Absolute quantification of Uridine and 4Thiouridine

h1. Protocols

h2. 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&region=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.  

h2. 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.

h2. Literature: