Buccal cells were collected for genetic analysis by rubbing the inside of each child participant’s cheek with two swabs. The Qiagen DNA Micro Kit (Qiagen, Valencia, CA) was used to extract genomic DNA from buccal swab samples. Extracts were kept at 4 °C when being actively used for analysis, and were held at −80 °C for long-term storage. The extracted DNA was used to genotype subjects for the 5-HTTLPR polymorphisms. Polymerase chain reaction (PCR) was carried out using the Applied Biosystems thermal cycler Gene Amp 9700, and PCR products were separated on polyacrylamide gels, stained with ethidium bromide, and visualized and documented by a UV imaging system (Bio-Rad Labs, Richmond, CA). Genomic DNA was purified from buccal swab cellular extracts and stored according to manufacturer instructions (Qiagen). Following Chorbov et al. (2007) , the primers used for amplification were 5`-GGCGTTGCCGCTCTGAATGC-3` (forward) and 5`- GAGGGACTGAGCTGGACAACCAC-3`(reverse). The PCR conditions used were: 5 min of initial denaturation at 94 °C followed by 30 cycles of 30 s of denaturation at 94 °C, 20 s annealing at 58 °C and 20 s of extension at 72 °C, and a final extension of 5 min at 72 °C. 5-HTTLPR distributions were: S/S = 30 (30%), L/S = 80 (51%), and L/L =46 (19%); this distribution is in Hardy-Weinberg equilibrium (χ2 (1, N = 156) = 2.34, p =.13).
Dna micro kit
Manufactured by Qiagen
Sourced in Germany, United States
The DNA Micro Kit is a laboratory equipment designed for the extraction and purification of DNA from small sample sizes. It provides a reliable and efficient method for isolating high-quality DNA from various sources, such as tissue, cells, or bodily fluids. The kit utilizes a silica-based membrane technology to capture and purify the DNA, while removing impurities and contaminants. The DNA Micro Kit is a versatile tool for a wide range of applications that require high-purity DNA, such as genetic analysis, PCR amplification, and molecular biology research.
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Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Hiseq 4000, by Illumina (3 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Kapa hyper prep kit, by Roche (2 mentions)
Dna blood mini kit, by Qiagen (2 mentions)
Nextflex 96 barcodes, by PSC Biotech (2 mentions)
Nextseq2000 300 cycles kit, by Illumina (2 mentions)
S220 acoustic sonicator, by Covaris (2 mentions)
Seqcap ez exome v2 kit, by Roche (2 mentions)
Qiamp dna micro kit, by Qiagen (2 mentions)
Ez dna methylation kit, by Zymo Research (2 mentions)
Qiaamp dna micro kit, by Qiagen (2 mentions)
Rnase a, by Qiagen (2 mentions)
Proteinase k, by Roche (2 mentions)
Lc ms quality grade water, by Thermo Fisher Scientific (2 mentions)
Micromanipulation, by Narishige (2 mentions)
Uv imaging system, by Bio-Rad (1 mentions)
Dna rna mini kit, by Qiagen (1 mentions)
Geneamp 9700, by Thermo Fisher Scientific (1 mentions)
Ultraviolet imaging system, by Bio-Rad (1 mentions)
50 protocols using dna micro kit
1
Buccal Swab Genetic Analysis Protocol
2
Sperm and Brain DNA/RNA Extraction
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The rats were anesthetized with isofluorane until the toe-pinch hind leg reflex was not observed, and were quickly decapitated. The epididymis was isolated from male rats, punctured with small holes using a needle, and incubated at 37 °C in 350 µL of phosphate buffered saline (PBS) for 30 minutes, to permit swim out of the sperm. The supernatant was transferred to a new tube and additional 350 µL PBS was added to the tube containing the epididymis. The new tube was allowed to incubate at 37 °C for an additional 30 minutes and the supernatant was removed again. The two supernatants were pooled and centrifuged at 4 °C. A pellet was obtained and sperm DNA was extracted using the Qiagen DNA Micro kit (Qiagen, Germany). At the time of sacrifice for male pups, the brain was quickly removed, weighed, and the HPC was extracted and flash frozen on dry ice. DNA and RNA were extracted from the HPC using the RNA/DNA Mini kit according to manufacturer’s protocols (Qiagen, Germany).
