mRNA stability analysis

Forty-eight hours after plasmid transfection, Actinomycin D (Sigma) was added to the culture medium at a concentration of 10 µg/ml. Cells were then collected at certain time points after Actinomycin D addition and total RNA was extracted with RNAiso Plus (Takara). For each sample, 3 µg of RNA was used for reverse-transcription with GoScript™ Reverse Transcriptase (Promega). From the 20 µl of reverse-transcription reaction volume, 1 µl was subjected to the following SYBR qRT-PCR analysis of mRNA. Human GAPDH served as the internal control gene. The value of AXIIR was normalized to that of GAPDH and the value of each time point was further normalized to that of the start point, which was designated as 100%.