Endy:Northern blot, AlkPhos end-labeled probes

The following protocol for Northern Blotting is based on getting RNA from the RNA extraction method used by Sean Moore, Agarose (as opposed to polyacrylamide) gel electrophoresis, and the Turboblotter system from Whatman. This is largely a compilation of another previous Endy lab protocol and the Knight lab's protocol for RNA gels.

Materials

• RNA extracted from cells (should be ~1-5µg/µl).
• SYBR Gold for staining the gel (if you're going to view it under UV).
• HPLC grade or better DMSO

RNAse free water

• make lots for rinsing glassware and electrophoresis chambers (4L for a reaction?)
  • Add DEPC to final concentration of 0.1%.
  • Incubate 1hr at 37°C.
  • Autoclave for 15 mins at 15 psi.

Turboblotter Kit

This kit uses downward capillary transfer and therefore can run a transfer in <4hrs. It uses (+) charge nylon membranes, which bind RNA more tightly but also have higher background.

• From Whatman [[1]]
• Stack and Buffer trays. These can apparently be improvised using empty tip boxes (?).
• Blotting paper.
• (+)nylon membrane (Nytran SuPerCharge)
• buffer wick

10X BPTE electrophoresis buffer

This buffer has low ionic strength and does not remove glyoxal from RNA during electrophoresis and therefore does not have to be recirculated.

• The final pH of this 10x buffer is ~6.5.
• 100 mM PIPES
• 300 mM Bis-Tris
• 10 mM EDTA
• Prepare by adding the following to 90 ml of distilled H₂O. (might scale up to 500ml for first time).

1. • 3 g of PIPES (free acid)
   • 6 g of Bis-Tris (free base)
   • 2 ml of 0.5 M EDTA
2. Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
3. Autoclave.

Glyoxal

• Commercially available stock solutions of glyoxal contain both hydrated forms of glyoxal and oxidation products that can degrade RNA. These must be removed; we used the protocol as follows (See Sambrook and Russel 3rd Ed, App. 1-24):
• Run the Glyoxal over mixed-bed ion exchange resin. We used BioRad AG 501-X8 (D) resin beads (these beads need to be largely blue; if they're gold-orange, they've oxidized and should be disposed of as HazWaste. Our resin was a mix of blue and gold beads, so I ended up just using as much resin as I needed to to get the pH up. This seemed to work fine. ~~Felix Moser. 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

• 6mL DMSO
• 2mL deionized glyoxal
• 1.2mL of 10X BPTE electrophoresis buffer
• 0.6mL of 80% glycerol
• 0.2ml of either ddH2O or ethidium bromide, depending on how or whether you're staining it.
• (divide into small aliquots and store at -70°C)

RNA gel loading buffer

• 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
• got some from Sean Moore. Make sure final volume of buffer is no less than 50% volume of the sample.

"Denaturing" Buffer

• 0.5NaOH
• 1.5M NaCl
• For 1L: 20g NaOH, 87.66g NaCl, bring volume to 1L w/ ddH2O.

"Neutralizing" Buffer

• 0.5M Tris-HCl pH 7
• 1.5M NaCl
• For 1L: 60.56g Tris, 87.66g NaCl, 800ml ddH2O, adjust pH w/ conc. HCl and bring volume to 1L w/ ddH2O.

20x SSC "Transfer" buffer

• 3M NaCl
• 0.3M sodium citrate
• adjust pH to 7.
• For 1L: 175.5g NaCl, 88.2g Na citrate (dihydrate), 800ml ddH2O, adjust pH to 7 then bring to 1L w/ ddH2O.

Prewash Solution (0.1x SSC/ 0.1% SDS)

50 mL Total Volume

1. • 250 µL of 20x SSC
   • 500 µL of 10% SDS
   • 49.25 mL of nuclease-free H2O

100x Denhardt Solution

1. • 5 g of Ficoll 400
   • 5 g of polyvinylpyrrolidone (Not polyvinylpolypyrrolidone!)
   • 5 g of BSA Fraction V (stored at 4°C)
2. dissolve to 250 mL in nuclease-free H2O
3. Sterile filter and freeze in 5 mL aliquots

Pre-hybridization Buffer

50 mL Total Volume

1. • 5 mL of 100x Denhart's Solution
   • 15 mL of 20x SSC
   • 0.5 mL of 10% SDS
   • 29.5 mL of Nuclease-free H2O

Stringency Wash (6x SSC/ 0.1% SDS)

200 mL Total Volume

1. • 2 mL of 10% SDS
   • 60 mL of 20X SSC
   • 138 mL of nuclease-free H2O

Protocol

Running the Gel (2-6hrs, depending on size of 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 H2O treated with 0.1% DEPC

This protocol comes pretty much straight from Sambrook and Russel 3rd ed.:7-29.

