Denaturing Formaldehyde Gels for Verifying RNA size

RNA can be sized correctly on an agarose gel. However, it needs to be denatured and then remain denatured during the gel run. This can be achieved using a denaturing agarose gel containing formaldehyde, coupled with denaturing of sample prior to analysis. This approach is not suitable for purification of RNA (use denaturing polyacrylamide gel for this).

Materials and Reagents

Use filter tips. Keep all solutions, reagents and plastics to be used for RNA work separate from general supplies. Solutions need to be certified RNase free, unless specified below.

Equipment:

  • Agarose, and usual gel rig apparatus necessary to run an agarose gel (these do not need to be designated for RNA work, but use a clean 250ml flask for making up the gel).
  • 10x MOPS (VWR BP2900500, store at 4C)
  • Formaldehyde, 37% w/v
  • DEPC (diethyl pyrocarbonate) -treated water (see bottom for prep, Sigma Aldrich D5758)
  • Millennium markers RNA ladder (Life Tech AM7150, aliquot and store at -80C)
  • NorthernMax formaldehyde load dye (Life Tech AM8552)
  • RNase free strip tubes
  • Sybrgold (Thermo Fisher S11494, aliquot and store at -20C).

Protocol

GEL

  1. Dissolve 0.5g of agarose in 43.5ml of DEPC-treated water as you would for usual agarose gel.
  2. Allow this solution to cool as you would normally do before pouring. You can run it under the tap to speed this up.
  3. IN HOOD add 1.5ml of 37% formaldehyde and 5 ml of 10x MOPS.
  4. Pour in to cast with comb, leave to set.

SAMPLE PREPARATION

  1. Load 500ng of each RNA sample into a well of a PCR strip tube.
  2. Add water to reach Xul total volume
  3. Add 3 volumes of NorthernMax buffer.
  4. The Millennium Markers need to be denatured also. Use 2ul + 6ul NorthernMax buffer.
  5. Heat at 65C for 15 minutes. IMPORTANT When this has finished, immediately move samples to ice. If samples are allowed to slowly cool, some RNAs may bind to proteins/form secondary structure and will run weird.
RUNNING GEL
  1. Load samples as usual for a gel.
  2. The buffer for this run is 1x MOPS, diluted in water (does not have to be DEPC-H2O).
  3. Run at a reduced voltage of 80V in order to avoid heating the gel.

STAINING GEL

  1. Remove the gel from the rig when the blue loading dye is approximately 1/2-3/4 down the gel. If the predicted RNA size is 4kb or larger, leave gel til 3/4 to get good resolution, smaller RNAs can be left for the shorter time.
  2. Wash quickly once in 1x TAE, followed by a longer, 20 minute wash in 1x TAE. This will bring the pH to a more neutral value to allow Sybrgold to bind to RNA.
  3. Incubate gel in 1x Sybrgold (from 10k x stock) in 1x TAE for 30 mins.
  4. Image as usual. If signal to background is low, destain gel in 1xTAE for 15 mins and image again.

Bonus Feature - Preparation of DEPC water

For molecular biology applications, best to buy 1L bottles of Nuclease free H2O from BioSci store in NHB. When making large solutions that need to be RNase free, use the following preparation.

  1. IN HOOD Make a 1% solution of DEPC in water (make many liters)
  2. Shake to mix, unscrew cap as if for autoclaving, leave in fumehood overnight.
  3. Next morning, autoclave on liquid setting for >30 minutes to break down the DEPC and prevent interference with chemistry in reactions.

-- Main.SimonDAlton - 20 Feb 2018

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Topic revision: r6 - 09 Nov 2018 - 16:02:38 - Main.SimonDAlton
 
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