Phenol Extraction of rRNA (Rat liver)

Phenol Extraction of rRNA (Rat liver)

LEVEL II

*** READ THROUGH ALL CAUTIONS BEFORE TRYING THIS EXPERIMENT ***

Materials

• Rat liver (fasted rat)
• Liquid nitrogen
• p-Amino-salicic acid
• Phenol mixture
• Homogenizer or blender6
• Refrigerated preparative centrifuge
• NaCl
• 95% and 70% (v/v) ethanol

Procedure

Obtain a rat which 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, being careful not to allow the 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-amino-salicylate (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 tell-tale 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.

CLEAN UP ALL SPILLS IMMEDIATELY! NOTIFY YOUR INSTRUCTOR OF ANY SPILLS AFTER YOU HAVE THOROUGHLY RINSED AND WASHED AWAY ANY MATERIALS IN CONTACT WITH YOUR SKIN.

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 15,600 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 five minutes. Recentrifuge as in step 8 above, but for 10 minutes.

Separate the aqueous phase and add 2.3 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 7 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).

For the purposes of introduction to the technique, this exercise extracts RNA from rat liver using a phenol extraction which yields predominantly rRNA and tRNA. There is some mRNA present, but it is variable and should be considered as a background contaminant. There is also a good portion of sRNA caused by sheering of the RNA during homogenization, and by enzymatic digestion by RNAase during the extraction.