Abstract
This chapter provides a home-made method of obtaining RNA suitable for reverse transcription and PCR analyses from formalin-fixed and paraffin embedded (FFPE) specimens, even of autopsy origin. This protocol is based mainly on a deparaffinization step, a proteolytic digestion with Proteinase K, phenol purification, and alcohol precipitation, using a point-to-point protocol with notes and references to guide the researcher into the laboratory practice. A section of the chapter is specifically dedicated to RNA handling, because of the instability of this macromolecule. Specific precautions are suggested in order to prevent the degradation of RNA during the extraction and to avoid the accidental introduction of RNases from external sources.
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Notes
- 1.
DEPC is a carcinogen and should be handled with care under a fume hood.
- 2.
The solubilization of Proteinase K in 50% sterile glycerol keeps the solution fluid at −20°C, with a better preservation of the enzymatic activity.
- 3.
Guanidinium thiocyanate is a very strong protein denaturant, which is used to denature all the cellular proteins including RNases. It is harmful, so it must be handled under a fume hood.
- 4.
2-Mercaptoethanol is toxic by inhalation, ingestion, and through skin contact, and is a severe eye irritant. Use only with adequate ventilation, and wear gloves and safety glasses. Wasted 2-Mercaptoethanol must be placed in a chemical waste container.
- 5.
We strongly recommend purchase of saturated phenol from a commercial manufacturer.
- 6.
The pH of Phenol/H2O is around 5, because phenol makes the water acidic during the process.
- 7.
Clean the pipettes with alcohol or another disinfectant and leave them under the UV lamp for at least 10 min. Alternatively, it is possible to autoclave the pipette depending on the provider instructions.
- 8.
Wear gloves and change them frequently over the entire procedure. Do not talk while handling the sample to avoid introduction of saliva drops into the tubes. If possible, perform the extraction under a laminar flow hood. After the deparaffinization steps, the tubes must be kept on ice.
- 9.
Clean the microtome with xylene.
- 10.
RNA extraction from sections previously adhered on slides is also possible. In this case, displace the slides in a jar for histochemistry and deparaffinize the tissues by incubating twice with xylene for 10 min. After the EtOH washings bring the samples to water and scrape the sections from the slide by the use of a needle and transfer it into a tube with the digestion buffer as described in point 10. All the equipments used must be RNase-free.
- 11.
It is possible to bypass the deparaffinization step by adding the digestion buffer directly to the cut sections. In such case, it is better to add a short incubation time of 5 min at 65°C to rapidly melt the paraffin. Paraffin remains on the top of the solution [ 8 ].
- 12.
When working with xylene, avoid breathing fumes; it is better to perform the deparaffinization step under a fume hood.
- 13.
Wear gloves when isolating and handling RNA to minimize contamination with exogenous nucleases. Use autoclaved pipette tips and 1.5 ml microcentrifuge tubes.
- 14.
Xylene is harmful; the wasted xylene must be collected in a chemical waste container and discharged according to the local hazardous chemical disposal procedures.
- 15.
If the pellet is firmly lodged at the bottom of the tube, it is possible to dislodge it in the digestion buffer using a sterile toothpick.
- 16.
Longer duration of digestion (up to 48 h) increases the yield of the RNA, because it promotes a greater cross-link reversal. Optional incubation in the digestion buffer at 70°C for 20 min after the overnight digestion facilitates the disruption of cross-links, resulting in improved quantity and quality of RNA.
- 17.
Phenol is very toxic and should be handled in a fume hood. The wasted phenol must be collected with hazardous chemical waste.
- 18.
The extraction could be performed with 1 volume of phenol (H2O saturated)-chloroform in the same ratio (70/30). Alternatively it is also possible to use chloroform-isoamyl alcohol (24:1, v/v) instead of pure chloroform in the extraction. Usually, isoamyl alcohol is added to the chloroform to prevent foaming. Phenol is an inhibitor of PCR reaction, because of Taq Polymerase inactivation. A single chloroform-isoamyl alchol (24:1, v/v) extraction could be performed after the phenol (H2O saturated)-chloroform extraction in order to completely remove phenol traces.
- 19.
Alternatively, it is possible to perform the extraction using a monophasic commercial solution (i.e., Trizol, RNazol…). If this procedure is chosen, do not proceed with the successive steps of this protocol, but follow the manufacturer’s instruction.
- 20.
After the aqueous phase has been transferred into a new tube, it is possible to extract DNA from the organic phase and interphase by adding an equal volume of Tris 50 mM pH 8–8.5. At this point, follow the protocol described in the chapter “DNA extraction from FFPE tissues.” See also other specific chapters dedicated to DNA extraction.
- 21.
As LiCl is highly soluble in ethanol-containing solutions, the salt is not coprecipitated with the nucleic acid even at −70°C.
- 22.
It is better to store RNA extracts in small aliquots in order to prevent multiple thawing/freezing that may degrade the nucleic acid.
- 23.
The concentration of RNA expressed in μg/μl is obtained as follows: [RNA] = A260 × dilution factor × 40 × 10−3; for example, when diluting 1 μl RNA in 199 μl sterile water, the dilution factor is 200. A clean RNA preparation should have a A260/A280 ratio of 1.5–2. This ratio is decreased by the presence of proteins, oligo- and polysaccharides. Concentration estimation can be also affected by phenol contamination. Also phenol strongly absorbs at 260 nm and therefore can mimic higher DNA yield and purity.
References
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Bonin, S., Stanta, G. (2011). RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_12
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