Bitan:Filter binding for SELEX
RNA–protein incubation and filter binding for SELEX:
In this step, the RNA and protein are first incubated in solution and then the RNA sequences that bind to the protein are separated from the non-binders by filter binding and washing. As SELEX cycles progress, filter binding will give an indication of protein–RNA binding enrichment.
1) Dissolve the protein in 8 μl 60 mM NaOH and add 36 μl nuclease-free deionized water.
2) Sonicate the mixture for 1 min. Add 36 μl 2× RNA binding buffer (20 mM Tris, 300 mM NaCl, 10 mM MgCl2, pH 7.5).
3) Incubate the RNA at 90 °C for 10 min for denaturation and then incubate at room temperature for 10 min for slow RNA renaturation.
4) Mix the appropriate amount of RNA with 20 μl 10× RNA binding buffer and make up the volume to 200 μl by adding nuclease-free water. This is the negative control; label the tube negative control.
5) Mix 20 μl protein and the desired amount of RNA with 20 μl 10× RNA binding buffer and make up the volume to 200 μl by adding nuclease-free water. This is the positive control.
6) Mix and incubate the tubes for 30 min at room temperature. Meanwhile prepare the filters and the filter-binding setup for the next step.
7) Attach a 125-ml side-arm flask to a vacuum inlet. Place a pre-cleaned, porous glass support for the filter on the side-arm flask.
8) Equilibrate three filter membranes in 2–3 ml of 1× RNA binding buffer in a 35×10-mm Petri dish for 10–15 min. The first filter will be used for adjusting the vacuum suction, the second will be used for RNA-alone, negative-control filter binding, and the third will be used for filter binding of the RNA–protein mixture, the positive control.
9) After 30 min, centrifuge the negative- and positive-control for 5 min.
10) Turn on the vacuum, and place the first membrane on the porous glass. Using a micropipette, drip 0.5 ml of RNA binding buffer onto the membrane and adjust the vacuum to allow slow flow of each buffer drop through the membrane.
11) Place the second membrane onto the porous glass and using the same flow rate, apply the negative-control onto the membrane.
12) Apply 4×0.5-ml aliquots of 1× RNA binding buffer to wash the membrane and discard the flow through. Note that if pre-clearing is desired, the flow-through is kept for RNA extraction. Pre-clearing removes RNA sequences that bind to the filter.
13) Remove the negative-control membrane and place into a correspondingly labeled 1.6-ml Eppendorf for scintillation counting.
14) Replace the porous glass with a pre-cleaned second porous glass support.
15) Place the third disk onto the porous glass and apply the positive-control. Wash the filter as in step 12 and discard the flow-through. The number of washes can be increased in later SELEX cycles to increase the stringency of SELEX conditions.
16) Remove the positive-control filter disk and place into a correspondingly labeled 1.6-ml Eppendorf for scintillation counting.
17) Perform scintillation counting as before and note down the counts for the respective filters.
18) Calculate the level of the filter-bound radioactivity compared to the total amount of radioactivity applied to the membranes (% binding). This will give an indication of binding enrichment as SELEX progresses.