m6a rna methylation quantification kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit detects N 6-methyladenosine (m 6A) RNA methylation status directly using total RNA isolated from any species such as mammals, plants, fungi, bacteria, and viruses.		Read on a microplate reader at 450 nm within 2 to 15 min.

Upstream tips
This kit detects N 6-methyladenosine (m 6A) RNA methylation status directly using total RNA isolated from any species such as mammals, plants, fungi, bacteria, and viruses.
Downstream tips
Read on a microplate reader at 450 nm within 2 to 15 min.

Publication protocol

Total m6A content was measured in 200-ng aliquots of total RNA extracted from MDA-MB-231 and MCF-7 subclones using an m6A RNA methylation quantification kit (cat. no. P-9005; Epigentek) according to the manufacturer’s instructions. To measure m6A+ NANOG mRNA levels, m6A immunoprecipitation was performed as described before (47). A 1-µg aliquot of m6A antibody was conjugated to protein A-agarose slurry (Millipore) overnight at 4 °C. A 100-μg aliquot of total RNA was incubated with the antibody in immunoprecipitation buffer (50 mM Tris⋅HCl, 750 mM NaCl, and 0.5% Igepal CA-630) supplemented with RNase inhibitor for 3 h at 4 °C, RNA was eluted from the beads by incubation in 300 μL of elution buffer (5 mM Tris⋅HCl, 1 mM EDTA, and 0.05% SDS) with 4.2 μL of proteinase K for 1.5 h at 50 °C, and m6A+ RNA was purified by phenol/chloroform extraction and analyzed by RT-qPCR. The nucleotide sequences of the primers used for detection for m6A+ NANOG mRNA were as follows: 5′-ATG CAA CCT GAA GAC GTG TG-3′ and 5′-GAG ATT GAC TGG ATG GGC AT-3′.

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