Additional quantification of the level of LOY for validation was performed using previously described procedure [21 (link)]. Briefly, extracted DNA was digested for 15 min with HindIII enzyme (Thermo Fischer) in 37 °C. After this, 50 ng of DNA was added together with PCR primers and probes targeting a known difference between the AMELX and AMELY gene assay C_990000001_10 (Thermo Fisher). Droplets were then generated using an automated droplet generator (Bio-Rad) and amplified using PCR. Droplets fluorescence intensity in two channels FAM and VIC was measured using the Bio-Rad’s QX200 Droplet Reader. The data were analyzed in Bio-Rad’s software QuantaSoft (version 1.7.4.0917) as described elsewhere [21 (link)]. The LOY quantification was performed by calculating the ratio between the amounts of AMELY and AMELX in each sample.
Hindiii enzyme
Manufactured by Thermo Fisher Scientific
Sourced in Lithuania, United States
HindIII is a type II restriction enzyme isolated from the bacterium Haemophilus influenzae. It recognizes and cleaves the palindromic DNA sequence 5'-AAGCTT-3' and its reverse complement. HindIII is a widely used tool in molecular biology for DNA manipulation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Qx200 droplet reader, by Bio-Rad (1 mentions)
Quantasoft, by Bio-Rad (1 mentions)
Automated droplet generator, by Bio-Rad (1 mentions)
Nanodrop spectrophotometry, by Thermo Fisher Scientific (1 mentions)
Ddpcr supermix for probes no dutp, by Bio-Rad (1 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (1 mentions)
Ddpcr supermix for probes, by Thermo Fisher Scientific (1 mentions)
4 protocols using hindiii enzyme
1
Droplet-based Quantification of Chromosome Y Loss
2
BRO Biobank DNA Quality Control
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BRO Biobank quality control was carried out every 6 months. 5% of tissue samples and 5% of DNA samples of the BRO Biobank were randomly selected and used to assess the DNA quality. Several methods have been used for quality control analysis of DNA samples stored in the BRO Biobank: The yield and purity of DNA samples were assessed by measuring the optical density (OD260/OD280) ratio using NanoDrop spectrophotometry (Thermo Fisher Scientific). A DNA sample of good quality should have an OD260/OD280 ratio between 1.8 and 2.1 [27 (link), 28 (link)]. The DNA integrity was evaluated by 0.8% agarose gel electrophoresis, and a quality score was assigned for each DNA sample based on the OD260/OD280 ratio and the DNA electrophoresis profile as described in [28 (link)].
In addition, DNA quality was assessed by enzymatic digestion (HindIII enzyme, Thermo Fisher Scientific, Lithuania) followed by gel electrophoresis as described in [29 , 30 (link)]. DNA quality was also evaluated by PCR amplification of four different regions of the β-globin gene using the primers shown inTable 1 . Obtaining four amplification bands was the indication of a good-quality DNA [27 (link), 29 , 31 (link)].
In addition, DNA quality was assessed by enzymatic digestion (HindIII enzyme, Thermo Fisher Scientific, Lithuania) followed by gel electrophoresis as described in [29 , 30 (link)]. DNA quality was also evaluated by PCR amplification of four different regions of the β-globin gene using the primers shown in
3
Droplet Digital PCR for LOY Quantification
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Quantification of the level of LOY in analyzed samples was performed using previously described procedure12 (link). Briefly, DNA extracted from sorted fractions of cells was digested with HindIII enzyme (#FD0504, ThermoFisher Scientific) for 15 min in 37 °C. Next, 50 ng of digested and diluted DNA was thoroughly mixed with ddPCR supermix for probes no dUTP (Bio-Rad), PCR primers and probes targeting 6 bp difference between the AMELX and AMELY gene assay (C_990000001_10, ThermoFisher Scientific). For the processing of samples and fluorescent measurement of the signal in channels FAM and VIC, we used the QX200 Droplet Digital PCR System (Bio-Rad). For the generation and analysis of the data we used the dedicated software QuantaSoft (version 1.7.4.0917).
4
Absolute mtDNA Copy Number Quantification
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Absolute quantification of mtDNA-CN was performed by droplet digital PCR based method, as described previously [6] . Briefly, amplification was first performed in a 20 μl multiplex reaction containing 1 ng of purified DNA from whole blood, 900 nM of primers and 250 nM of probes, 2X ddPCR supermix for probes (no UTP) and 5U/reaction Hin-dIII enzyme (Thermo Scientific, Hudson, NH, USA), and incubated for 20 min at room temperature to allow digestion with restriction enzyme (HindIII). Samples were subjected to droplet generation by an automated droplet generator and end-point PCR was performed [6] . The PCR plate was incubated overnight at 4 • C. This additional step significantly improved droplet recovery to maximum (19,000-20,000 droplets). Finally, droplets were read on a droplet reader and data were analysed using QuantaSoft™ Software, which determines the numbers of droplets that are positive and negative for each fluorophore in each sample. The fraction of positive droplets was then fitted to a Poisson distribution in QuantaSoft™ Software to determine the absolute copy number in units of copies/μl. DNA preparation and PCR experiments were performed in separate designated rooms and each run included negative and positive controls. No significant hazards or risks are associated with the reported work.
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