M6a rna methylation quantification kit

The global RNA methylation levels of 6-methyladenosine were quantified by the m6A RNA methylation quantification kit (Epigentek, Farmingdale, NY, USA). Two hundred nanograms RNAs were coated on per assay well, and the m6A content was captured and detected according to the manufacturer’s instructions.
Shortly, the methylated fraction of total RNA, through ELISA-like reactions, was recognized by the m6A antibody and quantified in a microplate spectrophotometer by reading the absorbance at 450 nm.
In each experiment, the percentage of m6A was calculated using the second-order regression equation of a standard curve that was constructed by mixing equivalent molar concentrations at different ratios of full unmethylated and methylated control DNA. Each sample was analyzed in triplicate.

The cells were cultured in six‐well culture dishes until they reached approximately 80% confluence and were then treated with 1 μg/ml Porphyromonas gingivalis LPS (InvivoGen, San Diego, CA, USA) for 3, 6, 12 and 24 hrs. Cells without LPS stimulation were used as controls.
Total RNA was extracted, and total m6A content was determined using an m6A RNA methylation quantification kit (EpiGentek, Farmingdale, NY, USA) according to the manufacturer's instructions.

Total RNA was isolated using TRIzol reagent (Cwbiotech). The m⁶A level was measured using an m⁶A RNA methylation quantification kit (EpiGentek), according to the manufacturer’s instructions.

Total m⁶A modification level was measured in 200 ng RNA extracted from RA-FLSs transfected with METTL3/14 siRNA. M⁶A levels of total RNA were measured using an m⁶A RNA methylation quantification kit (P-9005, EpiGentek, USA) according to the manufacturer’s instructions.

Total RNA from 18 pairs of pterygium and normal conjunctiva tissues from discovery phase was used for quantification of m6A modifications using an m6A RNA Methylation Quantification Kit (P-9005, EpiGentek). First, we prepared a negative control and also a positive control using six different concentrations of m6A ranging from 0.01 to 0.5 ng/μL, as recommended by the manufacturer. Then 2 μL positive control, 2 μL negative control, or 200 ng total RNA were bound in the 96-well plates. After removing the binding solution from each well, the samples were washed three times. The wells were incubated for 1 h with the addition of anti-m6a antibody and washed four times. Then, add the detection antibody to the wells, incubate at room temperature for 30 min and wash four times. Add the enhancer solution, incubate at room temperature for 30 min and then wash five times. Finally, add the developer solution and incubate at room temperature until the developer solution turns blue and then add the stop solution, the samples were analyzed on a SYNERGY 2 (BioTek) microplate reader at 450 nm; the absolute amount of m6A was quantified and the percentage of m6A in RNA was established as%m6A in total RNA.

The m⁶A levels in the total RNA were detected using a commercial m⁶A RNA methylation quantification kit (#P9005, Epigentek, NY, USA). Briefly, 250 ng of total RNA was added to each well, and capture antibody solution and detection antibody solution were added according to the manufacturer's protocol. The absorbance of each well at a wavelength of 450 nm was measured to determine the m⁶A level, and then calculations were performed based on the standard curve.