Rneasy lipid tissue rna extraction kit

Viral/total RNA was extracted from plasma/sera and tissues, using Qiamp viral RNA (Qiagen) and RNeasy lipid tissue RNA extraction kit (Qiagen), respectively. Protocol as outlined by the kit was followed, except for tissue harvested from CTR rabbits. For these samples, Qiazol (Qiagen) was substituted with Trizol (Invitrogen). Tissue was homogenized in 1 mL of Qiazol or Trizol using an Omni-tip homogenizer (Omni) or stainless steel beads with a mixer mill (Retsch, Inc., Newtown, PA, USA), respectively. The remaining steps were performed according to the manufacturer’s protocol. Additional DNase I digest in solution was performed on extracted RNA, followed by additional RNA clean up with RNeasy RNA extraction kit (Qiagen). The absence of genomic DNA was confirmed by performing PCR without reverse-transcription on a selection of RNA samples from each extraction batch. Quality and concentration of extracted RNA was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific). RNA samples were kept at −80 °C.

Samples representing the GM and WM of the human superior frontal gyrus (SFG) were obtained from the Sydney Brain Bank following ethical approval from the Human Research Ethics Committee of the University of New South Wales. The GM and WM tissues were obtained from three individuals (aged 79, 94 and 98) without significant neuropathology. The post mortem interval (PMI) of the samples ranged from 8–24 h, and the pH ranged from 5.77-6.65. For RT-qPCR experiments, another two SFG samples were used that were matched to the previous samples regarding age, PMI and pH.
Total RNA was extracted from approximately 100 mg for each case using Qiagen’s RNeasy Lipid Tissue RNA Extraction Kit. The RNA integrity numbers (RINs) were determined using an Agilent 2100 BioAnalyzer RNA Nano Chip. The RIN values ranged from 4.9-7.2. This RIN range was previously shown to have little effect on relative gene expression ratios [46 (link)].