Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 233

Add lysate and lysis buffer in a total of 1 mL to 50 m L of Dynabeads (Invitrogen) that
have been washed twice in 100 m L of WB buffer (0.1 M NaPO 4 , 0.1% Tween 20,
pH 8.2). Rotate at 4 Cfor1h.
4. Immunoprecipitation
Wash 50 m L of Dynabeads per sample with 100 m LWB buffer. Block beads by
resuspending in 180 m L of WB buffer and 20 m Lof10 m g/ m L sheared, salmon-
sperm DNA (ssDNA). Incubate on rotator at RT for 30 min. Add 5 m g of antibody
and incubate on rotator at RT for 10 min. Wash antibody-bead complex twice with
100 m L of conjugation buffer (20 mM NaPO 4 , 150 mM NaCl). Incubate beads in
250 m L bis(sulfosuccinimidyl)suberate (BS 3 ) conjugation buffer (5 mM BS 3 in
conjugation buffer containing 0.5 m g/ m L ssDNA) at RT for 30 min with rotation.
Quench cross-linking reaction by adding 12.5 m L of 1 M Tris-Cl pH 7.4 and
incubating at RT for 15 min with rotation. Wash beads twice with 100 m L of lysis
buffer. Add antibody-cross-linked beads to the precleared lysate from the previous
step, and rotate at 4 C overnight.
Save the supernatant from the immunoprecipitation reaction for IP efficiency anal-
ysis by western blotting. Wash beads twice for 1 min in a Thermomixer R (Eppendorf)
set to 4 C with: (1) wash buffer [1 PBS pH 7.4, 0.1% SDS, 0.5% sodium deoxycho-
late, and 0.5% NP-40], (2) high salt wash buffer [5 PBS pH 7.4, 0.1% SDS, 0.5%
sodium deoxycholate, and 0.5% NP-40], and (3) PK buffer [100 mM Tris-Cl pH 7.4,
50 mMNaCl, and 10 mM EDTA]. Resuspend beads in 100 m L of PK buffer. Remove a
15 m L aliquot of bead solution for IP efficiency analysis by western blotting. Remove
excess PK buffer and resuspend beads in 100 m L of proteinase K solution (5 mg of
proteinase K in 1 mL of PK buffer) at 37 C for 20 min in a Thermomixer R (Eppendorf)
set to 1200 rpm. Quench reaction by adding 100 m L of PK buffer containing 7 M urea,
and incubating the reaction at 37 C for 20 min at 1000rpm.
5. RNA Extraction
Extract total and immunoprecipiated RNA by adding 800 m L of Trizol (Invitrogen)
directly to an aliquot of input protein lysate or the proteinase K bead solution and
extracting as described above in Section I. Resuspend extracted RNA in 120 m Lof
DEPC H 2 O. Treat with DNase by adding 15 m Lof10 RQ1 DNase Buffer and 15 m L
of RQ1 DNase to each sample and incubating at 37 C for 1 h. Extract RNA a second
time as described above in the second RNA-extraction step of Section I. Resuspend
RNA samples in 10 m LofddH 2 O.
6. RT-PCR
Add 9 m L of immunoprecipitated RNA or 2.5 m g of total RNA in a 9 m L volume to
1 m L of random primers (250 ng/ m L), 1 m L of 10 mM dNTPs, and 1.2 m L of ddH 2 O.
Incubate at 65 C for 5 min and 4 C for 1 min. Add 4 m Lof5 buffer (Invitrogen),