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Version 3 (Ilian Atanassov, 02/24/2015 09:33 AM) → Version 4/6 (Ilian Atanassov, 02/24/2015 09:44 AM)
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®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.
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:
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®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.
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: