Skip to main content
NCBI home page
Brain Pathology logoLink to Brain Pathology
. 2006 Apr 5;14(2):175–182. doi: 10.1111/j.1750-3639.2004.tb00050.x

Comparison of Ethanol Versus Formalin Fixation on Preservation of Histology and RNA in Laser Capture Microdissected Brain Tissues

Jack Meng Fen Su 1, Laszlo Perlaky 1, Xiao‐Nan Li 1, Hon‐Chiu Eastwood Leung 1, Barbara Antalffy 2, Dawna Armstrong 2, Ching C Lau 1,✉
PMCID: PMC8096012  PMID: 15193030

Abstract

Although RNA can be retrieved from formalin‐fixed, paraffin‐embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol‐fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin‐fixed tissues. RNA from ethanol‐fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT‐PCR using RNA from ethanol‐fixed tissues was consistently successful. RNA from FFPE tissues composed of low‐molecular weight fragments, and their use in RT‐PCR failed repeatedly. The yield and quality of RNA from ethanol‐fixed brains were unaffected after immersion at 4°C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol‐fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol‐fixed tissues for RT‐PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.

Full Text

The Full Text of this article is available as a PDF (571.6 KB).

REFERENCES

  • 1. Ahram M, Flaig MJ, Gillespie JW, Duray PH, Linehan WM, Ornstein DK, Niu S, Zhao Y, Petricoin EF 3rd, Emmert‐Buck MR (2003) Evaluation of ethanol‐fixed, paraffin‐embedded tissues for proteomic applications. Proteomics 3:413–421. [DOI] [PubMed] [Google Scholar]
  • 2. Auerbach C, Moutschen‐Dahmen M, Moutschen J (1977) Genetic and cytogenetic effects of formaldehyde and related compounds. Mutation Research 39:317–362. [DOI] [PubMed] [Google Scholar]
  • 3. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1998) Preparation and analysis of RNA. In: Current protocols in molecular biology, John Wiley & Sons, New York . [Google Scholar]
  • 4. Ben‐Ezra J, Johnson DA, Rossi J, Cook N, Wu A (1991) Effect of fixation on the amplification of nucleic acids from paraffin‐embedded material by the polymerase chain reaction. J Histochem Cytochem 39:351–354. [DOI] [PubMed] [Google Scholar]
  • 5. Bresters D, Schipper MEI, Reesink HW, BoeserNunnink BDM, Cuypers HTM (1994) The duration of fixation influences the yield of HCV cDNA‐PCR products from formalin‐fixed, paraffin‐embedded liver tissue. J Virol Methods 48:267–272. [DOI] [PubMed] [Google Scholar]
  • 6. Brown PO, Botstein, D (1999) Exploring the new world of the genome with DNA microarray. Nat Genet 21:33–37. [DOI] [PubMed] [Google Scholar]
  • 7. Dubeau L, Chandler LA, Gralow JR, Nichols PW, Jones PA (1986) Southern blot analysis of DNA extracted from formalin‐fixed pathology specimens. Cancer Res 46:2964–2969. [PubMed] [Google Scholar]
  • 8. Fend F, Emmert‐Buck MR, Chuaqui R, Cole K, Lee J, Liotta L, Raffeld M (1999) Immuno‐LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am J Pathol 154:61–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Finke J, Fritzen R, Ternes P, Lange W, Dolken G (1993) An improved strategy and a useful housekeeping gene for RNA analysis from formalin‐fixed, paraffin‐embedded tissues by PCR. Biotechnology 14:448–453. [PubMed] [Google Scholar]
  • 10. Foss RD, Guha‐Thakurta N, Conran RM, Gutman P (1994) Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin‐embedded tissue. Diag Mol Pathol 3:148–155. [DOI] [PubMed] [Google Scholar]
  • 11. Giannella C, Zito FA, Colonna F, Paradiso A, Marzullo F, Alaibac M, Schittulli F (1997) Comparison of formalin, ethanol, and Histochoice fixation on the PCR amplification from paraffinembedded breast cancer tissue. Eur J Clin Chem Clin Biochem 35:633–635. [PubMed] [Google Scholar]
  • 12. Gillespie JW, Best CJ, Bichsel VE, Cole KA, Greenhut SF, Hewitt SM, Ahram M, Gathright YB, Merino MJ, Strausberg RL, Epstein JI, Hamilton SR, Gannot G, Baibakova GV, Calvert VS, Flaig MJ, Chuaqui RF, Herring JC, Pfeifer J, Petricoin EF, Linehan WM, Duray PH, Bova GS, Emmert‐Buck MR (2002) Evaluation of non‐formalin tissue fixation for molecular profiling studies. Am J Pathol 160:449–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Goelz SE, Hamilton SR, Vogelstein B (1985) Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun 130:118–126. [DOI] [PubMed] [Google Scholar]
  • 14. Goldsworthy S, Stockton PS, Trempus CS, Foley JF, Maronpot R (1999) Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Mol Carcinog 25:86–91. [PubMed] [Google Scholar]
