Allprep dna rna protein kit

DNA was isolated from fresh frozen samples using the AllPrep DNA/RNA/Protein Kit (Qiagen). Quantity and quality of isolated DNA was assessed by spectrophotometry (NanoDrop Technologies, Inc). Per the manufactory’s instructions, 5 samples yielded inadequate quality and/or concentrations of genomic DNA that precluded further analysis. One sample yielded DNA of sufficient quality and concentration, but the sample failed to amplify despite multiple attempts. As such, a copy number analysis was performed on a total of 26 ACC samples using the TaqMan Copy Number Assays (Applied Biosystems) with primers and probes specific to target gene SLC12A7 and house-keeping gene Ribonuclease P RNA Component H1 (RPPH1). Normal adrenal tissue was used as a diploid reference control. The assay was performed in quadruplicates. Copy calls were predicted using CopyCaller software v2.0 (Applied Biosystems).

All murine livers were cut to 25 mg per sample and RNA was extracted using the Qiagen AllPrep DNA/RNA/Protein kit (Gaithersburg, MD) according to the manufacturer’s instructions. For each reaction, Taqman primers (Invitrogen, Carlsbad, CA) and reagents (Agilent Technologies, Santa Clara, CA) were used according to the manufacturer’s instructions with 200 ng RNA. A 2-step cycling RT-PCR protocol was used in an ABI One Step Plus cycler. An initial reverse transcription step of 30 min at 50°C and 10 min at 95°C was followed by an amplification step consisting of 15 s at 95°C and 1 min at 60°C cycled 40 times. Target gene expression was normalized to the GAPDH gene and compared to the chow-fed control group using the 2−ΔΔCt method.

Cecum content was collected from CON mice kept on the MD, switched to WD or treated with 200 μl of oleic acid and analyzed 6 hours, 12h and 24h after the switch or the treatment. Isolated cecum content samples were immediately shock frozen in liquid nitrogen and stored at -80°C. DNA was extracted using the AllPrepDNA/RNA/Protein Kit (Qiagen) with the following changes in the disruption and homogenization steps: 600 ml of RLT buffer and 3 x 4 mm metal beads were added to the tube containing the cecum content and bead-beated at 10 Hz for 2 min using the Retsch M400. To separate fibers from bacteria, samples were centrifuged at 700g for 2 min at 4°C. Supernatants containing the bacteria were transferred to a tube containing 0.9 mg of 0.1 mm of zirconia beads (OPS Diagnostics) and samples were homogenized at 25 Hz for 5 min. Before the transfer of the lysates to the QIAshreeder column (Qiagen), lysates were pre-heated to 25°C. The QIAshreeder columns were centrifuged at 13,000g for 3 min at room temperature for further homogenization. The flow-though was centrifuged for 3 min full-speed to pellet the cell debris. The supernatants were loaded on the DNA columns and DNA was extracted according the manufacturer’s instructions with the modification that DNA was eluted in 100 μl EB.