Iscript reverse transcriptase kit

For RNA extraction, brain tissue was flash frozen in liquid nitrogen then pulverized using a glass tissue grinder (Wheaton). TRIzol reagent (Thermofisher) was added to resulting tissue particulate and RNA extraction was performed using the Purelink RNA Mini Kit (Thermofisher), following the manufacturer’s instructions. To remove any genomic DNA carryover, the samples were treated with DNase I (Qiagen) for 30 min at 37°C, followed by heat inactivation for 5 min at 65°C. Then, 1 μg of total RNA was used to synthesize cDNA with the Bio-Rad iScript reverse transcriptase kit (Bio-Rad), following the manufacturer’s instructions. The control reaction was set up using all components of the reaction mixture but without the reverse transcriptase enzyme (i.e., no reverse transcriptase).

For RNA extraction, C. neoformans planktonic or sonicated biofilm-derived cells were suspended at a density of 5 x 10⁸ in 5 mL of PBS and homogenized with 0.5-mm-diameter zirconium-silica glass beads (Thermo Fisher) using a beater for 4 min to ensure complete lysis. Cell debris was removed by centrifugation at 10,000 rpm for 10 min at room temperature. RNA extraction was performed using the Ambion RNA purification kit (Thermo Fisher), following the manufacturer’s instructions. To remove any genomic DNA carryover, the samples were treated with DNase I (Qiagen) for 30 min at 37°C, followed by heat inactivation for 5 min at 65°C. Then, 1 μg of total RNA was used to synthesize cDNA with the Bio-Rad iScript reverse transcriptase kit (Bio-Rad), following the manufacturer’s instructions. The control reaction was set up using all components of the reaction mixture but without the reverse transcriptase enzyme (i.e., no reverse transcriptase).

After receiving a sham or GA exposure, P21 rat pups were deeply anesthetized with isoflurane and transcardially perfused with ice cold phosphate buffered saline (PBS). Brains were rapidly extracted and the thalamus was exposed and removed for RNA extraction using dissection forceps. Samples were flash frozen in liquid nitrogen and stored at −80 degrees C. We isolated RNA from the thalamus using an RNeasy kit (Qiagen, Germantown, MD), after which cDNA was synthesized with an iScript reverse transcriptase kit (Bio-Rad, Hercules, CA). RNA and cDNA were measured for yield and purity spectrophotometrically.
PCR plates were run using primers for Gria1, Gria2, Gria3, Gria4 and housekeeping gene Gapdh. Primers were obtained from Bio-Rad and melting curves were checked on each plate to assess specificity. A CFX Connect rt-PCR Detection System (Bio-Rad) was used for quantification of sample cDNA. Each reaction well contained 100 ng of sample cDNA template, 7 μL nuclease-free water, 1 μL 20xPrimePCRAssay (primer), and 10 μL 2x SsoAdvanced universal SYBR Green supermix (Bio-Rad) for a final reaction volume of 20 μL. A PCR protocol was run consisting of the following parameters:
$$\mathit{Activation}:1\mathrm{cycle}\mathrm{for}2\mathrm{minutes}\mathrm{at}{95}^{{}_{°}}\mathsf{C}$$