Purelink rna mini

Brain tissue or FACS-sorted microglia were collected in TRIzol (Invitrogen), and total RNA was extracted using PureLink RNA Mini or Micro kits (Life Technologies) per the manufacturer’s instructions. Purified RNA was then treated with amplification-grade DNase I (Invitrogen) to remove contaminating DNA and reverse transcribed into cDNA by using an iScript cDNA Synthesis kit (Life Technologies). Real-time PCR was performed using SYBR Green (Applied Biosystems) and cDNA template or water (nontemplate negative control) at an optimized annealing temperature (Table S1) with a CFX96 Real-Time PCR machine (Bio-Rad Laboratories). All reactions were conducted in triplicate, and PCR products were subjected to melt analysis to confirm purity after DNA amplification. For each gene, expression values were normalized to a β-actin control. The resulting relative gene expression was then expressed as a fold-change from the naive control samples. Primers were designed and obtained from Integrated DNA Technologies (IDT), and sequences are provided in Table S1.

Human embryonic kidney (HEK293T) cells were transiently transfected in triplicate with 1.2 µg of OPN1LW-pMT4 recombinant expression vector using Attractene (Qiagen, Chadstone, VIC, Australia) in six-well cell culture plates. After 48 h, transfected cells were harvested using Trypsin-EDTA 1X (Sigma-Aldrich, Castle Hill, NSW, Australia), and washed four times with PBS (1X; 138 mM NaCl, 2.70 mM KCl, 10 mM NaPO₄, 1.8 mM KPO₄, pH 7.4). Total RNA was extracted using the PureLink RNA Mini with the TRIzol kit (Thermo Fisher Scientific, Scoresby, VIC, Australia), before the generation of oligo dT-primed cDNA using 5 µl (1–2 µg) of total RNA incubated with 5 µl oligo dT (500 ng) and 20.5 µl sterile RNase-free water for 15 min at 85 °C, followed by 2 min on ice. Subsequently, 10 µl of 5X First-Strand Buffer (Genesearch, Arundel, QLD, Australia), 5 µl (0.1 M) of DTT (Genesearch, Arundel, QLD, Australia), 2.5 µl (10 mM) dNTP mix (Bioline, Alexandria, NSW, Australia), and 1 µl RNase murine inhibitor (40 U/µl; Genesearch, Arundel, QLD, Australia) was added and incubated for 2 min at 25 °C. Then, 1 µl of M-MuLV Reverse Transcriptase (Genesearch, Arundel, QLD, Australia) was added and incubated for a further 5 min at 25 °C, followed by 1 h at 42 °C. Finally, a further 1 µl of M-MuLV Reverse Transcriptase (Genesearch, Arundel, QLD, Australia) was added and incubated for another 1 h at 42 °C.