The following protocol is recommended for the isolation of siRNA using 50 units of p19 siRNA Binding Protein. The composition of solutions that are not provided with p19 siRNA Binding Protein are provided. A magnetic rack (NEB #S1506, #S1509) is needed to separate the beads from solution.
- Pretreatment of chitin magnetic beads with BSA (reduces non-specific binding):
a. Transfer a 200 µl suspension of chitin magnetic beads (NEB #E8036) into a sterile microfuge tube.
b. Pull the beads to the side of the tube using a magnetic rack and remove the supernatant.
c. Add 200 µl bead pretreatment buffer to the pellet (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1mM TCEP, 1 mg/ml BSA, pH 7.0 at 25°C).
d. Vortex and remove supernatant
e. Add 200 µl of fresh bead pretreatment buffer and rotate the tube at 4°C overnight.
Note: The pretreated beads can be stored at 4°C for at least four months.
- Binding of siRNA to p19 siRNA Binding Protein:
a. Add 50 units of p19 siRNA Binding Protein to RNA extract containing siRNAs or double stranded miRNAs in 1X p19 siRNA Binding Buffer.
b. Incubate for 1 hour at room temperature with shaking to form siRNA/p19 complex.
- Binding of siRNA/p19 complex to chitin magnetic beads:
a. Aliquot 20 µl of the pretreated beads suspension into a sterile microfuge tube.
b. Pull the beads to the side of the tube with a magnetic rack and remove the supernatant.
c. Add the siRNA/p19 complex to the pellet and mix by shaking or laying the tube on a magnetic stir plate for one hour at room temperature.
- Remove unbound RNA:
a. Carefully remove supernatant containing unbound RNA.
- Wash step:
a. Wash the beads with 500 µl of p19 siRNA Washing Buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM TCEP, pH 7.0 at 25°C).
b. Vortex after wash.
c. Repeat three times. After third wash, remove as much buffer as possible without disturbing pellet.
- Elution of siRNA:
a. Add 30–40 µl p19 siRNA Elution Buffer to bead pellet (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM TCEP, 0.5% SDS, pH 7.0 at 25°C).
b. Incubate at 37°C for 10 minutes.
c. Mix on a stir plate for 10 minutes at room temperature.
Note: Elution step can be repeated and eluants can be combined if necessary.