3
Telomere Length Measurement in Ear Samples
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Ear samples (5–7/group were collected at the end of the study). The Qiagen DNA Micro kit (Qiagen, Germany) was used to extract genomic DNA (gDNA) as described in Cawthon, 2002 (link); Hehar and Mychasiuk, 2016 (link). gDNA samples had mean 260/230 and 260/280 spectral ratios of 2.24 and 1.90, respectively. TL analysis was conducted on all diluted DNA samples (20 ng/μL) using a similar protocol to that previously described (Sun et al., 2019 (link)). RT-qPCR reactions were conducted by adding 1 μL DNA, 1 × SYBR Green FastMix with Rox, and primers so that the total volume in each well was 20 μL. Each reaction was performed in duplicate, including no template controls (NTC) to ensure that the reactions were not contaminated. Primer final concentrations were (forward/reverse): 270 nM/900 nM for Tel; and 300 nM/500 nM for 36B4 (Hehar and Mychasiuk, 2016 (link)). Absolute quantitative PCR was used to determine the ratio of telomeres to a single copy gene (36B4), calculated as [2Ct(telomeres) / 2Ct(36B4)]−1 = −2−ΔCt. This was then used in the following formula to determine TL: TL = 1910.5 (−2−ΔCt) + 4,157 (Cawthon, 2002 (link); Hehar and Mychasiuk, 2016 (link); Sun et al., 2019 (link)).
4
Telomere Length Analysis in Rat Tissue
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Seven days after the final assigned sham or mTBI, a sample of tissue was taken from each rat's ear and stored at −80 °C (Hehar and Mychasiuk, 2016 (link)). The Qiagen DNA Micro kit (Qiagen, Germany) was used to extract genomic DNA (gDNA) as described in Hehar and Mychasiuk, 2016 (link). gDNA samples had mean 260/230 and 260/280 spectral ratios of 2.14 and 1.89, respectively. TL analysis was conducted on all diluted DNA samples (20 ng/μL) using a similar protocol to that previously described (Cawthon, 2002 ; Hehar and Mychasiuk, 2016 (link)). RT-qPCR reactions were conducted by adding 1 μL DNA, 1 × SYBR Green FastMix with Rox and primers so that the total volume in each well was 20 μL. Each reaction was performed in duplicates, including no template controls (NTC) to ensure that the reactions were not contaminated. Primer final concentrations were (forward/reverse): 270 nM/900 nM for Tel; and 300 nM/500 nM for 36B4 (Hehar and Mychasiuk, 2016 (link)). Absolute quantitative PCR was used to determine the ratio of telomeres to a single copy gene (36B4), calculated as [2Ct(telomeres) / 2Ct(36B4)]−1 = −2−ΔCt. This was then used in the following formula to determine TL: TL = 1910.5 ∗ (−2−ΔCt) + 4157 (Cawthon, 2002 ; Hehar and Mychasiuk, 2016 (link)).
5
Genotyping 5-HTTLPR in Buccal Samples
6
Genome-wide CNV Detection Protocol
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Genomic DNA of all available individuals was extracted from whole blood samples using the Qiagen DNA micro kit (QIAgen, Hilden, Germany). The quantity and quality of the isolated DNA were verified spectrophotometrically using the NanoDrop 2000 (Thermo Fisher Scientific, Prague, Czech Republic). Genotyping was performed using Illumina HumanOmni2.5 Exome BeadChips (San Diego, CA) at The Microarray Facility of The Centre of Applied Genomics of The Hospital for Sick Children in Toronto according to manufacturer protocol. Raw data were uploaded into Illumina GenomeStudio version 2011.1 for genotype calling. All samples with a genotype call rate >99 % were subjected to further analysis. Relatedness of investigated subjects was assessed from obtained genotypes in PLINK (Purcell et al. 2007) .
Extended homozygosity regions >3 Mb were detected using the 3.2.0 Illumina cnvPartition CNV Analysis Plug-in within GenomeStudio software.
CNVs were identified using PennCNV (Wang et al. 2007) and the above-mentioned cnvPartition CNV Analysis Plug-in. Only gains and losses containing a minimum of 10 probes were reported. Gene content of CNVs of interest was functionally annotated in GeneDistiller (Seelow et al. 2008 ) and population frequencies of the identified changes were assessed in the curated catalogue of human genomic structural variations known as DGV (http://dgv.tcag.ca/dgv/app/home).
Extended homozygosity regions >3 Mb were detected using the 3.2.0 Illumina cnvPartition CNV Analysis Plug-in within GenomeStudio software.
