Home › Analytical Chem › A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues
Analytical Chem JoVE (Open Access) Citable · DOI

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

DOI: 10.3791/54672-v
What you'll learn
  • ✓ Perform RNA isolation, qualification, and quantification for methylation analysis
  • ✓ Execute modified northern blotting to detect N6-methyladenosine in RNA samples
  • ✓ Interpret immunoblotting results to confirm m6A modification patterns
Protocol

Biopharma Insights A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

Difficulty
advanced
Total time
~1–2 days (including RNA isolation, gel electrophoresis, transfer, and detection)
Biosafety
BSL-1

Steps

1
Isolate, qualify, and quantify RNA samples

Extract total RNA from cells or tissues and assess quality and concentration using standard analytical methods to ensure suitable material for downstream methylation detection.

▶ 01:10
2
Perform gel electrophoresis of RNA

Load qualified RNA samples onto agarose gels and run electrophoresis to separate RNA by size before transfer to membrane.

▶ 02:29
3
Transfer RNA to membrane

Use northern blotting transfer techniques to move separated RNA from gel to membrane for subsequent probe hybridization and detection.

▶ 03:51
4
Detect N6-methyladenosine modifications

Apply methylation-specific probes or detection reagents to the membrane to identify and visualize N6-methyladenosine modifications across target RNA molecules.

▶ 06:03
5
Perform immunoblotting for validation

Use antibodies or immunological methods to confirm m6A modification patterns and validate northern blotting results.

▶ 06:50
6
Analyze and interpret methylation results

Examine band patterns and signal intensities from detection and immunoblotting to assess N6-methyladenosine modification profiles in experimental samples.

▶ 07:59
💬 Comments coming soon