Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 235

of Trizol LS and follow the RNA-extraction method described in Section I. Refer to
Section II for a description of miRNAs detection by northern blotting. Use 10-20 m L
of fraction sample for western analysis of known RISC components with appropriate
antibodies.
N. EMSA of miRNPs
EMSA can be used to qualitatively identify large ribonucleoprotein complexes,
for example, holo-RISC ( Pham et al., 2004 ), or to detect miRNA-binding proteins
that may only form small complexes with their substrates ( Chan et al., 2008 ). Below,
we show a basic EMSA setup using radiolabeled miRNAs and crude cell extracts.
However, the design of EMSA should depend on the purpose of the experiment and
the parameters, such as the gel, buffer, running temperature, and/or the presence of
competitor or heparin, have to be optimized.
1. Cell Extract Preparation
See above.
2. Radiolabeled Synthetic miRNA Preparation
Obtain synthetic miRNAs from Dharmacon Research, Inc. or Integrated DNA
Technologies, Inc. Mix 1 m Lof50 m M synthetic miRNA (50 pmol), 5 m Lof
gamma- 32 P ATP (6000 Ci/mmol, NEN), 2.5 m Lof10 T4 PNK buffer, 2.5 m Lof
T4 polynucleotide kinase, and 14 m L of RNase-free water. Incubate the reaction
mixture at 37 C for 30 min. Remove free gamma- 32 P ATP by passing the mixture
through a Sephadex G-25 column (Roche) according to the manufacturer ' s
instruction.
3. Native Gel Preparation
Prepare a 16 cm 16 cm gel of 0.8 mm thickness with 1 TBE and 5%
acrylamide. The ratio of acrylamide/bis-acrylamide depends on the experiment.
Use 40:1 for large complexes and 19:1 for small complexes.
4. miRNA Binding Reaction and Gel Running
Prepare miRNA binding reaction mixtures (10 m L) that contain 20 mM Tris pH
7.5, 1 mM magnesium acetate, 1 mM calcium chloride, 0.01% Nonidet P-40, 2 mM
ATP, 250 ng/ m L E. coli tRNA, 5 10 5 c.p.m radiolabeled miRNA and cell extract
that can be used in different amounts. Incubate the reaction at RT for 45 min. Mix
with 2 m Lof6 native gel loading dye (30% glycerol, 0.25% BPB, and 0.25%
xylene cyanol). Run the gel at 100 Vat 4 C until the BPB dye migrates to 3 cm from