Endy:Northern blot, AlkPhos end-labeled probes

The following protocol for Northern Blotting is based on the RNA extraction method used by Sean Moore, Agarose gel electrophoresis, and the Turboblotter system from Whatman.

Materials

• RNA extracted from cells (should be ~1-5µg/µl).
• RNAse free water (make lots for rinsing glassware and electrophoresis chambers)
  • Add DEPC to final concentration of 0.1%.
  • Incubate 1hr at 37°C.
  • Autoclave for 15 mins at 15 psi.
• 10X BPTE electrophoresis buffer (The final pH of this 10x buffer is ~6.5.)
  • 100 mM PIPES
  • 300 mM Bis-Tris
  • 10 mM EDTA
• HPLC grade or better DMSO
• Glyoxal
  • Commercially available stock solutions of glyoxal contain both hydrated forms of glyoxal and oxidation products that can degrade RNA. These must be removed by running the Glyoxal over mixed-bed ion exchange resin (See Sambrook and Russel 3rd Ed, App. 1-24). We used BioRad AG 501-X8 (D) resin beads. Equal volumes of resin and glyoxal were placed in an epp tube; the glyoxal's pH was measured w/ pH paper strips after a few minutes. Repeat this until the glyoxal's pH is >5.5.
• Glyoxal reaction mixture (divide into small aliquots and store at -70°C)
  • 6mL DMSO
  • 2mL deionized glyoxal
  • 1.2mL of 10X BPTE electrophoresis buffer
  • 0.6mL of 80% glycerol
• RNA size marker
• RNA gel loading buffer --> got some from Sean Moore. Make sure final volume of buffer is no less than 50% volume of the sample.
  • 95% deionized formamide
    • Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
  • 0.025% (w/v) bromophenol blue
  • 0.025% (w/v) xylene cyanol FF
  • 5 mM EDTA (pH 8.0)
  • 0.025% (w/v) SDS

• SYBR Gold

Protocol

Preparing the Gel

For RNase free electrophoresis apparatus: Clean electrophoresis tanks and combs used for electrophoresis of RNA with detergent solution, rinse in H20, dry with ethanol, and then fill with a solution of 3% H2O2. After 10 minutes at room temperature, rinse the electrophoresis tanks and combs thoroughly with H2O2 treated with 0.1% DEPC

1. Set up the glyoxal denaturation reaction by combining 1-2 ul of RNA (up to 10 ug) with 10 ul of glyoxal reaction mixture. Also treat the RNA ladder (?). Run molecular weight markers for staining with sybrGold(?).
2. Incubate the RNA solutions for 60 minutes at 55°C. Chill the samples for 10 minutes in ice water and then centrifuge them for 5 seconds to deposit all of the fluid in the bottom of the microfuge tubes.
3. While the samples are incubating, clean electrophoresis tank if necessary, and pour a 1.5% agarose gel in 1X BPTE (1.05 g agarose in 70 mL buffer). When set, cover the gel with sufficient buffer.
4. Add 2.5 ul of 6X gel loading buffer to the glyoxylated RNA samples, and without delay, load the glyoxylated RNA samples into the wells of the gel.
5. Carry out electrophoresis at 70 Volts.
6. Trim away areas of the gel to be stained with sybrGold. (Membrane should be cut to match the size of the gel.) Wrap gel to be stained in saran wrap and store at 4°C until post-transfer gel is ready to be stained.
7. Soak gel 30 min in 0.05 M NaOH/1.5 M NaCl (~400 mL) without agitation.
8. Soak gel 20 min in 0.5 M Tris (pH 7.4)/ 1.5 M NaCl (~400 mL)

7 and 8 are optional steps for improving the transfer of long RNAs, esp from >1% gels.

Prepare the Membrane (5-15 minutes)

NOTE: Be gentle with the membrane. The number of times a membrane can be stripped and re-probed is usually limited by physical damage to the blot.

1. Cut a piece of membrane to the dimensions of the agarose gel. Max dimensions for hybridization in 50mL tubes: 8 x 9 cm (circumference x diameter). If membrane is larger, sandwich between sheets of nylon mesh to allow buffer to penetrate overlap.
2. Wet the membrane by carefully laying it on top of Milli-Q water in a shallow tray. (Do not immerse the Immobilon-Ny+ membrane in liquid on the first liquid exposure. If you wet both sides, air can become trapped in the pores and form bubbles.)
3. Agitate the tray gently once the membrane is wet to completely immerse the membrane.
4. Transfer the membrane to a second tray containing transfer buffer (20 x SSC).
5. Equilibrate the membrane at least 5 minutes.

Transfer of RNA onto Membrane by Turboblotter Capillary Transfer (3-4 hours)

NOTE: Refer to Fig. 1 when setting up the TurboBlotter System.

1. Place stack tray of transfer device on bench, making sure it is level.
2. Place 20 sheets of dry GB004 blotting paper (thick) in stack tray.
3. Place 4 sheets of dry GB002 blotting paper (thin) on top of stack.
4. Place one sheet of GB002 blotting paper, prewet in transfer buffer on stack.
5. Place transfer membrane on stack.Smooth bubbles by rolling a clean glass pipette over the surface. Do not touch with gloves.
6. Cover the membrane with agarose gel, cut the gel to the size of the membrane, making sure there are no air bubbles between the gel and the membrane.
7. Wet the top surface of the gel with transfer buffer and place 3 sheets of GB002 Blotting Paper, presoaked in transfer buffer on top of the gel.
8. Attach the buffer tray of the transfer device to the bottom tray using the circular alignment buttons to align both trays.
9. Fill the buffer tray with 125 ml transfer buffer for 7 x 8 cm to 11 x 14 cm transfers; (200 ml for 12 x 21 cm to 20 x 25 cm transfers).
10. Start the transfer by connecting the gel stack with the buffer tray using the precut buffer wick (included in each blotter stack), presoaked in transfer buffer. Place the wick cover on top of the stack to prevent evaporation. Make sure the edges of the wick are immersed in the transfer buffer.
11. Continue the transfer for 3 hr. Additional transfer time may be required for gels thicker than 4 mm or larger-size nucleic acids. (Try 4 hr, since gel is 1.5%, cmc 3.25.05)
12. Disassembly: mark edges of gel and lane borders onto blot with pencil.

NOTE: Do not place any other weight on top of the wick cover during transfer. This is unnecessary and may inhibit transfer by crushing the pore structure of the agarose gel.

RNA Fixation with UV Cross-Linking (5 minutes)

1. It is not necessary to allow the blot to dry completely prior to UV cross-linking.
2. Place the blot on a sheet of clean filter paper to prevent contamination if you plan to place the UV light source above the blotted RNA. (If you plan to place the membrane on a UV transilluminator, clean the surface with Milli-Q water and a Kimwipe.)
3. Expose the side of the blot with the bound RNA to a UV light source (254 nm). Using Pabo Lab UV lamp, above blot, ~5 min. This lamp has not been calibrated, but multiple users have had success with 2-5 minutes exposure.