br Sample preparation for quantitative PCR qPCR br Frozen serum
Sample preparation for quantitative PCR (qPCR)
Frozen serum samples were thawed at room temperature on ice. The sample preparation for qPCR was done accord-ing to the method described by Iqbal et al. . A prepara-tion buffer containing 2.5% Tween-20, 50 mmol/L Tris, and 1 mmol/L EDTA was constituted. To each 20 µl of serum sam-ple, 20 µl of the preparation buffer was added to deactivate proteins that bound to template DNA or DNA polymerase which could invalidate qPCR results. To the mixture, 20 µg of Proteinase K (Inqaba Biotec, South Africa) was subsequently added for protein INCB018424 at 56 °C for 50 min followed by heat inactivation at 95 °C for 5 min. The mixture was centrifuged at 1000 × g for 5 min and 0.2 µl of the supernatant was used as template for each qPCR reaction. sets of ALU primers were designed: the primer set for the 115 bp amplicon (ALU115) amplified both shorter (truncated by apoptosis) and longer DNA fragments, whereas the primer set for the 247 bp amplicon (ALU247) amplified only longer DNA fragments. The sequences of ALU115 primers were forward: 5 -CCTGAGGTCAGGAGTTCGAG-3 and reverse: 5 -CCCGAGTAGCTGGGATTACA-3 ; ALU247 primers were forward: 5 -GTGGCTCACGCCTGTAATC-3 and reverse: 5 - CAGGCTGGAGTGCAGTGG-3 . β -actin was used as normal-izer for all qPCR assays. The sequences of β -actin primers used were forward: 5 -GACCTCTATGCCAACACAGT-3 and reverse: 5 -AGTACTTGCGCTCAGGAGGA-3 .
DNA integrity determination
DNA integrity was calculated as the ratio of ALU247-qPCR to ALU115-qPCR. ALU115-qPCR values represented the to-tal amount of free serum DNA. ALU247-qPCR values repre-sented the total amount of DNA released from non-apoptotic cells. Since the annealing sites of ALU115 are within the ALU247 annealing sites, the qPCR ratio (DNA integrity) is 1.0 when the template DNA is not truncated and 0.0 when all template DNA is completely truncated into fragments smaller than 247 bp .
Results were analyzed statistically using Statistical Package for Social Sciences version 20 statistical software for windows. Data was expressed as mean and standard deviation. Dif-ferences in the study parameters between groups were as-sessed using student t-test. A p-value ≤ 0.05 was considered as significant. The mean ALU values of two replicates each of two independent tests were computed and reported as Mean ± SD to two decimal places.
qPCR conditions and quantification of ALU fragments
The reaction mixture for each direct qPCR consisted of a tem-plate sequence, 0.2 µmol/L of the forward and reverse primers (Inqaba Biotec, South Africa) for both ALU115 and ALU247, one unit of iTaq DNA polymerase, 0.02 µL of fluorescein cal-ibration dye and 1 × concentration of SYBR Green in a total reaction volume of 20 µL with 5 mmol/L Mg2+ . Real-time PCR amplification was performed with precycling heat activation of DNA polymerase at 95 °C for 10 min, followed 35 cycles of denaturation at 95 °C for 30 s, annealing at 64 °C for 30 s and extension at 72 °C for 30 s using QuantStudio5 Real-time PCR. The absolute equivalent amount of DNA in each sam-ple was determined by a standard curve with serial dilutions (10 ng – 0.01 pg) of gently prepared genomic DNA obtained from peripheral blood leukocytes of healthy donor volunteers. The purpose of using DNA from peripheral blood leukocytes of a healthy donor was to serve as external standard. A neg-ative control (sample without targetable DNA) was performed in each plate.
To achieve the highest sensitivity for DNA quantification, a qPCR assay that utilizes primer sets was applied to amplify the consensus ALU sequences (ALU115 and ALU247). Two
Approval for the conduct of the study was given by the Ethical and Protocol Review Committee of the School of Biomedi-cal and Allied Health Sciences, College of Health Sciences, University of Ghana (SBAHS: MD./10550649/AA/5A/2016– 2017) and the Institutional Review Board (IRB) of the Korle-Bu Teaching Hospital (STC/IRB/000100/2016).