text: Apart from mass spectrometry and chromatography, other two validation techniques have been developed, namely 
  #Pre- and post-labelling techniques: 
  *Pre-labelling → involves the use of P: cells are grown in P containing medium, thus allowing the incorporation of [α-P]NTPs during transcription by T7 RNA polymerase. The modified RNA is then extracted, and each RNA species is isolated and subsequently digested by T2 RNase. Next, RNA is hydrolyzed into 5' nucleoside monophosphates, which are analyzed 2D-TLC (two-dimensional thin-layer chromatography). This method is able to detect and quantify every modification but will not contribute to the characterization of the sequence.
  *Post-labelling → implicates the selective labelling of a specific position within the sequence: these techniques rely on the Stanley-Vassilenko approach principles, that has been adjusted to achieve a better validation quality. First, RNA is cleaved into free 5’-OH fragments either by RNase H or DNAzymes, by sequence specific hydrolysis. The polynucleotide kinase (PKN) then performs the 5’ radioactive post-labelling phosphorylation using [γ-P]ATP. At this point, the labelled fragments undergo a size fragmentation, that can be performed either by Nuclease P1 or according to the SCARLET method. In both cases, the final product is a group of 5’ nucleoside monophosphates (5’ NMPs) that will be analyzed by TLC. 
  **SCARLET: this recent approach exploits not just one, but two sequence selection steps, the last of which is obtained during the splinted ligation of the radioactive-labelled fragments with a long DNA oligonucleotide, at its 3’-end. After degradation, the labelled residue is purified together with the ligated DNA oligonucleotide and finally hydrolyzed and therefore released thanks to the activity of the Nuclease P1. 
  This method has proven to be very useful in the validation of modified residues in mRNAs and lncRNAs, such as m6A and Ψ
  #Oligonucleotide-based techniques: this method includes several variants
  *Splinted ligation of particular modified DNAs, that exploits the ligase sensitivity to 3’ and 5’ nucleotides (so far used for m6A, 2’-O-Me, Ψ)
  *Microarray modification identification through a DNA-chip, that exploits the decrease in duplex stability of cDNA oligonucleotides, due to the impediment in conventional base-pairing caused by modifications (ex. m1A, m1G, m22G)
  *RT primer extension at low dNTPs concentration, for mapping of RT arrest signals.