*3.3. RNA Extraction and Quality Evaluation*

In the in vitro immunotoxicity screening test using Jurkat T cells, DMM was found to have significant immunotoxicity. Therefore, we further systematically evaluated the immunotoxicity of DMM on human Jurkat T cells, and the mechanism of action was also expounded by using comparative transcriptomics. To fully elucidate the underlying mechanism, a transcriptome analysis based on RNA-seq was performed. During transcriptomic studies, selecting a proper pesticide exposure concentration is very important, because too high a concentration could cause cell death, and too low a concentration might not be cytotoxic [37]. As shown in Figure 3A and Table 1, when the exposure concentration was EC50, the RNA bonds were unclear, and the RIN was below 8, which indicated that the total RNA degraded to a degree that it was not suitable for transcriptome analysis [38]. The RNA bonds under the EC25 treatment (named M21), the EC10 treatment (named L4), and the control (named Ctr) were clear, and there was no contamination of other impurities. The RIN values were higher than 9.5, which indicates good RNA quality. Moreover, according to the procedure of the ISO 10993-5 standard, a tested material that is incubated for at least 24 h with precultured cells and has a decreased viability of under 70% of the control is considered cytotoxic [39]. As shown in Figure 1, the cell viability under the EC10 and EC25 treatments exposure was 97.8% and 81.8%, respectively, which indicated that samples M21 and L4 could be used in the following transcriptomic analysis.

**Table 1.** RNA quality assessment.


RIN: RNA Integrity Number.
