tRNA Modification Analysis by LC-MS

In the first step, the total RNA was fractionated on an AdvanceBio SEC column (300 A pore size, 2.7 µm particle size, 7.8 by 300 mm; Agilent) at 40°C using isocratic elution with 0.1 M NH4OAc buffer at pH 7 to separate the total tRNA fraction from larger RNA species. The tRNA was collected in 1 mL and lyophilized in the SpeedVac, until 50 µL remained in the vial. By adding 5 µL ammonium acetate (5 M) and 125 µL ice-cold and pure ethanol (100%), the tRNA was incubated overnight and precipitated by centrifugation (12,000g, 4°C, 30 min.). The tRNA pellets were resolved in 30 µL MilliQ water and their concentrations were measured using an IMPLEN nano-photometer (Implen). A maximum of 500 ng tRNA was processed and analyzed by LC-MS using an MRM method as previously described (Heiss et al. 2017 (link); Ramos et al. 2019 (link)). The level of each modified nucleoside (I, m1A) was normalized to an average tRNA. The amount of injected tRNA was calculated by the detected amount of C in pmol and the average content of C in an average tRNA.

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Ramos J., Proven M., Halvardson J., Hagelskamp F., Kuchinskaya E., Phelan B., Bell R., Kellner S.M., Feuk L., Thuresson A.C, & Fu D. (2020). Identification and rescue of a tRNA wobble inosine deficiency causing intellectual disability disorder. RNA, 26(11), 1654-1666.

Publication 2020

AdvanceBio SEC column IMPLEN nano-photometer

Ammonium acetate Buffer Centrifugation Cold Ethanol Nucleoside Pellets Trna

Corresponding Organization :

Other organizations : University of Rochester, Science for Life Laboratory, Uppsala University, Linköping University

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Variable analysis

independent variables

Fractionation of total RNA on an AdvanceBio SEC column (300 A pore size, 2.7 µm particle size, 7.8 by 300 mm; Agilent) at 40°C using isocratic elution with 0.1 M NH4OAc buffer at pH 7

dependent variables

Levels of modified nucleosides (I, m1A) normalized to an average tRNA
Amount of injected tRNA calculated by the detected amount of C in pmol and the average content of C in an average tRNA

control variables

Temperature of fractionation (40°C)
PH of elution buffer (pH 7)
Maximum of 500 ng tRNA processed and analyzed

controls

No positive or negative controls were explicitly mentioned in the protocol.

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