Minibest universal genomic dna extraction kit

Genomic DNA was extracted from pereonites of the samples (Table 1) using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit. Parts of the mitochondrial cytochrome c oxidase subunit I (COI) were amplified using the primers LCO1490 and HCO2198 (Folmer et al. 1994 (link)). PCR amplifications were performed with an initial denaturation at 95 °C for 3 min, followed by 35 cycles of 30 sec at 94 °C, 30 sec at 50 °C, and 1 min 72 °C, with a final extension at 72 °C for 10 min. The PCR products were sequenced by using an ABI3730XL DNA Analyzer (Applied Biosystems). All sequences were deposited in DDBJ (DNA Data Bank of Japan), with accession numbers listed in Table 1.

Genomic DNA for the analysis of mtDNA content was isolated from human BMNC cells or mouse LSK cells using a TaKaRa MiniBEST Universal Genomic DNA Extraction kit (TaKaRa Bio Inc.). The relative mtDNA copy number was determined by qPCR with primers for the mitochondrial 16S rRNA gene and the nuclear Actin gene as previously described^{19 (link)}. All PCRs were performed in triplicate. The primers used to amplify 16S rRNA were 5′-GGTGCAGCCGCTATTAAAGG-3′ (16S rRNA, forward) and 5′-ATCATTTACGGGGGAAGGCG-3′ (16S rRNA, reverse).
For the measurement of Bcl-2 and Bax mRNA levels, the primers used were 5′-GACTGAGTACCTGAACCGGCATC-3′(Bcl-2, forward), 5′-CTGAGCAGCGTCTTCAGAGACA-3′(Bcl-2, reverse), 5′-ATGCGTCCACCAAGAAGC -3′(BAX, forward), and 5′-CAGTTGAAGTTGCCATCAGC-3′(BAX, reverse), according to a previous study^{20 (link)}. The relative mRNA levels of Bcl-2 and Bax were normalized to ACTB, the primers used for ACTB were 5′-TGACGTGGACATCCGCAAAG-3′ (ACTB, forward); and 5′-CTGGAAGGTGGACAGCGAGG-3′ (ACTB, reverse).

The cellular genomic DNA was extracted using a MiniBEST Universal Genomic DNA Extraction Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. PrimeSTAR® Max DNA Polymerase (Takara) and HIV-1 specific primers (Supplementary Table S1) were used to amplify the genomic DNA using PCR. The PCR products were purified using a MiniBEST Agarose Gel DNA Extraction Kit (Takara) and were detected for T7 Endonuclease I (T7EI)-based mutations using an EnGen® Mutation Detection Kit (NEB, Ipswich, MA, United States) following the manufacturer’s protocol. The purified PCR products were cloned into a TA cloning vector and sequenced for subsequent analysis.

Genomic DNA was isolated from peripheral blood leukocytes according to standard procedures using MiniBEST Universal Genomic DNA Extraction Kit (Takara Biomedical Technology Co., Ltd., Dalian, China). Relative mean telomere length of leukocytes was determined by a quantitative real-time PCR method which compares telomere repeat copy number (T) to single-copy gene copy number β-globin (S) (T/S ratio) as described previously [25 (link), 26 (link)]. In brief, each sample was measured in triplicates on an ABI 7500 Real-Time PCR System (Applied Biosystems) and the mean relative T/S ratio was calculated. A reference calibrator sample was included with each measurement to control inter-assay variability, and the average inter-plate coefficients of variability for the telomere and β-globin assays were <5.0%. A standard curve was also examined by using serially diluted reference DNA (1.56-100 ng; 2-fold dilution; seven points) with good linearity (R²> 0.97) for both the telomere and the β-globin measurement. The primers were as the following: for telomeres, forward 5’-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTT-GGGTT-3’ and reverse 5’-GGCTTGCCTTACCCTTA- CCCTTACCCTTACCCTTACCCT-3’; for β-globin measurement, forward 5’-GCTTCTGACACAACTG- TGTTCACTAGC-3’ and reverse 5’-CACCAACTTC- ATCCACGTTCACC-3’. In this study, 20% of samples were randomly chosen to test the reproducibility of measurements.

The pathogens used in this study were listed in Table 1. The genomic DNA of bacteria and mycoplasma strains were extracted from culture by using MiniBEST Universal Genomic DNA Extraction Kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol. Each virus’s genomic RNA was extracted from 200 μL of virus suspension or clinical samples using MiniBEST Universal RNA Extraction Kit (TaKaRa, Dalian, China) according to the manual. The extracted DNA/RNA were eluted in 30 μL of distilled water. The RNA was synthesized to cDNA via reverse transcription using the PrimerScriptTM cDNA Synthesis Kit (TaKaRa, Dalian, China) with random primers (Nona-deoxyribonucleotide mixture) according to the manual, then quantified at 260 nm using a Nano Drop 2000 (Thermo Fisher Scientific, Waltham, USA). All the DNA/RNA were stored at -70°C until used.