Materials
- Rat liver (fasted rat)
- Liquid nitrogen
- p-Amino-salicic acid
- Phenol mixture
- Homogenizer or blender
- Refrigerated preparative centrifuge
- NaCl
- 95% and 70% (v/v) ethanol
Procedure
- Obtain a rat that has been fasted for 24 hours (to remove glycogen from
the liver), decaptitate, exsanguinate, and remove the liver as rapidly as
possible.
- Weigh the liver don’t allow it to dehydrate.
- Immediately drop the liver into a container of liquid nitrogen.
Caution: Liquid nitrogen will cause severe frostbite!
- Using the weight of the liver as an indication of the volume (1 gm of liver
equivalent to 1 mL), add 15 volumes of freshly prepared 6% para-aminosalicylate
(pAS) to a chilled blender or homogenizer.
- Add an equal volume (equal to the pAS) of phenol mixture to the blender
and turn on the blender for a short burst to mix the pAS and phenol.
Caution: Phenol is extremely caustic.
Phenol causes severe skin burns, yet it is a local anesthetic. You will be
unaware of the burn at first, except for telltale discoloration of the skin and
blisters. You will become aware of the burn as the anesthetic properties
wear off. Phenol also readily dissolves most countertops and all rubber
compounds.
- Stop the blender and add the frozen liver (handle the liver with long
forceps, or tongs). Blend the entire mixture (pAS, phenol, and liver) for
30 seconds at full speed. Do not blend for longer periods or you will sheer
the RNA.
- Carefully transfer the homogenate to a beaker and continue to stir the
mixture for 10 minutes at room temperature.
- Transfer the homogenate to nalgene centrifuge tubes and centrifuge the
mixture at 15600 xg at 4°C for 20 minutes.
- Remove the centrifuge tubes and carefully separate the upper aqueous
layer from the lower phenol layer. Take care that none of the white
interphase material is mixed into the aqueous layer. The upper layer can
most efficiently be removed by using a large hypodermic equipped with a
long, large-bore, square-tipped needle. Should some of the interphase
material be stirred into the aqueous phase, it will be necessary to repeat
step 8.
- Measure the volume of the aqueous layer and discard the phenol layer and
interphase material.
- Add 3.0 grams of NaCl per 100 mL of aqueous phase and stir until
dissolved.
- Add 0.5 volumes of phenol mixture to the aqueous phase, place into a
suitable flask, and shake vigorously for about 5 minutes. Recentrifuge as
in step 8 above, but for 10 minutes.
- Separate the aqueous phase and add 2 to 3 volumes of cold 95% ethanol.
Allow the mixture to stand in the freezer until a precipitate forms.
- Collect the RNA precipitate by centrifugation, wash once in 70% ethanol
and store in 70% ethanol at 0–5°C.
Notes
Knowledge of transcription is based on our ability to extract “native” or functional
RNA molecules from cells, with subsequent use of those molecules “in
vitro.” One of the earliest methods for this type of analysis is a phenol-detergent
extraction of RNA coupled with separation of the various-sized molecules of
RNA with centrifugation in a gradient.
This basic procedure remains useful today, although there have been myriad
additions and alterations to the procedure using a host of extraction techniques
and separation procedures (such as electrophoresis or column chromatography).