CNVs were identified using PennCNV (Wang et al. 2007) and the above-mentioned cnvPartition CNV Analysis Plug-in. Only gains and losses containing a minimum of 10 probes were reported. Gene content of CNVs of interest was functionally annotated in GeneDistiller (Seelow et al. 2008 ) and population frequencies of the identified changes were assessed in the curated catalogue of human genomic structural variations known as DGV (http://dgv.tcag.ca/dgv/app/home).
7
Methylation Analysis of PRDM1 Overexpression
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Bisulfite-converted DNA from PRDM1 overexpressed T cells or control T cells was extracted by using DNA micro kit (QIAGEN, 74004) and transformed by using EZ DNA Methylation-Gold kit (ZYMO RESEARCH, D5006) according to manufacturer’s instructions. Illumina BEADLAB SYSTEM was use for analyzing MethylationEPIC BeadChip array. For pyrosequencing, methylated regions were amplified by PCR containing 10 ng of bisulfite-treated DNA, 10 μM forward and reverse primers, EpiTap PCR Buffer, 25 mM MgCl2, 2.5 mM dNTP mixture and 5 U/μl EpiTap HS in a final volume of 50 μl reaction according to the manufacturer’s instructions (TaKaRa R110A). Amplification and sequencing primers were according to a published article (20 (link)). Amp4: r-AACCCTCAAACCTAACTCATAC; q-GGAGGTGATAGTAAAGAAAGGA; Amp5: p-TGTTTGGGGGTAGAGGATTT; o-TATCACCCCACCTAAACCAA.
8
Extracellular Vesicle Characterization Protocol
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Supernatants of infected cells were centrifuged and treated with 10μg RNase (EN0531, ThermoFisher) or 10 units of recombinant DNase I (Roche 04716728001) in incubation buffer at 37°C for 30min before transferring to recipient cells. Supernatants that were heated at 70°C were cooled to room temperature before treating with DNase as mentioned above. Mock was treated with DNase Incubation buffer.
For analysis by fragment analyzer, EVs were treated with DNase I (18068-015, ThermoFisher) according to manufacturer’s instructions. DNase I was inactivated with EDTA at 65°C for 10min. The sample was divided in 4 parts and treated with 1% NP-40 for 15min on ice or 600μg/ml Proteinase K for 20min at 40°C and inactivated at 70°C for 20min. The samples were then subjected to DNase treatment as mentioned above before DNA isolation using the Qiagen DNA micro kit (56304).
For analysis by fragment analyzer, EVs were treated with DNase I (18068-015, ThermoFisher) according to manufacturer’s instructions. DNase I was inactivated with EDTA at 65°C for 10min. The sample was divided in 4 parts and treated with 1% NP-40 for 15min on ice or 600μg/ml Proteinase K for 20min at 40°C and inactivated at 70°C for 20min. The samples were then subjected to DNase treatment as mentioned above before DNA isolation using the Qiagen DNA micro kit (56304).
9
Extracellular Vesicle Characterization Protocol
10
Cytogenetic Analysis of Tumor Samples
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Genomic DNA was isolated from the FNAB specimen using DNA Microkit (Qiagen, Valencia, CA). DNA samples were processed for amplification, fluorescent labeling and hybridization to a high-throughput SNP array (AffymetrixCytoscan HD). Mean fluorescence for each SNP locus was compared to a normal reference (HapMap) and copy number was inferred by genomic segmentation (ChAS v2.0 Affymetrix). Copy number and heterozygosity were reported for chromosomes 3,6 and 8. The techniques used for cytogenetic analysis of the tumors have been described previously.1 , 2 (link), 10 (link), 11 , 12 (link)
All tumor samples underwent analysis for chromosome 3 (disomy/partial loss/loss) and 39 tumors underwent analysis for additional chromosomes 6 (6p disomy/loss/gain, 6q disomy/loss/gain) and 8 (8p disomy/loss/gain, 8q disomy/loss/gain). Alterations in chromosome 3 and 8 were considered high-risk cytogenetic features predictive of increased risk for systemic metastasis, based on previous publications.1 , 2 (link), 10 (link), 11 , 12 (link)
All tumor samples underwent analysis for chromosome 3 (disomy/partial loss/loss) and 39 tumors underwent analysis for additional chromosomes 6 (6p disomy/loss/gain, 6q disomy/loss/gain) and 8 (8p disomy/loss/gain, 8q disomy/loss/gain). Alterations in chromosome 3 and 8 were considered high-risk cytogenetic features predictive of increased risk for systemic metastasis, based on previous publications.1 , 2 (link), 10 (link), 11 , 12 (link)
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