Table 1.

Overview of techniques to isolate the RNA-bound proteome to study RNP dynamics.

Method	RNA Target	Advantage	Disadvantage	Cell Type	Cell Number
Affinity-Based Separation
RIC [3,61]	Poly(A) tailed RNA	Isolates only mRNA complexes (if subset is of interest) Widely used protocol	Isolates only mRNA complexes Low signal-to-noise ratios Additionally, co-purification of non-cross-linked (free) RNA Can purify off-target RNA containing poly(A) stretches within its sequence	All poly(A) tailed containing organisms	7500 cm2 HeLa cells [3] 107 cells [39] 108 cells [63]
e/cRIC [7,9]	Poly(A) tailed RNA	Isolates only mRNA complexes (if subset is of interest) Better signal-to-noise ratios than RIC	Isolates only mRNA complexes Additionally, co-purification of free RNA Can purify off-target RNA containing poly(A) stretches within its sequence	All poly(A) tailed containing organisms	1–1.3 × 108 cells [7] 3 × 15 cm dishes at 80% confluence [9]
CARIC [64]	Newly transcribed RNA	All RNA types RNP monitoring through time	Use of nucleoside analogs Potential co-purification of naturally biotinylated proteins Additionally, co-purification of free RNA	Limited to cell cultures receptive for nucleoside analogs	4 × 107 cells
RICK [65]	Newly transcribed RNA	All RNA types RNP monitoring through time	Use of nucleoside analogs Potential co-purification of naturally biotinylated proteins Additionally, co-purification of free RNA	Limited to cell cultures receptive for nucleoside analogs	Not specified
Solid Phase Separation
2C [66]	All RNPs	Fast and cost-effective method	Contamination of both free protein and free RNA Dependent on the scale of the silica columns A nucleotide size limitation can occur inherent to silica matrices	All cell types and tissue	Not specified
(PAR)-TRAPP [43]	All RNPs	Cost-effective method Scalable protocol	DNA is co-eluted Additionally, co-purification of free RNA A nucleotide size limitation can occur inherent to silica matrices	All cell types and tissue	750 mL of media containing cells at an OD600 of 0.5
VIR-CLASP [67]	Pre-replicated viral RNPs	Study of early-stage viral infection Theoretically adaptable to every type of in vitro transcribed RNA molecule Cost-effective method	The current field of application is a highly interesting but small niche SPRI beads can have size-selective artefacts	Limited to cell cultures receptive for nucleoside analogs	15 cm2 of cells
Organic Phase Separation
XRNAX [21]	All RNPs	All RNA types Little free RNA Cost-effective method Easily scalable Good starting point for more specific techniques	Glycoproteins and RNA–protein adducts cannot be distinguished Technically challenging Crude fraction	All cell types and tissue	1 × 108 cells
OOPS [22]	All RNPs	All RNA types Cost-effective method Easily scalable	Technically challenging Cannot be used as a starting point for more specific techniques	All cell types and tissue	28.2 cm2 of 90% confluence
PTex [23]	All RNP >30 bp	All RNA types Little free RNA Cost-effective method Easily scalable Good starting point for more specific techniques	Glycoproteins and RNA–protein adducts cannot be distinguished Technically challenging 25–30% recovery	All cell types and tissue	2 × 106 cells