RNA isolation and quantification Protocol

The acid-phenol extraction is a simple procedure to extract RNA from cells, and to separate RNA from DNA. When cells are lysed and extracted with phenol, protein and lipids always partition into the phenol layer whereas RNA partitions to the aqueous layer (regardless of the pH of the phenol). In contrast, DNA shows a pH-dependent partitioning. When the aqueous phase of the phenol is buffered above pH 7.5, DNA partitions with RNA into the aqueous layer. However, when the aqueous phase of the phenol is buffered at pH 4.0, DNA will partition into the phenol layer. The concentration of RNA can be conveniently measured due to its ability to absorb light at 260 nm. The ratio A₂₆₀:A₂₈₀ is an indication of the purity of the RNA. The ratio (for pure RNA) should be 1.8 -2.1. However, even if the ratio is less than 1.8, the RNA quality may be acceptable and can be confirmed by the subsequent gel electrophoresis.

Materials required:
TRIzol Reagent (Gibco BRL; DNA and protein can also be extracted from the samples using this reagent)
Chloroform       Isopropanol        75% ethanol         RNase-free water (i.e. DEPC treated)
 

Phenol - Caution Phenol is highly toxic, even in small amounts. If spilt on skin - wash immediately under cold water If spilt on clothing - remove immediately If spilt on bench, mop up immediately and consult a demonstrator. UV light -Caution Spectrophotometers are a source of UV radiation

RNA isolation
1. Discard the cell growth medium safely.
2. Wash the cell monolayer by carefully by adding 1 ml PBS to the side of the dish making sure you do not disrupt the cells. Pour off the PBS wash.
3. Add 1 ml TRIzol to the plate to lyse the cells. Pass the cell lysate several times through a pipette (insufficient TRIzol may result in contamination of the isolated RNA with DNA).
4. Transfer the lysate to a 1.5 or 2.0ml Eppendorf tube.
5. Incubate the homogenized samples for 5 min at room temperature (this allows complete dissociation of nucleoprotein complexes).
6. Add 0.2 ml of chloroform per 1 ml of TRIzol (Make sure the tube is tightly capped).
7. Shake tubes vigorously by hand for 15 sec and incubate at room temp for 2.5 min.

RNA/DNA separation
1. Centrifuge samples at no more than 12,000 rpm for 15 min at 4℃. Following centrifugation the mixture separates into a lower, phenol-chloroform phase (red), an interface, and an aqueous upper phase (colourless) Ð RNA remains exclusively in the aqueous phase.
2. Transfer the aqueous phase (i.e. top layer; »600 µl) to a fresh tube.
3. To precipitate RNA, add 1 ml isopropanol per 1 ml of TRIzol used for the initial lysis and mix by inversion, then incubate at room temp for 10 min.
4. Centrifuge at 12,000 rpm for 10 min at 4ûC (cold-room)
5. Discard the supernatant and wash the RNA pellet with 75% ethanol (use at least 1 ml of 75% ethanol per 1 ml of TRIzol used for the initial lysis) - mix by vortexing.
6. Centrifuge at 7,500 rpm for 5 min at 4℃ (cold-room).
7. Discard the supernatant and (briefly) air-dry the RNA pellet by inverting the Eppendorf tube for 5-10 min. It is important NOT to dry the RNA completely.
8. Dissolve RNA in about 20ml RNase-free water
9. Heat RNA at 55 ûC for 5-10 min (increases solubility of RNA). Store at -20℃ o/n.

RNA concentration measurement
1. Dilute 2 - 3 µl of RNA (e.g. approx. 1/10 of RNA from above) into 500µl H₂O.
2. Place in a quartz cuvette and measure the OD₂₆₀ and OD₂₈₀.
3. Calculate the RNA concentration using the following equation:

Appendix

Concentration measurement
RNA (and DNA) has an absorbance peak at 260nm. The ratio A₂₆₀:A₂₈₀ is an indication of the purity of the RNA. The ratio (for pure RNA) should be 1.8 - 2.1. However, even if the ratio is less than 1.8, the RNA quality may be acceptable and can be confirmed by the subsequent gel electrophoresis.
Alternatives
Trizol can be purchased from GibcoBRL. If you wish to avoid phenol, many companies sell RNA extraction kits (e.g. Qiagen, Hybaid, etc).

Handling RNA -the hazards of RNase
One of the major difficulties in working with RNA is its susceptibility to hydrolytic cleavage by the enzyme ribonuclease (RNase). RNase is literally everywhere - it is secreted from the skin and tends to coat tubes, benches, pipettes an so on. It is also very stable Ð it retains its activity even after autoclave procedures and cannot be removed by cleaning surfaces with ethanol or isopropanol. It even retains activity after exposure to chaotropic agents such as guanidium thiocyanate. The only way to eradicate the effect of RNase is to chemically inactivate it. This requires the use of the methylating agent, diethylpyrocarbonate (DEPC), which irreversibly modifies the active site histidine residue of the enzyme, thereby inactivating it. All water used for RNA work must be treated with DEPC before use. DEPC is dispensed in a fume hood and added to water in an autoclavable (Schott type) bottle containing a stir bar. The water is stirred for one hour in the fume cupboard and after treatment the solution is autoclaved to denature the DEPC. To avoid contamination by RNase, it is important to handle solutions and material for RNA use with extra care:
-Wear gloves at all times
-Maintain separate stocks of chemicals reagents for RNA work - do not use for other laboratory stocks. Many suppliers can provide RNase-free chemicals and reagents.
-Solutions that have been prepared for RNA work should be kept separate from the rest of the laboratory solutions- label these reagents appropriately.
-All pipette tips and Eppendorf tubes should not be handled by hand prior to autoclaving. If possible, purchase pre-packed RNase free materials.