Tesee precess 48 homogenizer

RNAlater—stored solid tissue was dried from excess RNAlater solution with Whatman filter paper, submerged in 0.5 mL Qiazol (Qiagen, Hilden, Germany) in TeSeE grinding tubes (Bio-Rad, Hercules, CA, USA) and homogenized using TeSeE PRECESS 48 Homogenizer (Bio-Rad). The total RNA from the homogenate was purified by Direct-zol RNA Miniprep column purification (Zymo Research, Irvine, CA, USA) according to the providers’ protocol. The RNA was eluted in 50 µL of nuclease-free water and stored in −80 °C for further use. Circulating RNA from plasma samples was purified using Norgen Total RNA kit (Norgen Biotek, Thorold, ON, Canada) according to the modified protocol for plasma samples by the manufacturer. Briefly, thawed plasma samples were centrifuged at 400× g for 2 min and 100 µL of cell-free plasma was transferred to a new nuclease-free eppendorf tube. After the denaturation step, 25 fmol synthetic C. elegans miRNA cel-miR-39-3p (Qiagen) was spiked in for normalization together with 0.7 µL MS2 RNA carrier (Roche). Instead of using the provided washing solutions, washing steps were done using 95% ethanol and the RNA was eluted and stored as above.

Tables 2 and 3 shows the tissue tested in each patient. Tissues samples were collected post mortem using disposable equipment, snap frozen, and stored at − 80 °C. Strict precautions were undertaken to prevent potential cross contamination of samples during collection, handling and storage. Frozen samples were homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) adjusted to 10% (w/v) using a TeSeE^™ Precess 48^™ homogenizer (Bio-Rad). Homogenates were then filtered through a 20 g needle with a syringe.