Skip to Content

Northern Blot Analysis

Prepare Gel

  1. For standard medium-sized gel, dissolve 1.5 g agarose in 108 ml DEPC H2O. Boil then cool to 60°C in a water bath.
  2. Place flask in fume hood once at 60°C and add 27 ml formaldehyde and 15 ml 10X MOPS. Swirl to mix, then pour gel and allow to set.
  3. Keep gel tank in fume hood due to presence of formaldehyde. Pour running buffer (1X MOPS in DEPC H2O) in amount sufficient to cover gel.

Prepare Sample and Run Gel

  1. Adjust volume of RNA sample (10 µg) to a total of 5.5 ml with DEPC H2O, then add 2.5 ml 10X MOPS, 4.5 ml 12.3M formaldehyde (stock concentration) and 12.5 ml formamide.

    Prepare RNA marker at same time.

  2. Mix by vortexing and centrifuge briefly. Incubate at 15 minutes at 55°C.
  3. Add 4 ml formaldehyde loading buffer and 1µl 10 mg/ml ethidium bromide, vortex and quick spin to collect liquid and load into gel.
  4. Run the gel at 5 V/cm (for a medium gel: ~70 V) until the bromophenol blue dye has migrated halfway to 2/3 way down the gel. This usually takes ~3 hours.

    Note: People in this lab have run medium gels up to 120 V without problems, thus far.

  5. Photograph gel with a ruler on the side to measure placement of markers.

Transfer of RNA

  1. Soak gel in 5 volumes distilled DEPC H2O for 5 minutes, shaking. Repeat 4X.
  2. Place GeneScreen® membrane, cut to size, in DEPC H2O for a few seconds to hydrate.
  3. Soak membrane in 10X SSC for 15 minutes.
  4. Set up capillary blot using 10X SSC as the transfer solution. Be sure to remove all bubbles between the layers.

    Top of transfer

    Stack paper towels on top to pull the SSC through the gel.
    Place book or weight on top.

    3 pieces Whatman® paper

    Membrane
    (Mark wells and what will be top left corner of membrane with needle or pen.)

    Gel, bottom side up

    3 pieces Whatman paper, cut to size of gel, prewet in SSC

    Bottom of transfer


  5. Transfer 16-24 hours. 
  6. Carefully remove filter paper without disturbing membrane. 
  7. Lift membrane away from gel with plastic forceps. 
  8. Rinse the membrane briefly in 2X SSC to remove residual agarose. Fix RNA to the membrane using UV crosslinker. 
  9. Place the membrane RNA side up on filter paper to dry. You can rehydrate it in 2X SSC.

Hybridization of Probe

  1. Prehybridize membrane in a minimum volume at 42°C for 2-4 hours with agitation.

    Pre-heat prehyb solution before placing on membrane.

  2. Remove prehyb solution and add fresh pre-heated hybridization solution and ~5 X 105 dpm denatured probe.
  3. Hybridize O/N at 42°C with agitation.

Washes

  1. Wash blot 1X for 5 minutes at RT with 2X SSC.
  2. Wash 3X for 20 minutes each at 65°C with 0.2X SSC/0.1%SDS.
  3. Wash 1X for 5 minutes at RT with 2X SSC.
  4. Wrap in plastic wrap and place on phosphoimager or film.

Stripping Northern

  1. Pour boiling stripping solution onto membrane, then keep at 80-90°C for 5 minutes.
  2. Pour off this solution. You can repeat as necessary. Check blot on phosphoimager to ensure successful removal of probe.

Solutions

DEPC H2O

  • 0.1% DEPC in H2O
  • Let sit O/N at 37°C
  • Autoclave

10X MOPS in DEPC H2O

  • 0.4M MOPS, pH 7.0
  • 0.1M Sodium Acetate
  • 0.01M EDTA
  • Store covered in foil up to 3 months at 4°C.

500 ml

  • Add 20.8 g MOPS to 350 ml DEPC H2O
  • pH to 7 with 10N NaOH before adding remaining ingredients
  • 25 ml of 2M stock made with DEPC H2O
  • 10 ml of 0.5M stock, preferably made with DEPC H2O
  • Store covered in foil up to 3 months at 4°C.

Formaldehyde Loading Buffer

  • 1mM EDTA
  • 0.25% (w/v) Bromophenol Blue
  • 0.25% (w/v) Xylene Cyanol
  • 50% (v/v) Glycerol
  • Store up to 3 months at RT

10 ml

  • 20 ml 0.5M EDTA
  • 0.25 g
  • 0.25 g
  • 5 ml
  • Store up to 3 months at RT

10X SSC

  • 3M NaCl
  • 0.3M Sodium Citrate
  • Autoclave.

100X Denhardt's

  • 1 g Polyvinylpyrrolidone (PVP; MW=40.000)
  • 1 g BSA
  • 1 g Ficoll 400
  • Add dH2O to 50 ml and filter sterilize
  • Store at 4°C or aliquot and store frozen

Prehybridization Solution

  • 5X SSC
  • 50% (w/v) Deionized Formamide
  • 5X Denhardt's Solution
  • 1% SDS
  • 10% Dextran Sulfate, Sodium Salt (MW=500,000)

100 ml

  • 20 ml 20X
  • 50 ml
  • 5 ml 100X
  • 10 ml 10%
  • 10 g
  • 100 mg/ml denatured sheared, nonhomologous DNA, i.e., salmon sperm DNA
  • Shear by pushing through syringe and then boil before adding to prehyb solution

Hybridization Solution

  • 5X SSC
  • 50% (w/v) Deionized Formamide
  • 5X Denhardt's Solution
  • 1% SDS
  • 10% Dextran Sulfate, Sodium Salt (MW=500,000)

100 ml

  • 20 ml 20X
  • 50 ml
  • 5 ml 100X
  • 10 ml 10%
  • 10 g

Stripping Solution

  • 1% SDS
  • 0.1X SSC
  • 40 mM Tris, pH 7.5
  • Store up to 1 year at RT

*Combination of protocols from Current Protocols in Molecular Biology and GeneScreen® Plus Protocols

© 2014 The University of Texas MD Anderson Cancer Center