Protocols¶
Worm samples.¶
- Starting amounts 1 and 10 micrograms of RNA (both N2 and P6661 and P6662 worms)
- Enzymes:
- Snake Venom Phosphodiesterase - to cleave RNA - http://www.sigmaaldrich.com/catalog/product/sigma/p3243?lang=de®ion=DE
- Bacterial Phosphatase - to dephopshorylate nucleotides to nucleosides - http://www.carlroth.com/catalogue/catalogue.do?favOid=0000000b000320fd00010023&act=showBookmark&lang=en-nl&market=NL
- Digestion conditions
Original - 50mM Tris (pH 7.5 - 9)
Preferred due to LC-MS/MS compatibility - 0.1 M ammonium bicarbonate, pH 8.0 or 0.1M TEAB pH 8
- Sample precipitation for LC-MS/MS analysis
Ethanol precipitation using 1/10 sample volume 3M sodium acetate pH 5.3 and 3 times sample volume absolute EtOH (-20 deg). Incubation for >2h to overnight. Centrifugation at 13000 rmp (4 deg) and keep supernantant (nucleosides). Dry supernatant for LC-MS/MS analysis.
This last step precipitates the digestion enzymes and undigested material. Dephosphorylated nucleosides remain in the supernatant which is dried and later resuspended and analyzed directly by LC-MS/MS.
Worm culture samples.¶
- Take 300 ul from the worm culture and spin at 13000 rpm for 5 min to remove worms, E.coli, cell debris and large particles. Transfer supernatant to a new eppi.
- Add 1/10 sample volume 3M sodium acetate pH 5.3 and 3 times sample volume absolute EtOH (-20 deg). Incubation for >2h to overnight. Centrifugation at 13000 rmp (4 deg) and keep supernantant (nucleosides). Dry supernatant for LC-MS/MS analysis.