq, by Qiagen (1 mentions)
Gotaq 1pcr master mix, by Promega (1 mentions)
Rneasy, by Qiagen (1 mentions)
Agarose gel electrophoresis, by Avantor (1 mentions)
Pcr master mix, by New England Biolabs (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Quantstudio real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Taqman genotype software, by Thermo Fisher Scientific (1 mentions)
Dna blood mini kit, by Qiagen (1 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
60 protocols using qiaamp
1
RHD Genotyping from Whole Blood
2
Cecum Microbial DNA Isolation
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At the end of the experiments, cecum contents were collected in sterile Eppendorf tubes. Genomic bacterial DNA was isolated from cecum samples with fast DNA stool mini kits (QIAamp, QIAGEN) following the manufacturer’s instructions.
3
Detecting Influenza A Virus Resistance
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Viral RNA was isolated from clinical specimens using PureLink (Invitrogen/Thermo Fisher, Carlsbad, CA) or QIAamp (Qiagen, Hilde, Germany) RNA extraction kits, according to the manufacturers’ instructions. Quantitative detection of H5N1 RNA was done batch-wise using real-time RT-PCR as described previously [11 (link), 12 (link)]. The presence of mutations in the viral neuraminidase and matrix 2 (M2) genes that confer resistance to oseltamivir and adamantanes, respectively, was analyzed using pyrosequencing or Sanger sequencing [16 (link), 17 (link)]. Viral RNA was reverse transcribed as described previously [8 (link), 11 (link), 12 (link)] and amplified using gene-specific primers. Primer sequences are available upon request. Amplification was performed using Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) or HotStar Taq (Qiagen, Hilden, Germany) according to the manufacturers’ instructions. The ExoSAP-IT (Affymetrix, Inc., Santa Clara, CA) purification kit was used to purify the PCR products. Pyrosequencing was performed on the PyroMark ID instrument using Pyro Gold Reagents kits (Biotage AB, Uppsala, Sweden). For Sanger sequencing, DNA was sequenced using the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer’s instructions using a 16-capillary 3130xl Genetic Analyzer (Applied Biosystems).
4
Rapid Enterovirus Detection in HFMD Outbreaks
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For the Neijiang CDC, the application of RT real-time PCR to confirm HFMD cases was enacted in Jan 2012. According to the national guidelines, throat swab samples from 6513 probable cases (available for testing out of 12751 probable cases) from Jan 2012 to Dec 2017 were collected in Neijiang city. Samples were frozen and stored at -80°C at each site before being shipped in batches to the Neijiang Center for Disease Control (CDC), Sichuan CDC and Sichuan University for further testing. Enterovirus RNA was extracted from throat swab samples using a viral RNA extraction kit (QIAamp, Qiagen, Valencia, CA) following the manufacturer’s instructions. One-step RT-real-time PCR was conducted using a commercial kit (BioPerfectus Technologies, Beijing, China; Cat No. JC20303) to detect the enterovirus RNA. The commercial assay was developed for multiple detections based on the Taqman probe method, which contains three sets of primer pairs and probes to detect EV71, CV-A16, and pan-enterovirus. The result was considered positive if amplification produced a signal at a cycle number ≤35 and otherwise was considered negative.
5
Gene Expression and Mitochondrial DNA Analysis
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Total DNA and RNA were extracted from PBMCs using the QIAamp and RNeasy kits, respectively, according to the manufacturers’ instructions (QIAGEN, Germany), to assess the gene products by quantitative polymerase chain reaction (PCR). The purified RNA in mRNA samples was reverse transcribed into complementary DNA using the QuantiTect Reverse Transcription Kit (QIAGEN, Hilden, Germany). The PCR was performed in duplicate using the GoTaq1 PCR Master Mix (Promega, Madison, WI, USA) and the subsequent primers (Table 2 ) on a CFX Connect Real-Time System (Bio-Rad Labs, Hercules, CA, USA).
Relative mRNA expression was calculated after normalization using beta actin as internal controls utilizing the 2−ΔCT method. The mitochondrial DNA copy number was quantified as the ratio of DNA products of mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 normalized to ribosomal 18S-RNA serving as an internal control using the 2−ΔCT method, as described previously [37 (link)].
