Dnazol

The pellet of cell debris left from RNA purification was subjected to 1 mL DNAzol-ES following instructions from the vendor (Molecular research center Inc., Cincinnati, OH, USA). The mixture was subjected to centrifugation at 10,000× g for 10 min, and the supernatant was then transferred to a new centrifuge tube. Then 500 µL 100% ethanol was added to the supernatant and mixed at room temperature for 3 min. Genomic DNA was precipitated at 5000× g centrifugation for 5 min. The DNA pellet was washed twice with 1 mL ice cold 75% ethanol and air dried for 5 min. The pellet was dissolved using 20 µL TE buffer (10 mM Tris pH8.0, 1 mM EDTA). To clone the transcription factor IIIA (TFIIIA) fragment in Arabidopsis, two primers (AtTFIIIA genome p f: 5′-ggagacctcctgagaagctccagc-3′ and AtTFIIIA genome p r: 5′-gtccttatcacggttgtcattactatg-3′) were used. The PCR product was confirmed using agarose gel electrophoresis.

Fresh leachate samples were collected from 19 landfills from six different regions across the United States in triplicate (57 total samples) during the summer and fall of 2011 by on-site technicians (Figure 1). The metadata used in this study were also used to determine the potential impact of environmental parameters on the distribution of CECs detected within landfills (Masoner et al., 2014 (link)). Any pipelines or tubing used to collect leachate were purged with at least three volumes or for 5 min to remove stagnant leachate or other contaminants. All equipment and tubing were field rinsed with at least 1 L of leachate prior to sampling. Triplicate samples of biomass and particulate matter were collected through a sterile in-line polypropylene filter (Advantec; 47 mm diameter) pre-loaded with a nitrocellulose membrane filter (Whatman; 0.45 μm pore size, 47 mm diameter). Volumes of leachate filtered varied from 8 to 1500 mL at the discretion of the on-site technicians, based on restriction of flow due to filter obstruction (Table S1). After filtration, the nitrocellulose filter was removed from the holder using sterile forceps, transferred to 5 mL of DNAzol (Molecular Research Center, Inc., Cincinnati, OH, USA), shipped overnight to the University of Oklahoma, and stored at −80°C until DNA extraction.

DNAzol (Molecular Research Center) was used to isolate genomic DNA from T. brucei cells. Typically, 5x10⁷ cells were collected for DNA extraction using guidelines recommended by the manufacturer. Total RNA was extracted from samples (3x10⁷cells) using TRIzol (Invitrogen), following the manufacturer’s protocol.

Total DNA was extracted from ∼5×10⁶ cells using DNAzol (Molecular Research Center, Inc.) according to the manufacturer's instructions. To measure mitochondrial DNA (mtDNA) copy number, semi-quantitative real-time PCR was employed using the iQ5 real-time PCR detection system and the SensiFAST SYBR & Fluorescein One-Step Kit (Bioline) for amplicon detection. A fragment of each of the mitochondrial ND1 and ND4 genes was amplified using the primers ND1F (5′-cacccaagaacagggtttgt-3′) and ND1R (5′-tggccatgggattgttgttaa-3′), and MTND4F (5′-caaccttttcctccgacccc-3′) and MTND4R (5′-ctggataagtggcgttggct-3′). For internal loading controls, fragments of the nuclear-encoded mitochondrial gene β2-microglobulin and the 18SrRNA subunit were amplified, using the primers B2-MGF (5′-cactaggaccttctctgagc-3′) and B2-MGR (5′-ctacagcttgggaattcctgc-3′) for β2-microglobulin and 18SrRNAF (5′-tagagggacaagtggcgttc-3′) and 18SrRNAR (5′-cgctgagccatgcagtgt-3′) for 18S rRNA. The threshold cycle numbers obtained for the ND1 and ND4 genes were subtracted from those for the β2-microglobulin and 18S rRNA genes to give normalized measures for the relative mitochondrial genome copy number.

The APOE genotyping was carried out at the San Carlos Clinical Hospital in Madrid. DNA was extracted from whole blood in ethylenediamine tetra-acetic acid (EDTA), using standard DNA isolation methods (DNAzol®; Molecular Research Center, Inc., Cincinnati, OH, USA). APOE haplotype was determined by analyzing single-nucleotide polymorphisms (SNPs) rs7412 and rs429358 genotypes with TaqMan assays (C____904973_10 and C___3084793_20, respectively), using an Applied Biosystems 7500 Fast Real-Time PCR machine (Applied Biosystems, Foster City, CA).
APOE ε3/ε4 and APOE ε4/ε4 subjects were considered APOE ε4 + , while APOE ε2/ε3 and APOE ε3/ε3 subjects were considered APOE ε4 − .

As indicated in previous studies conducted in our laboratory [27 (link),30 (link)], genomic DNA was extracted from whole blood in EDTA using standard DNA isolation methods (DNAzol®; Molecular Research Center, Inc., Cincinnati, OH, USA) from all participants. Using TaqMan genotyping assays on an Applied Biosystems 7500 Fast real-time PCR instrument (Applied Biosystems, Foster City, CA, USA), two single nucleotide polymorphisms (SNPs), rs7412 and rs429358, were genotyped. In this way, the APOE haplotypes were established. Sample controls for each genotype and negative sample controls were included in each assay. Several intra- and interplate duplicates of DNA samples were also included.