Validating sRNA-seq Results by qRT-PCR

We randomly selected seven DEMs to validate the sRNA‐seq results using quantitative real‐time PCR (qRT‐PCR). cDNA samples were synthesized from remanent total RNA from the Section 2.4 with TransScript® miRNA First‐Strand cDNA Synthesis SuperMix (TransGen Biotech). Furthermore, qRT‐PCR analysis was performed using TransScript® Green miRNA Two‐Step RT‐qPCR SuperMix (TransGen Biotech) on a CFX96 real‐time PCR Detection System (Bio‐Rad), and the following thermal cycling conditions were described as our previous study (Ren et al., 2023 (link)). The snRNA U6 was served as an internal reference gene, and other primers were designed by Primer Premier 5 (Table S9). All reactions were performed in triplicate, and the relative expression was calculated using the 2−ΔΔct method (Livak & Schmittgen, 2001 (link)). Finally, the column chart of relative expression of qRT‐PCR and sRNA‐Seq data was produced using GraphPad Prism (version 9.0; GraphPad Software Inc.).