Faststart universal sybr green master mix

LNCaP cells were seeded to either 6 cm or 10 cm dishes at 800,000 or 2,200,000 cells/dish respectively. Cells were allowed to attach for 24 h and then subsequently treated with either enzalutamide (5 μM) or DMSO for specified time period. Cells were then harvested with Trizol reagent (Ambion) and RNA integrity was verified via agarose gel electrophoresis. Promega High Capacity cDNA Reverse Transcription Kit (Promega) was utilized following manufacturer instructions and as described previously [36] (link), [37] (link), [38] (link). RT-qPCR was performed with FastStart Universal SYBR Green Master Mix (Thermo Fisher Scientific) and detected on a QuantStudio 6 Flex with QuantStudio Real-Time PCR control software (Thermo Fisher Scientific). QuantStudio Design and Analysis software (Thermo Fisher Scientific) was used for data analysis. Technical triplicates were run for all samples, samples without detectable amplification were deemed undetected. Primer sets were validated via melt curve and agarose gel analysis of RT-qPCR product. AR primers were used as described previously [36] (link) and IVL primers were used as described previously [39] (link). All primer sequences utilized are described in Supplementary Table S1.

Total RNA was isolated using Trizol reagent (Ambion, USA), and RNA concentration and integrity were verified by agarose gel electrophoresis. A high capacity cDNA reverse transcription kit (Promega, USA) was used to synthesize cDNAs as described previously.¹⁹ qPCR was performed using FastStart Universal SYBR Green Master Mix (Thermo Fisher Scientific, USA) on the QuantStudio 6 Flex System and QuantStudio Real‐Time PCR Software (Thermo Fisher, Scientific). Each sample was run with two technical replicates for 40 cycles and the average CT value of the two technical duplicates for each sample was used for further analysis. Autoclaved double‐distilled H₂O was also run as a nontemplate control for each primer set, and agarose gel electrophoresis was run to confirm the amplicon size and specificity for each primer set. Primer sequences used for all genes are described in Table S1.

Total RNA was isolated from the spinal cords of rats in the SCI and SCI+EA groups 21 days after SCI using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the RevertAid™ First-Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), as per the manufacturer's instructions. Gene-specific RT primers were used for each miRNA.
RT-qPCR was performed for 45 amplification cycles with the following conditions: 95°C for 10 sec, 60°C for 60 sec, and 95°C for 5 sec. FastStart Universal SYBR Green Master mix (Thermo Fisher Scientific, Inc.) was used to amplify cDNA using the ABI Q6 detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). All reactions were set up in triplicate. Relative quantification and calculations were assessed using the comparative threshold cycle method (2^−ΔΔCq) (44 (link)). All primers were from Yingbiotech. Primer sequences and are listed in Table I. GAPDH and U6 were used as the internal control for mRNA and miRNA, respectively.

Tissues were homogenized in 1 ml Trizol solution (Thermo Fisher, # 15596026). Tissue homogenates were clarified by centrifugation at 10,000 rpm for 5 min and stored at −80 °C. RNA was extracted according to the Trizol protocol. RNA was quantified by Nanodrop 1000 (Thermo Fisher) and reverse transcribed with Superscript III (Invitrogen # 18080093) using random primers. FastStart Universal SYBR Green Master Mix (Thermo Fisher # 4913850001) was used for relative quantification of cDNA on the ViiA 7 Real-Time PCR System (Thermo Fisher). Primer information is included in Table 1.