Relative mRNA expression was calculated after normalization using beta actin as internal controls utilizing the 2−ΔCT method. The mitochondrial DNA copy number was quantified as the ratio of DNA products of mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 normalized to ribosomal 18S-RNA serving as an internal control using the 2−ΔCT method, as described previously [37 (link)].
6
Genetic Analysis of NF-κB1 Polymorphism
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The genomic DNA of patients was extracted from whole blood using standard methods (QIAamp, QIAGEN, Hilden, Germany). The −94ins/delATTG NF-κB1 insertion–deletion (−94ins/delATTG) promoter polymorphism (rs28362491) was determined restriction analysis. Therefore, the primer NF-κB1 insertion–deletion (−94ins/delATTG) polymorphism _del/ins_SE (5′-CTTGGATCCATGCCGACCC-3′) and NF-κB1 insertion–deletion (−94ins/delATTG) polymorphism _del/ins_AS (5′-TAGGGAAGCCCCCAG-GAAG-3′) were used to amplify a 158 bp PCR fragment, followed by digestion with restriction enzyme PFiMI (New England Biolabs, Ipswich, PA, USA). The PCR was performed at an annealing temperature of 60 °C in a 25 µL commercially available PCR master mix (New England Biolabs). The analysis was performed using agarose gel electrophoresis (PeqLab, Erlangen, Germany), resulting in a 158 bp fragment for D-allele and a 121 bp product for I-allele. Randomly chosen samples with known genotype were reanalyzed for result verification.
7
Genomic DNA Extraction and Genotyping
8
Genetic Analysis of Tuberous Sclerosis
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All patients have a statement attesting to the informed consent of a parent and/or legal guardian for participation in the study and their parents signed their written informed consent. The study was conducted in accordance with the Declaration of Helsinki and Institutional Review Board (Comité de Ética en Investigación, Instituto Nacional de Pediatría, México) approval was obtained (protocol reference number 060/2014). Total peripheral blood leukocytes or buccal swab cells were obtained from the 66 cases, their available parents and first-degree affected family members. Genomic DNA was obtained with a commercially available kit using a silica-based approach (QIAamp; Qiagen, Victoria, Australia) according to the manufacturer’s protocol. Specific primers were designed to enable PCR amplification of coding regions and intron-exon boundaries (±20 base pairs) of the TSC1 (NG_012386.1, NM_000368.4) and TSC2 (NG_005895.1, NM_000548.3) genes. Primer sequences and amplification conditions are available upon request.
9
Genomic DNA Extraction from Treponema pallidum
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To obtain T. pallidum genomic DNA, in general, lesions were gently squeezed to release an exudate, then swabbed and placed in a 1.5 mL tube. Swab shafts were subsequently cut, and lysis buffer AL added, following the QiaAMP protocol (Qiagen, Germantown, MD, United States). Samples were quality assessed by tp47 and β-globin qPCR, sequenced and genomes assembled as previously described (Lieberman et al., 2021 (link)) or, for samples from Colombia, Malawi, Guangzhou, China, and Chapel Hill, United States, consensus sequences were assembled as previously described with minor modifications (Chen et al., 2021 (link); Parr, 2021 (link)). Alignment, recombination masking and generation of the maximum likelihood phylogeny were performed as previously described (Lieberman et al., 2021 (link)).
10
Genotyping of liver disease variants
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Genomic DNA extraction from blood samples was performed using a QIAamp (Qiagen, Hilden, Germany) DNA Blood Mini Kit, following the manufacturer’s instructions. To evaluate the extracted DNA qualitatively and quantitatively, we used a Thermo Scientific Nanodrop 2000 spectrophotometer, Wilmington, NC, USA. Each DNA sample underwent genotyping analysis for the variants PNPLA3 rs738409 C>G, TM6SF2 rs58542926 C>T, HSD17B13 rs9992651 G>A, and GCKR rs1260326 T>C. Using TaqMan SNP Genotyping Assays (ThermoFisher, Waltham, MA, USA), the experiment was conducted on a QuantStudio Real-Time PCR System (Applied Biosystems, ThermoFisher, Waltham, MA, USA). TaqMan Genotype Software (Applied Biosystems, QuantStudioTM Design & Analysis Software v1.4.1) was used to analyze these data for genotype, and allelic discrimination plots were created to show the genotypes of all samples.
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