1. Set up the glyoxal denaturation reaction by combining 0.5-2 ul of RNA (up to 10 ug) with 10 ul of glyoxal reaction mixture. You MUST also treat your RNA ladder the EXACT SAME WAY so it runs the same as your samples if you want to use it for comparison.
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 the formamide loading buffer (>equal volume 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 (in 14cm gels); ie. run gel @ 5V/cm.
6. Trim away areas of the gel to be stained with sybrGold. DO NOT trim the area BELOW the loading dye, since many RNA's run much lower than the dyes; trim the gel later once you know where all your RNA is. (Membrane should be cut to match the size of the gel.) Wrap gel to be stained in sarran wrap and store at 4°C until post-transfer gel is ready to be stained.
7. Soak gel 30 min in "Denaturing Buffer" (0.5 M NaOH/1.5 M NaCl (~400 mL)). Agitate occasionally.
8. Rinse the gel in ddH2O and Soak gel 20 min in "Neutralizing Buffer" (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. These steps are the from the Turboblotter protocol

Stain gel (can't do if continuing to northern)

1. Prepare fresh 1:10,000 dilution in RNase free water of SybrGold or SybrGreen (SybrGreen recommended by Sean, since it's specific for RNA; ppbly doesn't make a big difference, though).
2. Ensure pH is 7.0-8.5.
3. Pour into staining tray.
4. Place gel in plastic staining container.
5. Shield from light.
6. Agitate gently for 10-40 mins at room temperature.
7. Image with gel box. (Place a clear ruler next to gel to more accurately assess length.)

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 20xSSC 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. IF the gel is smaller than the membrane and the surrounding blotting paper, cover the area surrounding the gel w/ PLASTIC (eg. Sarran wrap, cut-out parts of tip boxes) so that the transfer buffer does not soak through the areas where the blotting paper above the gel touches the blotting paper below it.
8. WET the top surface of the gel with transfer buffer and place 3 sheets of presoaked (in 20x transfer buffer) GB002 Blotting Paper, in on top of the gel.
9. Attach the buffer tray of the transfer device to the bottom tray using the circular alignment buttons to align both trays.
10. 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).
11. Start the transfer by connecting the gel stack with the buffer tray using the precut, PRESOAKED buffer WICK (included in each blotter stack), 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.
12. 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%)
13. 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). We used one of the Sauer lab's UV handheld light sources for 5min.

Hybridization (2 hours + overnight + >1 hour)

1. If blot dimensions are less than 8 x 9 cm, place blot in a 50 mL Falcon tube, RNA facing in. Make sure the blot doesn't overlap itself. 50 mL conical tubes fit in the Sauer Lab "Bambino" mini-hybridization oven. If blot is too large for a 50 mL Falcon tube, use large glass tubes and Baker Lab hybridization oven.
2. PREWASH Blot in 0.1x SSC/ 0.1% SDS for 1 hour at 65°C in hybridization oven. 10 - 15 mL of solution is required to cover the blot in a 50 mL tube, and sufficient for all steps. This incubation can be cut to 30 min if pressed (says Sean).
3. Remove Prewash.
4. Prehybridize blot for > 1 hour in 10 - 15 mL of PREHY solution at hybridization temp. Hybridization temp ~15°C below estimated Tm of probe. If reusing probe in hybridization solution, thaw probe as balance. NOTE: It is possible to store blots in prehybridization solution sort-term to indefinitely at 4°C or -20°C (Sean)
5. Remove prehybridization buffer if reusing probe, otherwise retain same buffer.
6. Add hybridization buffer with old probe to blot or add new probe to pre-hybridization buffer. Hybridize >6 hrs at hybridization temp.
7. Pour off probe either into 32P liquid waste (remember to record waste) or into tube for storage at -20°C for later reuse. NOTE: if storing probe in 13 mL conical tube, remember not to fill tube completely.
8. Wash blot in 10 - 15 mL of 6x SSC/ 0.1% SDS for 5 min at room temp in the hybridization oven (Leave door open to change temp quickly. Put shield up.)
9. Repeat wash twice for a total of three room temp washes. Dispose of the first wash in the 32P liquid waste. For subsequent washes pour off buffer in the sink. (Record waste.)
10. During washes, pre-erase PhosphorImager screen 20 min on light table.
11. Repeat wash a forth time for 20 min at 10°C below estimated Tm in hybridization oven.
12. Pour off final wash. Remove damp blot from tube and lay on clean saran wrap. Fold saran wrap to seal blot.
13. Image