RNase-Assisted RNA Interactome Profiling

RNase-assisted RNA chromatography with RNAse A/T1 was performed, using in vitro-transcribed pri-mir-30c-1 and total MCF7 cell extracts28 (link). RNA-bound proteins were separated using 4–12% NUPAGE bis–tris system (Invitrogen) and individual lanes were subjected to mass spectroscopy (BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews). Results were confirmed by western blot analysis with anti-SRSF3 (MBL), anti-PARP-1 (Santa Cruz), anti-DDX17 (Santa Cruz), anti-hnRNPA1 (Thermo Scientific) and anti-FUS (Abcam) specific antibodies, as indicated above (Western blot analysis section).

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Fernandez N., Cordiner R.A., Young R.S., Hug N., Macias S, & Cáceres J.F. (2017). Genetic variation and RNA structure regulate microRNA biogenesis. Nature Communications, 8, 15114.

Publication 2017

NUPAGE bis–tris system anti-PARP-1 anti-DDX17 anti-FUS

Antibodies Bis tris Chromatography Mass spectrometry Mcf7 cell Parp 1 Pri mir Proteins Rnase t1 Srsf3 Western blot analysis

Corresponding Organization : MRC Institute of Genetics and Molecular Medicine

Other organizations : University of Edinburgh

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Variable analysis

independent variables

RNase-assisted RNA chromatography with RNAse A/T1
In vitro-transcribed pri-mir-30c-1
Total MCF7 cell extracts

dependent variables

RNA-bound proteins
Western blot analysis with anti-SRSF3, anti-PARP-1, anti-DDX17, anti-hnRNPA1, and anti-FUS specific antibodies

control variables

4–12% NUPAGE bis–tris system (Invitrogen) for protein separation

controls

Positive controls: Not explicitly mentioned.
Negative controls: Not explicitly mentioned.

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