Abstract
The extraction of useful RNA from FFPE tissues is often compromised for the extraction efficacy. Moreover, because it is resistant to extraction due to cross-linking with proteins, RNA in FFPE is not completely available for RT-PCR reactions. RNA, as DNA, is indeed modified in FFPE tissues by the presence of methylol addition. Prolonged fixations could favor further reactions with the above-mentioned groups, resulting in irreversible artifacts. The presence of the methylol group doesn’t allow the reverse transcription reaction, but the presence of the majority of these groups could be removed by simply heating the RNA extracts in formalin-free buffers. This chapter provides two simple temperature treatments to demodify RNA obtained from FFPE. In our experience the demodification treatment seems to improve the RNA recruitment only in old samples. The methods described hereafter could be added as routine procedure to the RNA extraction protocols especially for very old samples.
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- 1.
DEPC is a carcinogen and should be handled with care under a fume hood.
- 2.
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.
- 3.
For this demodification method, it is not mandatory to use the RNA extraction protocol described in this book; any extraction procedure, even commercial kits, can be used.
- 4.
It is possible to protract this step up to 1 h; however, in our experience, the best results have been obtained by heating at 70°C for 20 min.
- 5.
It is better to store RNA extracts in small aliquots to prevent multiple thawing/freezing, which may degrade the nucleic acid.
- 6.
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 1.5–2.0. This ratio is decreased by the presence of proteins, phenol and oligo-, polysaccharides.
- 7.
For this demodification method, it is not mandatory to use the proteinase K digestion buffer described in this book; any proteinase K buffer could be used, even the ones provided in commercial kits or commercial solutions.
References
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© 2011 Springer-Verlag Berlin Heidelberg
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Bonin, S., Stanta, G. (2011). RNA Temperature Demodification. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_14
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DOI: https://doi.org/10.1007/978-3-642-17890-0_14
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