RNA analysis by total hydrolysis and quantification of released nucleosides

A method for analyzing a sample comprising RNA molecules transcribed from a template DNA with an expected sequence and length, comprising the steps of:

• a) providing a sample comprising RNA molecules, wherein the RNA molecules comprise RNA molecules that are capped and polyadenylated;
• b) completely hydrolyzing the RNA molecules, thereby releasing nucleosides;
• c) separating and quantifying the released nucleosides by HPLC, capillary electrophoresis or mass spectrometry;
• d) determining at least one of:
• (i) the capping degree of the RNA molecules within the sample by using formula 4:
• % cap=n(CA)/n(RNA)*100%,
• wherein % cap is the percentage of capped RNA molecules, n(CA) is the content of a cap analogue in the sample, and n(RNA) is the number of copies of the RNA molecules in the sample;
• (ii) the number of copies of the RNA (n(RNA)) molecules in the sample by using formula 1.1:
• n(RNA)=⅓*(n(C)/u(C)+n(G)/u(G)+n(U)/u(U)),
• wherein n(C), n(U), and n(G) is the measured content of the corresponding nucleoside and any modifications thereof in the sample, and u(C), u(U), and u(G) is the number of the corresponding nucleoside and any modifications thereof in the expected sequence of the RNA molecules;
• and/or
• (iii) the length of a homopolymeric element in the RNA molecules by using formula 5:
• lxm=n(X)/n(RNA)−[u(X)−lxe],
• wherein lxm is the calculated average length of the poly(X) stretch, n(X) is the measured content of a nucleoside X forming the homopolymeric element in the sample, n(RNA) is the number of copies of the RNA molecules in the sample, u(X) is the number of X in the expected sequence of the RNA molecules, lxe is the expected average length of the poly(X) stretch in the RNA molecules.