  • 15. Goodrich LV, Milenkovic L, Higgins KM, Scott MP (1997) Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277:1109–1113. [DOI] [PubMed] [Google Scholar]
  • 16. Guerrero RB, Batts KP, Brandhage DJ, Germer JJ, Perez RG, Persing DH (1997) Effects of formalin fixation and prolonged block storage on detection of hepatitis C virus RNA in liver tissue. Diag Mol Path 6:277–281. [DOI] [PubMed] [Google Scholar]
  • 17. Jackson DP, Quirke P, Lewis F, Boylston AW, Sloan JM, Robertson D, Taylor GR (1989) Detection of measles virus RNA in paraffin‐embedded tissue. Lancet 1:1391. [DOI] [PubMed] [Google Scholar]
  • 18. Kabbarah O, Pinto K, Mutch DG, Goodfellow PJ (2003) Expression profiling of mouse endometrial cancers microdissected from ethanol‐fixed, paraffin‐embedded tissues. Am J Pathol 162:755–762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Koopmans M, Monroe SS, Coffield LM, Zaki SR (1993) Optimization of extraction and PCR amplification of RNA extracts from paraffin‐embedded tissue in different fixatives. J Virol Methods 43:189–204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Koshiba M, Ogawa K, Hamazaki S, Sugiyama T, Ogawa O, Kitajima T (1993) The effect of formalin fixation on DNA and the extraction of high‐molecular‐weight DNA from fixed and embedded tissues. Path Res Pract 189:66–72. [DOI] [PubMed] [Google Scholar]
  • 21. Mizuno T, Nagamura Hiroko, Iwamoto Keisuke, Ito T, Fukuhara T, Tokunaga M, Tokuoka S, Mabuchi K, Seyama T (1998) RNA from decades‐old archival tissue blocks for retrospective studies. Diag Mol Pathol 7:202–208. [DOI] [PubMed] [Google Scholar]
  • 22. Mok SC, Chan WY, Wong KK, Cheung KK, Lau CC, Ng SW, Baldini A, Colitti CV, Rock CO, Berkowitz RS (1998) DOC‐2, a candidate tumor suppressor gene in human epithelial ovarian cancer. Oncogene 16:2381–2387. [DOI] [PubMed] [Google Scholar]
  • 23. Noguchi M, Furuya S, Takeuchi T, Hirohasi S (1997) Modified formalin and methanol fixation methods for molecular biological and morphological analyses. Pathol Int 47:685–691. [DOI] [PubMed] [Google Scholar]
  • 24. Rupp GM, Locker J (1988) Purification and analysis of RNA from paraffin‐embedded tissues. Biotechnology 6:56–60. [PubMed] [Google Scholar]
  • 25. Satoy, Mukai K, Furuya S, Shimosato Y (1991) The AmeX method: a multipurpose tissue‐processing and paraffin‐embedding method. III. Extraction and purification of RNA and application to slot‐blot hybridization analysis. J Pathol 163:81–85. [DOI] [PubMed] [Google Scholar]
  • 26. Shibata D. Martin WJ, Arnheim N (1988) Analysis of DNA sequences in forty‐year‐old paraffin‐embedded thin‐tissue sections: a bridge between molecular biology and classical histology. Cancer Res 48:4564–4566. [PubMed] [Google Scholar]
  • 27. Shibutani M, Uneyama C, Miyazaki K, Toyoda K, Hirose M (2000) Methacarn fixation: a novel tool for analysis of gene expressions in paraffin‐embedded tissue specimens. Lab Invest 80:199–208. [DOI] [PubMed] [Google Scholar]
  • 28. Simone NL, Bonner RF, Gillespie JW, Emmert‐Buck MR, Liotta LA (1998) Laser capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genetics 14:272–276. [DOI] [PubMed] [Google Scholar]
  • 29. Stanta G, Schneider C (1991) RNA extracted from paraffin‐embedded human tissue is amenable to analysis by PCR amplification. Biotechnology 11:304–308. [PubMed] [Google Scholar]
  • 30. Tokuda Y, Nakamura T, Satonaka K, Maeda S, Doi K, Baba S, Sugiyama T (1990) Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde. J Clin Pathol 43:748–751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. van Deerlin VM, Gill LH, Nelson PT (2002) Optimizing gene expression analysis in archival brain tissue. Neurochem Res 27:993–1003. [DOI] [PubMed] [Google Scholar]
  • 32. van Gelder RN, Von Zastrow ME, Yool A, Dement WC, Barchas JD, Eberwine JH (1990) Amplified RNA synthesized from limited quantities of heterogeneous. CDNA. Proc Natl Acad Sci U S A 87:1663–1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Weizsacker F, Labeit S, Kock HK, Oehlert W, Gerok W, Blum HE (1991) A simple and rapid method for the detection of RNA in formalin‐fixed, paraffin‐embedded tissues by PCR amplification. Biochem Biophys Res Comm 174:176–180. [DOI] [PubMed] [Google Scholar]

Articles from Brain Pathology are provided here courtesy of Wiley

RESOURCES