Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 236

the bottom of the gel. Lay the gel onto Whatman 3MM paper and dry it using a gel
dryer. Detect the signals by autoradiography or phosphoimaging.
5. UV-Cross-Linking of miRNPs
Prepare 150 m L of miRNA binding reaction mixture and separate the sample on a
nondenaturing gel (see above). Do not dry the gel. Instead, expose the gel wrapped in
a transparent plastic film to a BioMax MS film (Kodak) for 1 h. After autoradiogra-
phy, excise gel bands that contain radioactive signals corresponding to miRNPs of
interest. Place the gel slices on a clean glass plate that is sitting on ice and expose
them to 254 nmUV light in Stratalinker UV Crosslinker (Stratagene) with an energy
output of 2 J/cm 2 at a distance of approximately 10 cm from the light source. Mince
the gel slice to small pieces and incubate with 150 m L of RNase A (250 U/mL in
50 mMTris-HCl pH 7.5 buffer) at RT for 30 min. Add 50 m L of XT sample buffer 4X
(Bio-Rad) and agitate vigorously on a vortex mixer for 3 h. Analyze the eluted
proteins by denaturing gel electrophoresis using a Criterion XT Bis-Tris 4-12%
gradient gel (Bio-Rad) and autoradiography. To detect all UV-cross-linking signals
not specific to any individual complex, irradiate 20 m L of miRNA binding reaction
mixture placed in a 96-well plate on ice following RNase treatment and denaturing
gel electrophoresis as mentioned above.
O. Affinity Purification of miRNPs by Biotinylated 2 0 -O-methyl Oligonucleotides
Antisense 2 0 -O-methyl oligonucleotides block siRNA and miRNA function
in vitro and in vivo ( Hutvagner et al., 2004 ). In addition, immobilized 2 0 -O-methyl
oligonucleotides complementary to siRNAs or miRNAs capture Argonaute proteins
in C. elegans extract ( Hutvagner et al., 2004; Steiner et al., 2007; Yigit et al., 2006 ).
Below, we describe an adapted procedure to purify miRNPs from C. elegans crude
extract using biotinylated 2 0 -O-methyl oligonucleotides. The RNA or protein con-
tents in purified miRNPs are subjected to further investigation.
1. Cell Extract Preparation
Depending on the particular miRNA of interest, harvest synchronized worms of
the appropriate developmental stage. For example, to capture let-7 binding ribonu-
cleoprotein complexes, prepare cell extract from L4 stage worms. For one volume of
packed worms, use five volumes of homogenization buffer [10 mMTris-HCl pH 7.5,
100 mM KCl, 2 mM MgCl 2 , 0.5% (v/v) Nonidet P-40, 15% (v/v) glycerol, 5 mM
DTT, 50 U/mL SuperaseIn RNase inhibitor (Ambion) and 1 Roche Complete
protease inhibitor, EDTA-free].
2. Biotinylated 2 0 -O-Methyl Oligonucleotides
Obtain 5 0 -biotinylated 2 0 -O-methyl oligonucleotides from Integrated DNA
Technologies, Inc. In previous studies, five nucleotides complementary to the