Turbo dna free treatment

RNA was purified from the 21 individual prothoracic ganglion samples using the QIAGEN RNeasy Lipid Tissue Minikit (QIAGEN). Briefly, after removal from the cricket, each ganglion was homogenized by hand with a pestle. The ganglia were either stored at -20°C or immediately put through the remainder of the RNA extraction. Additional QIAzol lysis buffer was added to bring the volume to 1 mL. After 5 minutes of incubation at room temperature, 200 μl of chloroform was added. After 15 sec shaking, samples were incubated for 2 min at RT. Samples were centrifuged at 12,000g for 15minutes at 4°C. The aqueous phase was transferred to a new tube and 500 μl of 70% ethanol was added. QIAGEN RNeasy Mini Spin Columns were used to purify RNA. RNA was eluted from the column with 30 μl of RNase free water (the same 30 μl were put through the column twice). RNA concentrations were assessed before and after TURBO DNA-free treatment (Ambion by Life Technologies) with a nanospectrophotometer (Nanodrop, Thermo Fisher Scientific). An Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA) was used to further assess sample quality. RNA samples were stored at– 80°C until shipment on dry ice for sequencing.

A tissue panel (brain, heart, kidney, liver, lung, pancreas, ovary, skin, testis) was created from 23 samples donated from four anonymous euthanized dogs. Total RNA was extracted from ∼50 milligrams of tissue using the RNeasy mini kit (Qiagen) following the manufacturers instruction and with an additional Turbo DNA-free treatment (Ambion) to remove genomic DNA. RNA was quantified on a Bioanalyser (RIN between 7 and 9 for all samples) prior to cDNA synthesis using one microgram of total RNA and the Advantage RT-for-PCR kit (Clontech). No gDNA contamination was identified and so the expression of 22 novel loci were measured and normalised against ACTB (primers, Table S7). Loci were defined according to physical location, either intergenic or antisense, and tissue specificity, where tissue specific loci were required to be expressed at an FPKM>5.0 in a single tissue and <2.0 in all others.