I am trying to map N6-dimethyladenosine on rRNA using primer extension (low dNTPs assay) method. But i am not able to detect map my position. I went through some articles they mentioned like they hydrolysed the RNA but they did not mention exactly by which hydrolysis method (enzyme, alkaline). can you please help me?
Great. I used (10 g) of Total RNA for primer extension reaction with 100,000 cpm [- 32P]ATP-labeled oligonucleotide. After annealing at 55–60 °C for 15 min and chilling on ice for 2 min, 1 unit of reverse transcriptase (Expand RT, Roche) and 1 unit of RNase inhibitor were added and an extension reaction was performed at 42 °C for 90 min. The extension products were ethanol precipitated and analyzed on 6% polyacrylamide, 7 M urea gel. (DOI 10.1074/jbc.M308997200)
This method specialized for 2’o methylation on rRNA, my interest is N6-dimethyladenosine (M26-A). In the literature M26-A blocks the RT I did not get the extension. In this publication, they have mentioned the RNA was hydrolyzed but hydrolysis has many different types (alkaline, enzyme) (https://doi.org/10.1091/mbc.E15-02-0073)
