Affinity purification of SL-let-7g in either the presence or the absence of a WCE. Aliquots from the ARiBo purification either with (A) or without (B) incubation with an NT2 WCE are analyzed on SYBR-Gold stained 7-M urea PAGE gels (LE, loading eluate; W1–3, washes; WCE, NT2 WCE eluate; E1–2, RNA elutions; NaCl, matrix regeneration with 2.5 M NaCl) in amounts shown, where 1× corresponds to the volumes of affinity purification eluates (or washes) equivalent to a maximum of 50 ng of ARiBo-tagged SL-let-7g from the transcription reaction or 19 ng of SL-let-7g to be purified (Di Tomasso et al. 2011, 2012a). The protocols for the RNA pull-down and the RNA purification have been optimized such that for the RNA pull-down (A), there is one wash after immobilization of the RNA on the resin (W1) and one wash after (W2) incubation with the NT2 WCE, whereas for the RNA purification (B), there is a total of three washes (W1–3) after immobilization of the RNA on the resin (Di Tomasso et al. 2011, 2012a). In addition, standard amounts of ARiBo-tagged SL-let-7g from the transcription reaction, purified SL-let-7g and SL-let-7g resulting from addition of GlcN6P in the transcription reaction were loaded for quantitative analysis, as previously described (Di Tomasso et al. 2011, 2012a). The red box highlights the purified RNA.
