Isolation and Purification
The first step for the analysis of RNA modification density is the isolation of total RNA from the target cells. The total RNA will contain all different kinds of RNA, mainly ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA). To purify the RNA of interest our lab uses two principle techniques:
- Separation by size (top right corner) or
- Separation by targeted oligohybridization (bottom right corner)
Mass spectrometric analysis
The purified RNA is digested to the nucleoside level using different enzymes. After addition of an internal standard (from metabolic isotope labeling), the RNA digest is analyzed on our own sensitive LC-MS/MS system ( Agilent 1290 LC with 6470 QQQ mass spectrometer). After signal integration and data evaluation the quantity of modified nucleoside per canonical or per RNA is plotted revealing the modification profile of the analyzed RNA.
Dynamically monitoring RNA modifications
Bacteria, yeast and cells produce their own nucleic acids from nutrients. By feeding them with certain nutrients we can achieve the incorporation of heavy isotopes into the RNA of the organism. Different nutrients result in different labeling position within the smallest building blocks of the nucleic acid (nucleoside).
We use this feeding strategy in elegant pulse chase experiments and in combination with the techniques shown above, we ca follow the fate of modified nucleosides in these organisms. We have applied this technique successfully in unstressed yeast. We are now curious to use this technique to unravel the mechanisms which allow the adaptation of tRNA modification density to overcome stress.