Gdnf and Manf mRNA Expression in Microglia

To determine whether Gdnf and Manf mRNA is expressed in microglial SIM-A9 cells, total RNA was extracted from cells in TRI reagent (T9424, Sigma-Aldrich), digested with DNase I and converted to cDNA using iScript gDNA Clear cDNA Synthesis Kit (172-5034, Bio-Rad Laboratories, Hercules, CA, USA). To check whether target genes were amplified from contaminated genomic DNA, negative control without reverse transcriptase was included. Mouse hypothalamic cDNA was used as a positive control. PCR was performed for 40 cycles at 95 °C for 3 s and 60 °C for 30 s. PCR products along with a 50 bp DNA ladder (10416014, Thermo Fisher Scientific, Waltham, MA, USA) were separated on 3% agarose gel in 1X TAE and visualized under UV light.
Expression levels of mRNA were measured by real-time PCR using the ABI 7500 Fast thermal cycler (Applied Biosystems, Foster City, CA, USA) as described previously [29 (link)]. All primer pairs (Table 1) were designed using the NCBI Primer-Blast tool. Relative mRNA levels were determined using ΔΔCt method by normalizing to hypoxanthine guanine phosphoribosyl transferase (Hprt) mRNA levels. All experiments were performed in triplicates and the coefficient of variation (CV) was less than 5% for each triplicate.