High capacity cdna synthesis kit with rnase inhibitor

RNA was generated from each well of a 6-well treated plate of human neural progenitor cells. After media aspiration, wells were washed with 1 mL of PBS and then lysed with 1 mL TRIzol (Thermo Fisher). The cells were incubated at room temperature for 5 min and RNA was extracted with the DirectZol RNA MiniPrep Kit (Zymo Research #R2052). RNA was stored at −80°C until ready for use. cDNA was generated from 1200ng RNA using the High Capacity cDNA Synthesis Kit with RNase inhibitor (Thermo Fisher #4368814). cDNA was used immediately or stored at −20°C until ready for use. Before use, cDNA was diluted 1:4 with water. qPCR was conducted on the Roche 480 Light Cycler in a 384-well plate. Into each well 5 μL of TaqMan Gene Expression Master Mix (Thermo Scientific #4369510), 0.5 μL of 20X commercial TaqMan primer probe for each gene described (Thermo Scientific: GRN, Hs00963707_g1; FXN, Hs00175940_m1; BDNF, Hs00380947_m1; EGR1, Hs00152928_m1; CDK5, Hs00358991_g1; SYT1, Hs00194572_m1; SYP, Hs00300531_m1), 0.5 μL of DNase/RNase free water, and 4 μL of above diluted cDNA was added. C_T values (n=9 technical replicates) were calculated with LightCycler 480 software. Results were normalized to GAPDH. Replicate mean values and standard error of the mean are reported.

RNA was generated from each well of a 6-well treated plate of NPCs or neurons. Media was aspirated. The wells were washed with 1 mL of DPBS and then lysed with 1 mL TRIzol Reagent (ThermoFisher #11596026). The cells in TRIzol were incubated at room temperature for 5 minutes and RNA was extracted with the DirectZol RNA MiniPrep Kit (Zymo Research #R2052). All RNA was stored at −80°C until ready to use.
cDNA was generated using the High Capacity cDNA synthesis Kit with RNase inhibitor (ThermoFisher #4368814) with 1200 ng RNA. cDNA was used immediately or stored at −20°C until ready to use. Before use, cDNA was diluted 1:4 with DNase/RNase-free H₂O (Invitrogen #10977-015).
qPCR was conducted on the Roche 480 Light Cycler in a 384-well plate. Into each well was added 5 μL of TaqMan 2X Gene Expression Master Mix (ThermoFisher #4369510), 0.5 μL of 20X commercial TaqMan primer probe (ThermoFisher, GRN: Hs00963707_g1, FXN: Hs00175940_m1, GAPDH: Cat.#432924E), 0.5 μL of DNase/RNase-free H₂O, and 5 μL of above diluted cDNA. Results were normalized to GAPDH and replicate mean values and standard error of the mean are reported. Significance was determined with unpaired t-tests with GraphPad Prism software.