Blood samples were collected from the antecubital vein after the subjects had fasted for ≥8 h. Part of the collected samples was used to examine biochemical indicators such as serum lipid levels, whereas the other part was transferred into a test tube containing anticoagulant solution to extract DNA. DNA was extracted using Tiangen Blood Genomic DNA extraction kits (Tiangen Biotech, Beijing, China), we use a PCR-LDR method to genotyping analysis as we described in detail in our previous study [27 (link)].
Tiangen blood genomic dna extraction kit
Manufactured by Tiangen Biotech
Sourced in China
Tiangen Blood Genomic DNA extraction kits are a set of laboratory equipment designed to isolate and purify genomic DNA from blood samples. The kits utilize a spin column-based method to efficiently extract high-quality DNA for downstream applications such as PCR, sequencing, and molecular biology experiments.
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5 protocols using tiangen blood genomic dna extraction kit
1
Genetic Profiling from Fasted Blood
2
Genotyping Blood Samples by PCR-LDR
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Genomic DNA was extracted from the blood cells using a standard phenol/chloroform extraction method, centrifuged, and stored at − 80 °C. All genomic DNA samples were analyzed by electrophoresis. DNA was extracted using Tiangen Blood Genomic DNA extraction kits (Tiangen Biotech, Beijing, China) and sent to Shanghai Jierui Biological Engineering Co., Ltd., for genotyping analysis using the polymerase chain reaction (PCR)-ligase detection reaction (LDR) method (Generay Biotech Company, Shanghai, China). For this part, we have covered this in detail in previous articles [24 ]. For quality control, we randomly chose 10% of samples for re-genotyping, and the concordance was 100%.
3
Genotyping of Five Key SNPs
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Five SNPs in BCO2, TRIB1, and PCSK9 were selected using the HapMap website (http://www.hapmap.org ) and the Haploview 4.2 software (Broad Institute, Cambridge, MA, USA). The SNPs included rs10431036 and rs11214109 in BCO2, and rs17321515 and rs2954029 in TRIB1, rs11583680 in PCSK9. The minor allele frequencies (MAFs) of these five SNPs were greater than 5%, and the linkage disequilibrium (LD) was r2>0.8.
A refrigerator at −80℃ was selected to store 5 mL of whole blood from each fasted individual, which was anticoagulated with EDTA. DNA was extracted using Tiangen Blood Genomic DNA extraction kits (Tiangen Biotech, Beijing, China) and sent to Shanghai Jierui Biological Engineering Co., Ltd. (Shanghai, China), for genotyping analysis using the PCR-ligase detection reaction method (Generay Biotech Company, Shanghai, China). For quality control, we randomly chose 10% of samples for regenotyping, and the concordance rates were 96.8% (rs10431036), 97.7% (rs11214109), 98.9% (rs17321515), and 99.2% (rs2954029).
A refrigerator at −80℃ was selected to store 5 mL of whole blood from each fasted individual, which was anticoagulated with EDTA. DNA was extracted using Tiangen Blood Genomic DNA extraction kits (Tiangen Biotech, Beijing, China) and sent to Shanghai Jierui Biological Engineering Co., Ltd. (Shanghai, China), for genotyping analysis using the PCR-ligase detection reaction method (Generay Biotech Company, Shanghai, China). For quality control, we randomly chose 10% of samples for regenotyping, and the concordance rates were 96.8% (rs10431036), 97.7% (rs11214109), 98.9% (rs17321515), and 99.2% (rs2954029).
4
Heteroplasmic mtDNA Mutation Analysis
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A total of 48 human peripheral blood samples (45 samples from unaffected individuals and three samples from patients carrying the m.3460G>A, m.11778G>A and m.14484T>C mutations) were collected from the Children's Hospital of Chongqing Medical University. Three positive samples were confirmed to be heteroplasmic mutations by DNA sequencing. The study was approved by the Children's Hospital of Chongqing Medical University review board, and informed consent was obtained from all the participants. All blood samples were handled anonymously according to ethical standards and the Helsinki declaration. Total genomic DNA from peripheral blood was extracted using a TIANGEN Blood Genomic DNA Extraction Kit (Tiangen, Beijing, China), quantified by a NanoDrop Microvolume Spectrophotometers (Thermo Fisher, Waltham, MA, USA), and was diluted to 5 ng/μl in Easy dilution buffer (Takara, Dalian, China). All DNA samples were randomly ordered and double-blind tested.
5
European Mouflon Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted from the venous blood of a single (sex unknown) European mouflon (Daqingshan Wild Animal Park, Hohhot, Inner Mongolia, China) with the Tiangen Blood Genomic DNA Extraction Kit (Tiangenbiotech, Beijing). Paired-end libraries with short-insert sizes from 244 bp to 700 bp were constructed with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, United States), while mate pair libraries with long-insert sizes from 1 kb to 15 kb were constructed with Illumina Nextera Mate Pair Library Preparation Kit (Illumina, United States; Supplementary Table 5 ). Sample processing was according to the manufacturer’s instructions. Illumina HiSeq 4000 platform (Illumina, CA, United States) were used for sequencing with the PE-150 module (Quail et al., 2012 (link)). Then the correction steps were performed with SOAPec_v2.01 (Luo et al., 2012 ) using the parameters: −k 21, −i 450,000,000.
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