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Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens

Kiyohiro Hamatani, Hidetaka Eguchi, Keiko Takahashi, Kazuaki Koyama, Mayumi Mukai, Reiko Ito, Masataka Taga, Wataru Yasui and Kei Nakachi

Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan (KH,HE,KT,KK,MM,RI,MT,KN), and Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan (WY)

Correspondence to: Kiyohiro Hamatani, Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima-shi, Hiroshima 732-0815, Japan. E-mail: hamatani{at}rerf.or.jp

Recently, in addition to DNA, RNA extracted from archival tissue specimens has become an invaluable source of material for molecular biological analysis. Successful amplification with PCR/RT-PCR is problematic when using amplicons of short size due to degradation of DNA or RNA. We established an improved method for efficient RT-PCR amplification of RNA extracted from archival formalin-fixed, paraffin-embedded tissue by the elimination of RNA modification and the restoration of RNA template activity. Namely, the preheating in citrate buffer (pH 4.0) of RNA extracted from long-term preserved tissue specimens resulted in significantly increased efficiency of RT-PCR. (J Histochem Cytochem 54:773–780, 2006)

Key Words: preheating of RNA • archival formalin-fixed and paraffin-embedded tissue • citrate buffer • modification of RNA by formalin • RT-PCR amplification

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