Directionality of noncoding human RNAs: how to avoid artifacts.
Directionality of noncoding human RNAs: how to avoid artifacts.
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非编码人类 RNA 的方向性：如何避免伪影。
DOI：
10.1016/j.ab.2013.03.031
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发表时间：
2013
影响因子：
2.9
通讯作者：
I. Dotan
中科院分区：
文献类型：
--
作者：
Sivan Tzadok;Yarden Caspin;Y. Hachmo;D. Canaani;I. Dotan
通过表观遗传沉默使肿瘤抑制基因和转移抑制基因失活是人类癌症中常见的事件。最近的研究显示了表观遗传沉默的新机制，基于长非编码启动子跨越反义和/或义rna （lncRNAs）的发生，它们构成了染色质沉默复合物的一部分。利用逆转录聚合酶链反应（RT-PCR），我们已经开始扫描“三阴性”和her2过表达的乳腺癌细胞系，通过已知在体内表观遗传沉默的肿瘤抑制基因和转移抑制基因的启动子进行定向/双向转录。令人惊讶的是，我们发现在没有外源引物的情况下，rt - pcr扩增产物的频率很高。这些扩增产物是通过启动子或上游跨越区域的转录物进行RT引物而产生的。因此，这种启动推翻了方向性的确定，并导致错误的检测-鉴定这些lncrna。我们表明，这种普遍的“无引物”伪影可以通过用高碘酸盐处理RNA制剂，在高温下进行RT反应，或两者结合来消除。这些实验改进能够确定单个跨启动子的长链非编码rna的存在和方向性。BRMS1转移抑制基因，以及RAR-β2和CST6人类肿瘤抑制基因，在乳腺癌细胞系的例子。
Inactivation of tumor suppressor and metastasis suppressor genes via epigenetic silencing is a frequent event in human cancers. Recent work has shown new mechanisms of epigenetic silencing, based on the occurrence of long noncoding promoter-spanning antisense and/or sense RNAs (lncRNAs), which constitute part of chromatin silencing complexes. Using reverse transcription polymerase chain reaction (RT–PCR), we have started to scan “triple negative” and Her2-overexpressing breast cancer cell lines for directional/bidirectional transcription through promoters of tumor suppressor and metastasis suppressor genes known to be epigenetically silenced in vivo. Surprisingly, we found that RT–PCR-amplified products were obtained at high frequency in the absence of exogenous primers. These amplified products resulted from RT priming via transcripts originating from promoter or upstream spanning regions. Consequently, this priming overruled directionality determination and led to false detection–identification of such lncRNAs. We show that this prevalent “no primer” artifact can be eliminated by treating the RNA preparations with periodate, performing RT reactions at highly elevated temperatures, or a combination of both. These experimental improvements enabled determination of the presence and directionality of individual promoter-spanning long noncoding RNAs with certainty. Examples for the BRMS1 metastasis suppressor gene, as well as RAR-β2 and CST6 human tumor suppressor genes, in breast carcinoma cell lines are presented.
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非编码 RNA 对人类细胞基因表达的表观遗传调控。
DOI：
10.1016/b978-0-12-415795-8.00003-9
发表时间：
2011
影响因子：
--
作者：
Knowling,Stuart;Morris,KevinV
通讯作者：
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作者：
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通讯作者：
Sidransky, David
影响因子：
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作者：
Wang KC;Chang HY
通讯作者：
Chang HY
影响因子：
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作者：
Janowski, Bethany A.;Younger, Scott T.;Corey, David R.
通讯作者：
Corey, David R.