Puregene blood core kit b

Malaria infections were diagnosed by microscopy of Giemsa-stained thick blood smears, and by Real-Time PCR amplification of a species-specific segment of the multicopy 18SSU rRNA gene of human malaria parasites. The Giemsa-stained smears were evaluated by experienced microscopists, according to the malaria diagnosis guidelines of the Brazilian Ministry of Health. For Real-Time PCR, genomic DNA was extracted from either whole blood samples collected in EDTA, or from dried blood spots on filter paper using the Puregene blood core kit B (Qiagen, Minneapolis, MN, USA) or the QIAmp DNA mini kit (Qiagen), respectively, according to manufacturers’ instructions. Real-Time PCR was performed as previously described [25 (link)].

DNA was extracted from peripheral blood samples using PureGene Blood Core Kit B (Qiagen). For the derivation cohort, 28 DNA samples were tested using next generation sequencing (NGS) of the NF1 (NM_000267.3) and SPRED1 (NM_152594.3) genes. For the validation cohort, all 505 samples were tested using next generation sequencing (NGS) of 14 RASopathy‐associated genes (Table S1) as previously described (Pugh et al., 2014). Briefly, NGS was performed by oligonucleotide hybridization‐based DNA capture (SureSelect; Agilent) followed by sequencing using the MiSeq‐M01450 instrument (150‐base paired end mode; Illumina). Sequence reads were aligned to the reference sequence (GRCh37) using bwa‐mem v0.7.10, followed by variant calling using GATK, version 1.0.4705 (McKenna et al., 2010). For the validation cohort, Sanger sequencing was used to confirm all clinically significant variants and fill in regions with insufficient coverage. Methods used for polymerase chain reaction and Sanger sequencing have been previously described (Zimmerman et al., 2010). Copy number variants (CNVs) were identified via an NGS‐based detection tool (VisCap) (Pugh et al., 2016), but were only available for 23 patients in the derivation cohort and 281 patients in the validation cohort due to NGS data quality. Confirmation of CNVs was done using ddPCR as previously described (Ceyhan‐Birsoy et al., 2015).

At the time of blood collection, all individuals were submitted to a finger-prick for malaria diagnosis by light microscopy. The Giemsa-stained thick blood smears were prepared and examined by experienced local microscopists, according to the malaria diagnosis guidelines of the Brazilian Ministry of Health (2009) [41 ]. Species-specific PCR assays targeting different plasmodial targets (18S rRNA gene and non-ribosomal Pvr47/Pfr364 sequences) were carried-out essentially as previously described [42 (link)]. For this, genomic DNA was extracted from either whole blood samples collected in EDTA, or from dried blood spots on filter paper using the Puregene blood core kit B (Qiagen, Minneapolis, MN, USA) or the QIAmp DNA mini kit (Qiagen), respectively, according to manufacturers’ instructions.

The isolation and modification of genomic DNA was performed using Puregene Blood Core Kit B and EpiTect Bisulfite Kit (QIAGEN, Duesseldorf, Germany) as described previously [15 (link), 16 (link)]. Real-time quantitative methylation-specific PCR (RT-qMSP) was first used to evaluate SLIT2 promoter methylation with AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Piscataway, NJ). The primers for SLIT2 promoter methylation detection were as reported [12 (link)]. Relative SLIT2 promoter methylation was counted using the 2− ^∆∆CT formula as referred to ALU methylation.

Genomic DNA was extracted from 2 mL whole blood obtained from patients before induction of anesthesia using a Puregene Blood Core Kit B (Qiagen, Hilden, Germany). The quality and quantity of DNA were checked using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Samples with an absorbance ratio lower than 1.6 at 260 and 280 nm were excluded from further analysis. The samples were stored at 4 °C until use.