The DNA was extracted from the collected blood samples using the blood genome extraction kit (No. DP348-03) from Tiangen Biochemical Technology (Beijing, China) Co., Ltd. according to the manufacturer’s instructions. The extracted DNA was placed under an ultraviolet spectrophotometer to detect its purity and concentration. The DNA concentration was >20 ng/µL, and the OD 260/280 was between 1.7 and 1.9, which met the experimental requirements. The samples were then stored at −20 °C after extraction.
Blood genome extraction kit
Manufactured by Tiangen Biotech
Sourced in China
The blood genome extraction kit is a laboratory tool designed to isolate and purify genomic DNA from whole blood samples. The kit includes the necessary reagents and protocols to efficiently extract high-quality genomic DNA, which can be used for various downstream molecular biology applications.
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6 protocols using blood genome extraction kit
1
Extraction and Quantification of High-Quality DNA
2
Genomic DNA Extraction from Blood
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The genomic DNA was extracted from the peripheral venous blood in both groups using the blood genome extraction kit (Tiangen, Beijing, China) in strict accordance with the instructions of the kit. According to the volume of sample, an appropriate amount of protease K solution was added into the centrifuge tube, and peripheral venous blood samples and buffer were also added. The mixture was mixed evenly using a vortex oscillator and incubated at 65°C for 8–10 min. Then, 2 mL of absolute alcohol was added into the samples, mixed evenly, and transferred into an absorption column. We added 2 mL of buffer into the absorption column, followed by centrifugation at 3000 rpm for 1 min. Finally, 200 μL of elution buffer was added into the absorption column, and the resulting solution was the genomic DNA.
3
Genetic Profiling of Hydrosalpinx Patients
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Genomic DNAs were extracted from peripheral blood of hydrosalpinx patients and those carrying fallopian tube patency/obstruction, according to the instruction of Blood Genome Extraction Kit (TIANGEN, Germany). Polymerase chain reaction (PCR) was carried out under following conditions: 1) 95°C for 5 min, 2) 35 cycles of 95°C for 40 s, 60°C for 40 s, and 72°C for 60 s, and 3) 72°C for 5 min. PCR reaction system (25 ml) was composed of DNA template (1 ml), upstream primer (1 ml), downstream primer (1 ml), 2 × PCR TaqMix (12.5 ml), and ddH2O (9.5 ml). The primers for SNPs (Table S1 ) were synthesized by Sangon Biotech (Shanghai, China). After digestion at 37°C overnight, genotypes of the SNPs were identified utilizing 2.5% agarose gel electrophoresis and GoldViewI nucleic acid staining.
4
Comprehensive Genetic Profiling in ALL
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Pre-treatment bone marrow mononuclear cells were harvested via density gradient centrifugation. DNA was extracted using a blood genome extraction kit (Tiangen company, Beijing, China). ALL second-generation sequencing gene chips (Yuanqi Biomedical Technology company, Shanghai, China) and MISEQ second-generation sequencer (Illumina Inc., San Diego, CA, USA) were used to detect sixteen mutation genes, including NOTCH1, FBXW7, IKZF1, TP53, PAX5, FLT3, IL7R, CREBBP, JAK1, JAK2, JAK3, CRLF2, PHF6, PTEN, NT5C2, and SH2B3.
5
Comprehensive Genetic Analysis of Transporter and Metabolic Factors
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DNA was extracted through TIANGEN Blood Genome Extraction Kit (TIANGEN, Beijing, China) based on the manufacturer's instructions. All 194 tag SNPs (Supporting Information Table S1 ), involved in transporters, metabolic enzymes, inflammatory, immunological and autophagy-related factors, were selected with HaploView 4.2 and analyzed by using a previously published Agena MassARRAY System technique (Agena, CA, USA)24 (link).
6
Genotyping of TP63 and CD40 Polymorphisms
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Three microliters of fasting and EDTA anti-coagulated peripheral blood samples were collected from each subject. Genomic DNA was extracted using a blood genome extraction kit (Tiangen, Beijing, China). The concentration and purity of the extracted DNA were determined using a UV spectrophotometer. The DNA samples were stored at -20°C until analysis.
The polymorphisms rs6790167 in the TP63 gene and rs1535045 in the CD40 gene were genotyped using allele-specific polymerase chain reaction (AS-PCR). Primers were designed using the Gene Quest software (primer sequences are presented in Table 1). Each AS-PCR system comprised a total volume of 20 µL, which included 10 µL of 2 × TaqMan Master Mix, 1 µL of the forward primer (F), 1 µL of the reverse primer (R), 1 µL of the allele-specific primer (RA or FG or RC or RT), 2 µL of genomic DNA (equivalent to 50 ng), and 5 µL of ddH 2 O. The reaction conditions included a pre-denaturation step at 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 59°C for 30 s. and 72°C for 60 s, and a final extension at 72°C for 10 min. The PCR products were analyzed via electrophoresis in a 2% agarose gel.
All samples underwent duplicate pyrosequencing using the PyroMark ID system (Qiagen, Hilden, Germany) with the SNP sequencing program mode, following the manufacturer's manual. The SNP analysis software was used for sequencing and interpretation.
The polymorphisms rs6790167 in the TP63 gene and rs1535045 in the CD40 gene were genotyped using allele-specific polymerase chain reaction (AS-PCR). Primers were designed using the Gene Quest software (primer sequences are presented in Table 1). Each AS-PCR system comprised a total volume of 20 µL, which included 10 µL of 2 × TaqMan Master Mix, 1 µL of the forward primer (F), 1 µL of the reverse primer (R), 1 µL of the allele-specific primer (RA or FG or RC or RT), 2 µL of genomic DNA (equivalent to 50 ng), and 5 µL of ddH 2 O. The reaction conditions included a pre-denaturation step at 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 59°C for 30 s. and 72°C for 60 s, and a final extension at 72°C for 10 min. The PCR products were analyzed via electrophoresis in a 2% agarose gel.
All samples underwent duplicate pyrosequencing using the PyroMark ID system (Qiagen, Hilden, Germany) with the SNP sequencing program mode, following the manufacturer's manual. The SNP analysis software was used for sequencing and interpretation.
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