M6a rna methylation quantification kit

Total RNAs were extracted from murine hearts by miRNeasy Mini Kit (QIAGEN, Cat# 217004) with DNase I on column treatment (QIAGEN, Cat# 79254). mRNA was isolated from total RNA via oligo(dT) polystyrene beads (Sigma, Cat#MRN10). To achieve sufficient enough mRNA concentration and make sure the quantity of purified mRNA, 2–3 mice left ventricular tissues or about (2–4) × 10⁶ NRCM were pooled and homogenized as one biological replicate. For mRNAs m⁶A relative quantification to determine the relative m⁶A RNA methylation status of two or three different RNA samples, m⁶A RNA Methylation Quantification Kit (Abcam, #ab185912) were used according to the manufacturer’s protocols. Briefly, 200 ng of mRNA was added to a 96-well plate. After RNA binding, m⁶A RNA capture, signal detection, relative quantification of m⁶A was analyzed as recommended by the manufacturer.

The quantification assay for m⁶A was performed as previously described^{20 (link)}. Total RNAs were isolated by TRIzol from CC cells according to the manufacturer’s instructions. For the m⁶A quantitation, m⁶A RNA methylation quantification kit (ab185912, Abcam) was performed. The m⁶A content level was quantified colorimetrically by reading the absorbance at 450 nm.

Total RNA from cell lines of different treatment were extracted using TRIzol reagent (Invitrogen, CA) and treated with deoxyribonuclease I (Sigma, Shanghai, China). After RNA quality inspection, the commercial m⁶A RNA methylation quantification kit (ab185912; Abcam, U.S.A.) was used to detect the total m⁶A level. Briefly, 250 ng RNAs were seeded in each well, followed by adding capture antibody solution and detection antibody solution according to the manufacturer’s instruction. The m⁶A levels were then measured colorimetrically by reading the absorbance of each well at a wavelength of 450 nm.

Total RNA from pulmonary cells or lung tissues was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The mRNA was purified from total RNA using the Dynabead mRNA Purification Kit (Invitrogen, Carlsbad, CA, USA). Two hundred nanograms of total RNA or mRNA were measured with a m⁶A RNA Methylation Quantification Kit (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. In brief, the test wells were coated with 200 ng total RNA or mRNA. Dilutions of the capture antibody solution and detection antibody solution were then added to each test well, and the optical absorbance of the samples was measured at 450 nm.

To analyze the relationship between m6A methylation levels and KIAA1429 expression in NSCLC cells, the m6A RNA Methylation Quantification Kit (Abcam) was carried out for m6A methylation of total RNA. Total RNA was isolated from cells using TRIzol (Beyotime). After binding 200 ng RNA to the wells for 90 min at 37°C, the samples were incubated for 60 min with capture antibody at room temperature according to the manufacturer's instructions. Then, the RNA mixture was incubated with the enhancement solution at room temperature for 30 min after the incubation with detection antibody. Once the detection signal transduction was complete, the m6A colorimetric quantification of m6A levels was performed at an absorbance of 450 nm on a microplate reader FlexStation III (Molecular Services, USA) within 2 to 15 min.
²⁴ (link)