The Procedure

ELECTROPHORETIC SEPARATION

RNA samples should be free of contaminating particles and DNA, with an A₂₆₀/A₂₈₀ ratio of 1.9-2.0.

1. For highly expressed genes, load 10µg total of RNA. For trace expression detection, load 10µg of poly A+ RNA. Ethidium may be added to the gel (4 µl of 10 mg/ml per 100ml gel = 0.4µg/ml).
2. Visualize the gel on UV and photograph with a phosphorescent ruler.

TRANSFER

1. To remove formaldehyde and EtBr which may hamper transfer and/or recovery, soak the gel in 3 changes of DI water for 10 minutes each.
2. Place a platform slightly larger than the gel in the center of a 9 X 13 X 2" tray. Fill the tray with 10X SSC until the buffer is 0.5 cm below the surface of the platform (An inverted gel mold or a sponge make good platforms. Rinse commercial sponges before first use, to remove surfactants).
3. Cut a strip of Whatman 3MM paper to the width of the platform and length of the tray. Lay it over the platform so that the ends rest in the buffer. Roll a pipette over the filter to remove any bubbles beneath.
4. Flood the filter paper surface with 10X SSC and lay the gel on the filter paper. Roll the gel with a pipette if needed to remove air bubbles.
5. Place strips of plastic wrap on the platform around the gel to prevent buffer from bypassing the gel. Alternatively, lay a piece of plastic wrap over the gel and platform, and cut away the plastic over the gel with a razor blade, leaving a "mask" on the tray.
6. Flood the surface of the gel with 10X SSC and carefully overlay the membrane onto the gel (Note: handle membrane with gloves or forceps and only at the corners).
7. Cut 3 pieces of Whatman 3MM to the size of the gel. Wet one piece and lay it over the membrane. Roll the paper and membrane with a pipette to remove any bubbles.
8. Inspect carefully - any gap between gel and membrane will not only block transfer, it may create a "hot spot" on the blot, making the interpretation more difficult.
9. Wet each remaining cut piece of 3MM paper, lay over the stack, and roll to remove bubbles.
10. Lay a stack (3-4" high) of paper towels, cut to the size of the gel, over the stack.
11. Place a glass plate and a 500g weight on top of the stack. Allow to transfer overnight.

POST TRANSFER PROCESSING

1. Disassemble capillary stack down to membrane, mark well positions with indelible pen or pencil before removing membrane from gel.
2. Rinse membrane in 5X SSC for 5 minutes at room temperature.
   Note: The gel will be flattened to approximately 2mm thickness. The gel can be stained with EtBr and checked under UV to determine extent of transfer.
3. Cross-link RNA to membrane: Nylon - expose to UV (approx. 150 mj/cm²). Nitrocellulose - Bake at 80°C for 120 minutes.
4. (Nylon filter only) Wash filter in 1X SSC + 0.1% SDS at 65° C for 1 hour. This will substantially reduce the background.

HYBRIDIZATION

SSPE (20X)

1. Perform probe purification.
2. Prehybridize in hybridization solution (see below) for 1 hour at 65°C, then replace with fresh solution containing probe for hybridization.
   NOTE: Many different protocols exist for hybridization reactions and each must be optimized for any given probe. General guidelines are given below.Hybridization solution (filter sterilize and store at -20°C once mixed):
   • 5X Denhardt's Solution (see below)
   • 100mg/ml Salmon or Herring Sperm DNA
   • 0.1% SDS
   • 5X SSPE (see below)
   • 50% formamide

   SSPE (20X):

   • 3M NaCl
   • 0.2M sodium phosphate, pH 7.4
   • 25mM EDTA
3. Select a hybridization temperature which will allow annealing of the probe, but prevent non-specific binding to nontarget sequences. Note that high stringency washing later in the protocol may not be able to compensate for too low a hybridization temperature. The result will be a large number of false positive bands.Calculation of theoretical melting temperature (Tm):Tm_DNA:RNA = 79.8°C + 18.5 (log10[Na]) + 0.58 (%GC) + 11.8 (%GC)² - 0.50 (%F) - (820/length)Notes:
   • Tm decreases by approximately 1° C for every 1% increase in mismatches.
   • Tm decreases by 0.5° C for every increase of 1% in formamide (%F).
   • A good hybridization temperature to begin with is 20°C below the calculated Tm.
   • Washes should be carried out at approximately 15°C below the calculated Tm.

STRINGENCY WASHES

After hybridization, non-specifically bound probe is removed by washing in low salt buffer at high temperature. The salt concentration and temperature must be optimized for each probe/sample combination. A good starting point is to wash one time for 20 minutes in 1X SSC and 0.1% SDS at 45° C, followed by three 20 minute washes in 0.2X SSC and 0.1 % SDS at 65° C.

AUTORADIOGRAPHY

Peform autoradiography as described previously on the following page.

STRIPPING

After autoradiography, the probe may be stripped from the blot, allowing the blot to be re-probed up to 5 times (nylon) or 2 - 3 times (Nitrocellulose). Incubate the blot in 50% formamide, 6X SSC at 65° for 30 - 60 minutes. Wrap stripped blot in plastic wrap and place on film overnight to confirm probe removal. Note: If blot is allowed to dry with probe bound to it, the probe will become permanently attached.

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