Bitan:Extraction of RNA from filters

RNA extraction from the filters

 

RNA is extracted from the filters to obtain the sequences that bind to the protein. These sequences are amplified for the next SELEX cycle.

 

1) After scintillation counting, remove the positive-control filter from the Eppendorf tube (from previous experiment) and place into a clean, dry, 35×10-mm Petri dish.

 

2) Use a clean scalpel and a pair of tweezers to cut the membrane in small pieces.

 

3) Using the tweezers, transfer the cut pieces of the membrane into the same labeled Eppendorf tube from Step 1.

 

4) Add 400 μl elution buffer (7 M urea, 3 mM ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), 100 mM sodium citrate, pH 5.0) and incubate the tube at 95 °C for 10 min.

 

5) Centrifuge the tube at top speed, aspirate, and collect the extraction butter into a new labeled Eppendorf tube.

 

6) Measure the remaining radioactivity counts in the tube containing the membrane pieces by scintillation counting to assess the extraction efficiency.

 

7) Repeat the extraction process (steps 4–6) thrice. The efficiency after 3 extractions is usually ~95–96%.

 

8) In the tubes containing the RNA extracts, add 1 volume (400 μl) of citrate-saturated phenol (pH 4.7):chloroform:isoamyl alcohol (125:24:1). Mix by a vortex for ~1 minute and centrifuge at 16,000 g for 2 minutes.

 

9) Transfer the upper, aqueous phase to a fresh tube or discard the bottom phase by aspiration using a micropipette.

 

10) Add 1 volume of chloroform:isoamyl alcohol (24:1), mix by a vortex for 1 minute and centrifuge at 16,000 g for 2 min.

 

11) Transfer the upper, aqueous phase to a fresh tube or discard the bottom phase by aspiration using a micropipette.

 

12) To precipitate the RNA, add 0.1 volume of 3 M sodium acetate (pH 5.2), 3–4 μl glycogen (10 μg/μl) as the coprecipitant, and 1 volume equivalent of propan-2-ol. Mix and place in a −20 °C freezer overnight.

 

13) Spin at top speed, preferably in a microcentrifuge at 4 °C, for 20–30 minutes to precipitate the RNA from step 12.

 

14) After centrifugation, aspirate the supernate carefully without dislodging the coprecipitant phase barely visible in the tube.

 

15) Wash the RNA pellet with 0.5 ml of 70% ethanol, centrifuge for 5 min a top speed and discard ethanol by aspiration without dislodging the coprecipitant phase barely visible in the tube.

 

16) Dissolve the RNA pellet in 50 μl STE buffer and proceed to G-50 purification.

 

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