Improved Method for Analysis of RNA Present in Long-Term Preserved Thyroid Cancer Tissue of Atomic Bomb Survivors

被引：3

作者：

Hamatani, Kiyohiro ^{[1
]}

Eguchi, Hidetaka ^{[1
,2
]}

Mukai, Mayumi ^{[1
]}

Koyama, Kazuaki ^{[1
]}

Taga, Masataka ^{[1
]}

Ito, Reiko ^{[1
]}

Hayashi, Yuzo ^{[3
]}

Nakachi, Kei ^{[1
]}

机构：

[1] Radiat Effects Res Fdn, Dept Radiobiol Mol Epidemiol, Minami Ku, Hiroshima 7320815, Japan

[2] Saitama Med Univ, Int Med Ctr, Translat Res Ctr, Saitama, Hidaka, Japan

[3] Facil Hidamari, Geriatr Hlth Serv, Hiroshima, Japan

来源：

THYROID | 2010年 / 20卷 / 01期

关键词：

CARCINOMA; REARRANGEMENT; AMPLIFICATION;

D O I：

10.1089/thy.2009.0098

中图分类号：

R5 [内科学];

学科分类号：

1002 ; 100201 ;

摘要：

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 (TM)) 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. Conclusion: 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.

引用

页码：43 / 49

页数：7