Fig. 3
Relative enrichment and mRNA expression of target transcripts in BC patients and controls. A MeRIP-RT-qPCR was used to analyze the mRNA enrichment of target genes, comparing the level of m6A modification in NK cell transcripts between BC patients and healthy controls. The dotted line represents the non-IP fraction (input control), which is set to 1. CT values were normalized to an exogenously added m6A-modified control (Gaussia luciferase), provided in the EpiMark® N6-methyladenosine Enrichment Kit (NEB, MA, USA). This RNA m6A-modified control was spiked into the samples before RNA fragmentation to monitor m6A enrichment. Quantitative m6A analysis reveals significant alterations in the m6A levels of NKG2D, ERK2, and PRF1 transcripts in NK cells from BC patients pools (n=3) compared to controls (n=3). In BC patients, m6A levels were higher in NKG2D mRNA and lower in PRF1 mRNA compared to healthy controls. Although ERK2 m6A levels were already very low in controls, they were even lower in BC patients. No significant changes were observed in the m6A levels of other target gene transcripts in NK cells between BC patients and controls. Results are expressed as the mean fold change in gene expression ± SD. Differences between groups were analyzed using an unpaired Student’s t-test. B RT-PCR was used to assess the mRNA levels of target genes in BC patients (n=16) compared to healthy controls (n=10). To measure relative mRNA expression levels, RPLP0 was used as an internal control (the housekeeping gene) to normalize the data across different samples. Results are expressed as the mean fold change in gene expression ± SD, and statistical differences were analyzed using an unpaired Student’s t-test