Improved method for analysis of RNA present in long-term preserved thyroid cancer tissue of atomic bomb survivors.
BACKGROUND
Since many thyroid cancer tissue samples from atomic bomb (A-bomb) survivors have been preserved for several decades as unbuffered formalin-fixed, paraffin-embedded specimens, molecular oncological analysis of such archival specimens is indispensable for clarifying the mechanisms of thyroid carcinogenesis in A-bomb survivors. Although RET gene rearrangements are the most important targets, it is a difficult task to examine all of the 13 known types of RET gene rearrangements with the use of the limited quantity of RNA that has been extracted from invaluable paraffin-embedded tissue specimens of A-bomb survivors. In this study, we established an improved 5' rapid amplification of cDNA ends (RACE) method using a small amount of RNA extracted from archival thyroid cancer tissue specimens.


METHODS
Three archival thyroid cancer tissue specimens from three different patients were used as in-house controls to determine the conditions for an improved switching mechanism at 5' end of RNA transcript (SMART) RACE method; one tissue specimen with RET/PTC1 rearrangement and one with RET/PTC3 rearrangement were used as positive samples. One other specimen, used as a negative sample, revealed no detectable expression of the RET gene tyrosine kinase domain.


RESULTS
We established a 5' RACE method using an amount of RNA as small as 10 ng extracted from long-term preserved, unbuffered formalin-fixed, paraffin-embedded thyroid cancer tissue by application of SMART technology. This improved SMART RACE method not only identified common RET gene rearrangements, but also isolated a clone containing a 93-bp insert of rare RTE/PTC8 in RNA extracted from formalin-fixed, paraffin-embedded thyroid cancer specimens from one A-bomb survivor who had been exposed to a high radiation dose. In addition, in the papillary thyroid cancer of another high-dose A-bomb survivor, this method detected one novel type of RET gene rearrangement whose partner gene is acyl coenzyme A binding domain 5, located on chromosome 10p.


CONCLUSIONS
We conclude that our improved SMART RACE method is expected to prove useful in molecular analyses using archival formalin-fixed, paraffin-embedded tissue samples of limited quantity.
Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan. hamatani@rerf.or.jp
Kiyohiro
K
Hamatani
Hidetaka
H
Eguchi
Mayumi
M
Mukai
Kazuaki
K
Koyama
Masataka
M
Taga
Reiko
R
Ito
Yuzo
Y
Hayashi
Kei
K
Nakachi
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D002291:Carcinoma, Papillary / Q000235:genetics*; D020134:Catalytic Domain / Q000235:genetics; D018076:DNA, Complementary; D015972:Gene Expression Regulation, Neoplastic; D015321:Gene Rearrangement / Q000528:radiation effects; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007564:Japan; D008565:Membrane Proteins; D008832:Microchemistry / Q000379:methods; D054043:Nuclear Weapons; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D051562:Proto-Oncogene Proteins c-bcr / Q000235:genetics / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis* / Q000302:isolation & purification; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*; D017741:Survivors; D013964:Thyroid Neoplasms / Q000235:genetics*; D013997:Time Factors; D014015:Tissue Banks
C548478:ACBD5 protein, human; D018528:ATP-Binding Cassette Transporters; D048868:Adaptor Proteins, Signal Transducing; D018076:DNA, Complementary; D007527:Isoenzymes; D008565:Membrane Proteins; D015514:Oncogene Proteins, Fusion; D012334:RNA, Neoplasm; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D051562:Proto-Oncogene Proteins c-bcr