Allprep dna rna protein mini kit

All blood samples were subjected to a Lymphoprep density gradient centrifugation according to the manufacturer's instructions. Peripheral blood mononuclear cells (PBMCs) were lysed in RLT buffer from the AllPrep DNA/RNA/Protein Mini kit (Qiagen, Hilden, Germany). Plasma and PBMC samples were stored at −80 °C until further processing.
Nucleic acid extraction from 1 mL (first five patients) or 4 mL of plasma (diluted 1 : 1 with 0.9% NaCl during density gradient centrifugation) was performed using the QIAamp Circulating Nucleic Acid kit (Qiagen) following the manufacturer's instructions. ctDNA was eluted in 50 μL of Buffer AE. The concentration of cell‐free DNA was measured using a NanoDrop spectrophotometer (median concentration 4 ng·μL⁻¹, ranging from below the detection limit to 79 ng·μL⁻¹). DNA was extracted from PBMCs using the AllPrep DNA/RNA/Protein Mini kit (Qiagen) according to the manufacturer's protocol. All samples were then stored at −80 °C until further analysis.

After homogenising 30 mg of thyroid tissue in 600 µL of Buffer RLT pre-added with β-mercaptoethanol using TissueRuptor (Qiagen, Hilden, Germany), genomic DNA (gDNA), RNA and protein were extracted from the tissue lysate using Qiagen AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The concentration and purity of the extracted gDNA were determined using Qubit dsDNA BR Assay kit (Life Technologies, Darmstadt, Germany) on Qubit 2.0 Fluorometer, and by Thermo Scientific NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific, Massachusetts, United States), respectively. The yield and purity of the nucleic acids were estimated using Nanodrop 2000c Spectrophotometer. The gDNA and RNA integrity tests were performed by 1% agarose gel electrophoresis.

RNA from prostate cell lines (source: ATCC, mycoplasma free) was extracted using the Qiagen RNeasy mini kit (#74104), and all other RNA samples were isolated using the Qiagen AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Manchester, UK #80004), according to the manufacturer’s instructions. RNA concentration and quality was confirmed using a NanoDrop 2000 (ThermoFisher, Loughborough, UK), and cDNA was synthesised using the Transcriptor 1st strand cDNA kit (Roche, Welwyn Garden City, UK, #04379012001) following the manufacturer’s instructions. QRT-PCR reactions were performed using 10 ng of cDNA and 2x qPCRBIO SyGreen Blue Mix (PCRBiosystems, London, UK, #PB20.16) following the manufacturer’s instructions in a QuantStudio 7 QRT-PCR machine with the following primers: AR (Exon 5-6): F 5′-CCTGGCTTCCGCAACTTACAC-3′, R 5′-GGACTTGTGCATGCGGTACTCA-3′, AR-v7: F 5′-CGTCTTCGGAAATGTTATGAAGC-3′, R 5′-GAATGAGGCAA-GTCAGCCTTTCT-3′ [41 (link)], GAPDH: F 5′-ACAGTTGCCATGTAGACC-3′, R 5′-TTGAGCACAGGGTACTTTA-3′) [42 (link)], AXIN2: F 5′-TCAAGACGGTGCTTACCTGT-3′, R 5′-TGCTGCTTCTTGATGCCATCA-3′ and ASCL2: F 5′-AAAGAACCCTTGACCTGGGG-3′, R 5′-AGATCTTGGCCAGCATGGA-3′.

Frozen post-mortem hippocampal tissues were processed for western blot analysis. A total of 18 subjects were included in the western blot analysis. Frozen hippocampal brain tissue (∼250mg) was prepared from controls (n = 6), PD (n = 6) and PDD (n = 6). All cases had a post-mortem interval of < 29h. Briefly, protein was extracted using the QIAGEN AllPrep DNA/RNA/Protein mini kit according to manufacturer’s instructions (QIAGEN, 80204). Protein was quantified using the Pierce BCA Protein Assay Kit according to instructions (ThermoFisher, 23227) and the concentration of each sample standardized. Protein samples were combined with 4X sample loading buffer (BioRad, 1610747) and made up to a final volume of 25 μL with water, boiled at 100°C for 5 mins and loaded onto 10% acrylamide gels and separated by SDS-PAGE. The separated proteins were transferred from the gel to the PVDF membrane (BioRad 1620177). The blots were subsequently incubated overnight at 4°C in TBST with 5% BSA with anti-GOAT (1:1000; Phoenix Peptide) and anti-GAPDH (1:5000, Sigma) antibodies. Blots were visualized using the chemiluminescence method (ECL Select, RPN2235; GE Healthcare) and levels were quantified using ImageLab Software v4.1 (ChemiDoc XRS, BioRad) and normalized to GAPDH.

Frozen hippocampus samples (100 mg) were homogenized using Allprep^® DNA/RNA/Protein Mini Kit (Quiagen, Hilden, Germany). The extracts were aliquoted and stored at −80 °C for Western blotting analysis.

Genomic DNA and total RNA were extracted from the mandibular lymph nodes using the AllPrep DNA/RNA/Protein Mini Kit (QUIAGEN) and following the manufacture’s recommendations. RNA was stored at −80 °C until subsequent mRNA expression analysis. Individuals were genotyped using the Illumina porcine SNP60 BeadChips^{71 (link)}, from which 61565 SNPs were obtained. The average genotyping rate was 0.92, varying across individuals from 0.50 to 0.99.

Tumor gDNA samples were available from The Swedish Childhood Tumor Biobank^{20 (link)} for all Karolinska University Hospital patients. Isolation of gDNA from tumors was performed using the AllPrep DNA/RNA/Protein Mini kit (Qiagen, Hilden, Germany) and from blood (minimum 1 ml) using the QIAamp® DNA Blood Midi/Maxi kit, vacuum protocol (Qiagen), according to the manufacturer’s instructions. WGS analysis was performed on paired tumor-normal data by The Swedish Childhood Tumor Biobank as previously described and used to detect driver genetic aberrations.^{18 (link)}