Maxwell rsc tissue dna kit

The obtained organs (bladder, muscle, liver, heart, lung, spleen, kidney, blood, brain, ear, tail, ovary, tooth, and bone marrow) were washed with 1 × PBS prior to genomic DNA extraction. Genomic DNA was extracted using an automated system (Maxwell® RSC, Promega, Madison, WI, USA) and a DNA extraction kit (Maxwell® RSC tissue DNA kit). These steps were performed according to the manufacturer’s instructions. Real-time PCR was performed using an ABI Step One Plus detection system (Applied Biosystems, Foster City, CA, USA) with SYBR Green (Luna Universal qPCR Master Mix, New England BioLabs Inc., Ipswich, MA, USA). The primer sequences used for the human Alu primer were as follows: forward 5′ CTG GGC GAC AGA ACG AGA TTC TAT 3′; reverse 5′ CTC ACT ACT TGG TGA CAG GTT CA 3′. qPCR was performed with 38 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 20 s.

Genomic DNA was either isolated from frozen liver tissue with a Maxwell RSC Tissue DNA Kit or from ETDA blood samples with the Maxwell RSC Whole Blood Kit using a Maxwell RSC instrument (Promega, Dübendorf, Switzerland).

For DNA extraction, tissues were initially homogenized in a homogenization solution (Promega) and then processed with Maxwell® RSC Tissue DNA Kit (Promega). Before loading samples onto Maxwell® RSC Cartridges, 300 μl of Lysis Buffer and 30 μl of Proteinase K were added to the homogenized samples, and further incubated for 20 min at 56°C. DNA was quantified with Qubit™ 4 fluorometer using Qubit™ dsDNA High Sensitivity Assay Kit (Invitrogen).

Frozen (−80°C) tumor tissue adjacent to that used for cytogenetic analysis and histologic examination was available for 21 myxomas (Table 1; Supplementary Table S1) and formalin-fixed, paraffin-embedded (FFPE) sample was used for case 14 (Supplementary Table S1). DNA was extracted from the frozen tumor samples, using the Maxwell RSC Tissue DNA Kit and the Maxwell RSC Instrument (Promega, Madison, WI, United States). For FFPE sample, DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). The DNA concentration was then estimated for each sample, using the QuantiFluor ONE dsDNA System using Quantus™ Fluorometer (Promega).

Genomic DNA was extracted in triplicate from 50 µL of each tissue digest (see Tissue digestion and mouse bioassay) obtained from piglets and from 50 to 80 mg from the brains of mice of the bioassay (see Tissue digestion and mouse bioassay) using a commercial DNA purification kit (Maxwell RSC Tissue DNA kit, Promega, Wisconsin, USA) following the manufacturer’s instructions.
Detection in the mouse brain from the bioassay was performed using 529 bp repetitive element (RE) template-based qPCR under PCR conditions as previously described (25 (link)). Detection and quantification of parasite DNA on target tissues from piglets was performed as described above with slight modifications in cycling parameters: preheating at 95°C for 10 min and then 40 two-step cycles of 95°C for 15 s and 65°C for 30 s. The standard curve was constructed using 10-fold serial dilutions of known copy numbers of the T. gondii tachyzoites (see Parasites and inoculum) and diluted in pig DNA (20 ng/µl). The slope (S between -3.5 and -3.2) and regression coefficient (R² > 0.991) of the standard curves were also calculated to estimate the efficiency of the assay.

DNA and RNA extractions from tumor samples were carried out using Maxwell RSC DNA FFPE kit (Promega, AS1450), Maxwell RSC Tissue DNA Kit (Promega, AS1610), Maxwell RSC RNA FFPE kit (Promega, AS1440) and Maxwell RSC Simply RNA tissue (Promega, AS1340). DNA and RNA were quantified using a Qubit fluorometer and adjusted to a final quantity of 50 ng of both DNA and RNA. Complementary DNA (cDNA) was obtained using SuperScript VILO Reverse Transcriptase (Thermo Fisher Scientific).

Blood samples were collected from the jugular vein of 1700 clinically healthy Thoroughbred horses (936 males, 78 geldings, and 686 females, aged 2–28 years, mean age ± SD = 4.0 ± 2.4) using heparinized tubes during annual laboratory tests for monitoring infectious diseases at the Ritto Training Center of the JRA and SSS from May 2018 to July 2022. The samples were stored at 4 °C, and leukocytes were collected within a couple of days.
eHRG polymorphisms were analyzed using genomic DNA purified from horse leukocytes. Briefly, blood was centrifuged at 1500 rpm for 15 min at 4 °C, and the plasma and blood cells were separated. Leukocytes isolated from buffy coats were used for DNA analysis, and plasma was stored at − 80 °C until protein analysis. DNA was extracted from leukocytes in the buffy coat using Maxwell® 16 Blood DNA Purification or Maxwell^® RSC Tissue DNA Kit (Promega, Madison, WI, UAS), according to the manufacturer’s protocol. Extracted DNA samples were used for polymerase chain reaction (PCR) with GoTaq® Master Mix (Promega) and a pair of primers (Forward: 5′-ACTCTGGTCGGCATGAGCATA-3′, Revers: 5′-TTTGTGTTTATTACTGGTCACATT-3′). The PCR products were separated using 2% agarose gel electrophoresis and visualized with ethidium bromide. Allele frequencies were estimated using the gene-counting method, and Hardy–Weinberg equilibrium was performed using allele frequencies.