Improved RT-PCR amplification for molecular analyses with long-term preserved formalin-fixed, paraffin-embedded tissue specimens.
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.
Hamatani
Kiyohiro
K
Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima-shi, Hiroshima 732-0815, Japan. hamatani@rerf.or.jp
Eguchi
Hidetaka
H
Takahashi
Keiko
K
Koyama
Kazuaki
K
Mukai
Mayumi
M
Ito
Reiko
R
Taga
Masataka
M
Yasui
Wataru
W
Nakachi
Kei
K
D002021:Buffers; D005404:Fixatives; D005557:Formaldehyde; D011905:Genes, ras; D006361:Heating; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016612:Paraffin Embedding; D051562:Proto-Oncogene Proteins c-bcr / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013964:Thyroid Neoplasms / Q000737:chemistry; D013997:Time Factors; D014021:Tissue Preservation
D002021:Buffers; D005404:Fixatives; D005557:Formaldehyde; D012313:RNA; D051562:Proto-Oncogene Proteins c-bcr