Maxima first strand cdna synthesis kit with thermolabile dsdnase

Whole RNA was isolated by a Quick RNA Miniprep kit (ZYMO Research) and quantified by a Q3000 microvolume spectrophotometer (Quawell, San Jose, CA, USA). RNAs were retrotranscribed on a Maxima First Strand cDNA Synthesis Kit with Thermolabile dsDNase (Thermo Fisher Scientific, Madrid, Spain). Real-time quantitative polymerase chain reaction (qPCR) was carried out using the PowerUp SYBR Master Mix (Thermo fisher Scientific, Madrid, Spain) and StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Madrid, Spain). Gene expression of GAP43 was quantified by the ΔΔCt method. Sample values were normalized to the threshold value of the housekeeping gene GAPDH.

Total RNA was extracted from ESCs using Quick RNA Miniprep kit (ZYMO Research); quantity and integrity were measured on a Q3000 micro volume spectrophotometer (Quawell). RNAs were reverse transcribed by a Maxima First Strand cDNA Synthesis Kit with Thermolabile dsDNase (Thermo Fisher Scientific). Real-time qPCR was carried out on a PowerUp SYBR Master Mix (Thermo Fisher Scientific) and ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). The reactions were run four times (independent biological experiments). The primers used for amplification are indicated in Supplementary Table 3. The fractional cycle number at which fluorescence passed the threshold (C_t values) was used for gene expression quantification by the comparative ΔΔC_t method. Sample values were normalized to the threshold value of Gapdh housekeeping gene.