Epiquik m6a rna methylation quantification kit

The m⁶A levels of total RNA were assessed by using an EpiQuikTM m⁶A RNA methylation quantification kit (Epigentek; Wuhan, China, CAT. No. p-9005) according to the manufactures’ protocol. Briefly, 200 ng aliquots of RNA were extracted from liver tissues. Negative control, positive control, and RNA samples were bound to strip wells using RNA high binding solution. Then, capture and detect antibodies were added into the mixed solution. After the detected signal was enhanced, the OD intensity of m⁶A was quantified at 450 nm absorbance in a microplate spectrophotometer (Thermo Fisher, Waltham, MA, USA).

Total RNA was extracted via TRIzol (Invitrogen, Carlsbad, CA, USA) and RNA quality was assessed by NanoDrop (Thermo Fisher Scientific, Farmingdale, NY, USA). The m6A modification level of total RNA was examined according to the instruction of EpiQuikTM m6A RNA methylation quantification kit (Colorimetric, EpiGentek, Farmingdale, NY, USA). In brief, 200 ng RNA was added to the assay well; then, the appropriate diluted concentration of detection antibody solution was added to the assay well. The m6A levels were quantified using the colorimetrical analysis of absorbance at 450 nm and calculated according to the standard curve.

Sequenced SARS-CoV-2-positive RNA samples (n = 60; 10 per variant) were randomly selected from adult (30–60 years old) patients (men and women). In addition, 10 negative samples were used as controls. RNA concentration and purity were measured with a spectrophotometer (Nanodrop, Thermo Fisher Scientific, Waltham, MA, USA Thermo). Because of the small amounts of RNA in nasopharyngeal samples, all samples were vacuum-lyophilized (LABCONCO, Kansas City, MO, USA) at −56 °C and 0.123 mBar, for 48 h. The samples were then resuspended in nuclease-free water for a final concentration of 60 ng/µL. The methylation assay was performed with the EpiQuik^TM m6A RNA Methylation Quantification Kit (Epigentek, Farmingdale, NY, USA), according to the manufacturer’s instructions. Absorbance at 450 nm was measured in a microplate reader (EPOCH2, BioTek, Thermo Fisher Scientific, Waltham, MA, USA). The standard curve was obtained and the relative methylation level m6A (%) was determined according to the following formula: $$m6A (\%)=\frac{\left(O{D}_{sample}-O{D}_{NC}\right)÷S}{\left(O{D}_{PC}-O{D}_{NC}\right)÷P}\times 100$$
where OD_sample is the optical density of the sample, OD_NC is the optical density of the negative control, OD_PC is the optical density of the positive control, S is the amount of RNA (ng) applied to the well, and P is the amount of RNA (ng) applied to the positive control well (Supplementary File S2).