Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan.
Thyroid. 2010 Jan;20(1):43-9. doi: 10.1089/thy.2009.0098.
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.
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.
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.
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.
由于许多来自原子弹（A-bomb）幸存者的甲状腺癌组织样本已经保存了几十年，作为未缓冲的福尔马林固定、石蜡包埋标本，因此对这些存档标本进行分子肿瘤学分析对于阐明 A-bomb 幸存者中甲状腺癌发生的机制是必不可少的。虽然 RET 基因重排是最重要的靶标，但使用从 A-bomb 幸存者宝贵的石蜡包埋组织标本中提取的有限量 RNA 来检查所有 13 种已知类型的 RET 基因重排是一项艰巨的任务。在这项研究中，我们使用从小量 RNA 中提取的少量 RNA ，建立了一种改良的 5' 快速扩增 cDNA 末端（RACE）方法，用于从存档的甲状腺癌组织标本中建立了一种改良的 RNA 转录物 5' 端切换机制（SMART）RACE 方法；一个组织标本带有 RET/PTC1 重排，一个带有 RET/PTC3 重排，作为阳性样本。另一个标本作为阴性样本，未检测到 RET 基因酪氨酸激酶结构域的可检测表达。
使用来自三个不同患者的三个存档甲状腺癌组织标本作为内部对照，以确定改良的 SMART RACE 方法 5' 端切换机制的条件；一个组织标本带有 RET/PTC1 重排，一个带有 RET/PTC3 重排，作为阳性样本。另一个标本作为阴性样本，未检测到 RET 基因酪氨酸激酶结构域的可检测表达。
我们通过应用 SMART 技术，从小到 10ng 的长期保存的、未缓冲的福尔马林固定、石蜡包埋的甲状腺癌组织中提取的 RNA 建立了 5' RACE 方法。这种改良的 SMART RACE 方法不仅鉴定了常见的 RET 基因重排，还从一个暴露于高辐射剂量的 A-bomb 幸存者的福尔马林固定、石蜡包埋的甲状腺癌标本中分离出一个包含罕见 RTE/PTC8 的 93bp 插入物的克隆。此外，在另一位高剂量 A-bomb 幸存者的乳头状甲状腺癌中，该方法检测到一种新的 RET 基因重排，其伙伴基因是位于 10p 上的酰基辅酶 A 结合域 5。
我们得出结论，我们改良的 SMART RACE 方法有望证明在使用有限数量的存档福尔马林固定、石蜡包埋组织样本进行分子分析时非常有用。
