Validation of Dysregulated mRNAs

Two dysregulated mRNAs (Filip1 and Nsmf) were randomly selected, and further validated by using RT-qPCR to verify the authenticity of microarray assay data, as previously described [42 (link)]. Briefly, cDNA was synthesized by using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) from a total RNA of 2.5 μg (with mixed primer of oligo d(T) and random hexamer). In the qPCR assays, the cDNA, PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA, USA), primers (listed in Supplementary Table S4), and pure water (Qiagen, Germantown, MD, USA) were mixed for reaction. PCR triplicates were used, and qPCR reactions were performed by using the QuantStudio™ 6 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). The specificity of the PCR reaction was checked with the melting curves of PCR product at the end of reaction. The mean cycle threshold (Ct) values from the PCR triplicates were used, and the raw data for mRNA expression were further normalized against endogenous control β-actin and finally analyzed by using 2^−ΔΔCt calculation.