Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 232

potential nonphysiological interactions that can happen upon extract preparation
( Mili and Steitz, 2004; Van Wynsberghe et al., 2011 ).
In addition to CLIP and RIP experiments, which are aimed at the identification of
RNA sequences that associate with proteins of interest, the characterization of the
protein components of miRNA ribonucleoprotein complexes (miRNPs) contributes
to the understanding of mechanistic aspects of miRNA pathways. Gel filtration and
electrophoretic mobility shift assay (EMSA) allow for the estimation of the size of
miRNPs both in wild-type organisms and in mutants or under designed experimental
circumstances. UV-cross-linking of miRNPs assembled with radio-labeled miRNAs
in EMSA experiments transfers the radioactive label to miRNA-binding proteins and
can be used to visualize them on SDS-PAGE. Ultimately, affinity selection using
biotinylated 2 0 -O-methyl oligonucleotides allows purification of miRNPs and fur-
ther identification of miRNP component proteins by mass spectrometry.
L. RIP
This protocol is adapted from the Dynabeads instruction protocol (Invitrogen) and
published immunoprecipitation protocols ( Van Wynsberghe et al., 2011; Zhang
et al., 2007; Zisoulis et al., 2010, 2011 ). Cross-linking of the antibody to the beads
and blocking of the beads with salmon-sperm DNA is recommended to decrease
background, and enhance reproducibility and specificity.
1. Worm Collection and Cross-Linking
At least 200 m L of worms collected at the appropriate stage are required for this
protocol. After collection, rock worms in M9 for 10 min to allow residual bacteria
to be digested. Resuspend worms in 200 m L of M9 and plate several drops onto 2-3
100-mm worm plates. Spread worms equally around the plate. UV cross-link worms
in a Spectrolinker XL-1000 UV Crosslinker with an energy output of 3 kJ/m 2 at a
distance of 10 cm from the light source. Collect cross-linked worms with M9.
Flash freeze worm pellet in a dry ice-ethanol bath.
2. Lysate Preparation
Crush worms in liquid nitrogen with mortar and pestle. Transfer worms to a
1.5 mL eppendorf tube containing three times the pellet volume of lysis buffer
[150 mM NaCl, 25 mM HEPES pH 7.5, 0.2 mM DTT, 10% glycerol, 0.025 U/ m L
RNAsin, 1% Triton X-100, and protease inhibitors (Roche)]. Spin sample at top
speed at 4 C for 15 min. Transfer supernatant to a new eppendorf tube and determine
the protein concentration by the Bradford protein assay (Bio-Rad).
3. Preclear Lysate
At least 500 m g of lysate protein is required per sample. Equal amounts of sample
should be used for each reaction (typically: antibody of interest and IgG control).