Bioanalyzer 2100 nano chip

All qPCR data shown is based on four biological samples each tested using three technical replicates. Cells were grown in liquid VSuTB for specified times at 30 °C with or without Cm. For RNA isolation, mycelium was harvested by vacuum filtration and 100 mg was flash frozen in liquid nitrogen. Total RNA was isolated using the RNeasy Kit from Qiagen following the manufacturer’s instructions for fungi (Qiagen Inc., Santa Clarita, CA, USA). RNA integrity was determined using a BioAnalyzer 2100 nano chip (Agilent Technologies, Mississauga, ON, Canada) following the supplier’s instructions. cDNA was reverse transcribed from RNA (1 μg/reaction) using superscript III (Invitrogen, Carlsbad, CA, USA) following the manufacture’s directions. qPCR (StepOnePlus, Applied Biosystems, Foster City, CA) used the synthesized cDNA and PowerSYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) with 5 ng of cDNA template and a primer concentration of 200 nM in a total sample volume of 10 μL in a 96-well reaction plate. Samples were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolysis enzyme [52 (link)], using the ΔΔCT method [53 (link)].

Total RNA was purified using the RNAqueous^®-Micro kit (AM1931, Ambion, Austin, TX, USA). The manufacturer’s instructions were followed, except that wash steps 2 and 3 were modified to include rolling of the tubes and incubation with the wash buffer for 1 min, and the ethanol added for precipitation was increased to 1.25× lysis buffer volume to include small RNAs. The RNA was eluted in 2 × 10 µl RNAqueous Elution Buffer. In the layer samples for miRNA microarray analysis, four of the tissue samples were extracted using the mirVana^™ kit (Ambion, Austin, TX, USA). The RNA was eluted in 2 × 50 µl mirVana Elution Buffer. We used the Norgen Total RNA purification kit (Norgen Biotek, Canada) to purify RNA from whole medial entorhinal tissue.
RNA yield was determined using the NanoDrop 1000 spectrophotometer or the Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad, CA), and the quality was assessed with the Agilent BioAnalyzer 2100 Nano chip. Only samples with a RIN value of 8.5 or above were included for further analysis. Isolated RNA samples were stored at −80 °C until further use.