Abstract
Bone tumors are very rare. They account for about 0.8% of all human neoplasms. Molecular techniques have a quite limited application in this field, because few entities show characteristic aberrations useful for diagnostic and therapeutical purposes. The prototype of these entities is the Ewing sarcoma, typically recognized by recurrent translocations. Often are described new aberrations, such as point mutations or gene amplifications that can occur in bone tumors. The major problem in this very specialized field is the kind of tissue, the bone, that needs decalcification, and this treatment can interfere with nucleic acid isolation. In the decalcified tissues, the affordability of molecular techniques is low (reported to be around 20%). For this reason, it is important to sample bone tumors before the decalcification process in order to store adequate tissue in biobanks for molecular purposes. A valid alternative choice could be to use adequate systems of decalcification that sufficiently preserve the quality of nucleic acids.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Notes
- 1.
The solubilization of Proteinase K in glycerol Buffer keeps the solution fluid at −20°C, with a better preservation of the enzymatic activity.
- 2.
Apply RNAse Away over the surface of glassware or plasticware to be treated. Unwanted RNase and DNA contamination are eliminated.
- 3.
Clean the pipettes with RNAse Away or leave them under the UV lamp for about 10 min to prevent contaminations. Alternatively, the pipette can be autoclaved, if possible, according to the manufacturer’s specifications.
- 4.
This step should be performed by two operators under a fume hood.
- 5.
Avoid breathing fumes when working with formalin. The wasted formalin must be collected in a chemical waste container and discharged according to the local hazardous chemical disposal procedures.
- 6.
For 1 l of decalcifying solution, mix 51ml of 98% formic acid (5% final concentration), 30.8ml of 65% nitric acid (2% final concentration), and 918.2 ml of distilled water. Formic acid and nitric acid are corrosive solutions. Wear gloves and handle the solutions under the chemical hood.
- 7.
Wear gloves when isolating and handling RNA or reagents for RNA isolation to minimize the contamination with exogenous RNAses. Use autoclaved pipette tips and 1.5 ml microcentrifuge tubes.
- 8.
Clean the microtome and blade with RNAse Away. It is recommended that the blade be changed after the cutting of each paraffin block to avoid any potential contamination.
- 9.
Avoid breathing fumes when working with xylene. It is better to perform the deparaffinization step under a chemical hood. Xylene is harmful; the wasted xylene must be collected in a chemical waste container and discharged according to the local hazardous chemical disposal procedures.
- 10.
Make sure that ethanol dilutions used to RNA isolation procedure, are performed in RNase-free water. This step is performed in an orbital shaker (shaking moderately).
- 11.
Use a sterile pipette tip or a glass pasteur to gently spread a small amount of Pinpoint solution over the selected tissue region. Generally, about 0.5 μl of Pinpoint solution is used per mm2 of tissue area. Usually, one drop of Pinpoint is adequate for 25 mm2 of tissue area. Four drops on tissue with appropriate cellularity (using one to four slides) allow good results.
- 12.
Leave the slides for about 30–45 min; if left under the chemical hood, 10–15 min are sufficient.
- 13.
Use a sterile blade or scalpel to cut and remove the embedded section from the slide. Transfer the sample to a 1.5 ml tube.
- 14.
Vortex the tube every 30 min to improve the digestion.
- 15.
Use one Column for each extraction tube.
- 16.
The isolated RNA can be used directly for RT-PCR amplification, or it can be stored at −70°C for future use.
- 17.
The concentration of RNA expressed in μg/μl is obtained as follows: [RNA] = A260  ×  dilution factor  ×  40  ×  10−3 (see Chap. 16). An appropriate RNA preparation from FFPE tissue should have a A260/A280 ratio of 1.40–1.80. The ratio variability is linked to the presence of proteins and oligo-, polysaccharides.
References
Shibtat Y, Fujita S, Takahashi H, Yamaguchi A, Koji T (2000) Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues. Histochem Cell Biol 113:153–159
Weizacher FV, Labeit S, Koch HK, Oehlert W, Blum HE (1991) A simple and rapid method for the detection of RNA in formaliln-fixed, paraffin-embedded tissue by PCR amplification. Biochem Biophys Res Commun 174:176–180
Greer CE, Lund JK, Manos M (1991) PCR amplification from paraffin-embedded tissue: recommendations on fixatives for long-term storage and prospective studies. PCR Methods Appl 1:46–50
Bonin S, Hlubek F, Benhattar J, Denkert C, Dietel M, Fernandez PL, Hofler G, Kothmaier H, Kruslin B, Mazzanti CM, Perren A, Popper H, Scarpa A, Soares P, Stanta G, Groenen PJ (2010) Multicentre validation study of nucleic acids extraction from FFPE tissues. Virchows Arch 457(3):309–317
Mangham DC, Williams A, KcMullan DJ, McClure J, Sumathi VP, Grimer RJ, Davies AM (2006) Ewing’s sarcoma of bone: the detection of specific transcripts in a large, consecutive series of formalin-fixed, decalcified, paraffin-embedded tissue samples using the reverse transcriptase-polymerase chain reaction. Histopathology 48:363–376
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Alberghini, M., Benini, S., Gamberi, G., Cocchi, S., Zanella, L. (2011). RNA Extraction from Decalcified and Non-decalcified 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_13
Download citation
DOI: https://doi.org/10.1007/978-3-642-17890-0_13
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-17889-4
Online ISBN: 978-3-642-17890-0
eBook Packages: MedicineMedicine (R0)