Rps18

To verify the reliability of transcriptome sequencing, the total RNA extracted from primary astrocytes of the x12, x12A, and x90 groups was used for quantitative real-time PCR (qRT-PCR) to detect expression level changes of selected genes. qRT-PCR was performed with the Bio-RAD CFX96 Real-Time PCR detection system using the RealStar Green Fast Mixture kit (GenStar, Beijing, China) according to the manufacturer’s instructions. Genes were normalized to RPS18 (Sangon Biotech, Shanghai, China). The primers used for qRT-PCR are listed in Table 1. The output Ct values were used for statistical analysis and were calculated as described previously (Chen et al., 2011 (link)):

1) ΔCt (Target gene Ct−averaged endogenous control Ct)

2) ΔΔCt (ΔCt_(sample)− ΔCt_{(Control group)})

3) Fold change (2^−ΔΔCt)

The t-test was used for statistical analysis, and the results are presented as the mean ± SD; n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001.

Cerebral RNA (n = 3) was extracted from 2 litters with different parents. HiScript II Q RT SuperMix for qPCR (with gDNA wiper) (Vazyme, Nanjing, China) was used to reverse transcribed equal amount of RNA of each sample to complementary DNA. Real-time PCR was conducted with AceQ qPCR SYBR^® Green Master Mix (Vazyme, Nanjing, China) on a LightCycler^® 480 (Roche Diagnostics, Basel, Switzerland).
The amplifying conditions for cDNA were as follows: denaturation at 95 °C for 10 min, 45 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by 95 °C for 10 s, 65 °C for 60 s, 97 °C for 1 s and 37 °C for 30 s. Quantitation of the relative levels of mRNA were carried out by the comparative 2^−ΔΔCt method and normalized to Ribosomal Protein S18 (RPS18) levels (B661301, Sangon Biotech, Shanghai, China). Primer sequences are listed in Table 1.