A high-throughput method to detect RNA profiling by integration of RT-MLPA with next generation sequencing technology.
A high-throughput method to detect RNA profiling by integration of RT-MLPA with next generation sequencing technology.
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RT-MLPA 与新一代测序技术相结合的高通量 RNA 分析检测方法
DOI：
10.18632/oncotarget.17551
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发表时间：
2017-07-11
期刊：
影响因子：
--
通讯作者：
Gao J
中科院分区：
文献类型：
--
作者：
Wang J;Yang X;Chen H;Wang X;Wang X;Fang Y;Jia Z;Gao J
福尔马林固定和石蜡包埋（FFPE）组织中的RNA提供了大量指示疾病阶段、组织学肿瘤类型和等级以及临床结果的信息。然而，由于RNA质量差，在福尔马林固定和石蜡包埋的样品中检测RNA表达水平是极其困难的。在这里，我们开发了一种高通量的方法，逆转录多重连接依赖性探针测序（RT-MLPSeq），以确定FFPE样品中多种转录本的表达水平。RT-MLPSeq结合RT-MLPSeq技术和新一代测序技术，克服了多重连接依赖性探针扩增技术中探针长度的限制，可以无定量限制地检测转录物的表达水平。我们证明了靶向相同转录物的不同RT-MLPSeq探针具有高度一致的结果，并且起始RNA/cDNA输入可以低至1 ng。RT-MLPSeq还呈现了与逆转录定量PCR一致的所选13个基因的相对RNA水平。最后，我们通过测量21个基因的mRNA表达水平来展示新的RT-MLPSeq方法的应用，这些基因可用于准确计算乳腺癌复发评分-这是一种广泛用于管理乳腺癌患者的指标。
RNA in formalin-fixed and paraffin-embedded (FFPE) tissues provides large amount of information indicating disease stages, histological tumor types and grades, as well as clinical outcomes. However, Detection of RNA expression levels in formalin-fixed and paraffin-embedded samples is extremely difficult due to poor RNA quality. Here we developed a high-throughput method, Reverse Transcription-Multiple Ligation-dependent Probe Sequencing (RT-MLPSeq), to determine expression levels of multiple transcripts in FFPE samples. By combining Reverse Transcription-Multiple Ligation-dependent Amplification method and next generation sequencing technology, RT-MLPSeq overcomes the limit of probe length in multiplex ligation-dependent probe amplification assay and thus could detect expression levels of transcripts without quantitative limitations. We proved that different RT-MLPSeq probes targeting on the same transcripts have highly consistent results and the starting RNA/cDNA input could be as little as 1 ng. RT-MLPSeq also presented consistent relative RNA levels of selected 13 genes with reverse transcription quantitative PCR. Finally, we demonstrated the application of the new RT-MLPSeq method by measuring the mRNA expression levels of 21 genes which can be used for accurate calculation of the breast cancer recurrence score – an index that has been widely used for managing breast cancer patients.
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影响因子：
2.1
作者：
Sun, Bing;Zhang, Feng;Song, San-tai
通讯作者：
Song, San-tai
影响因子：
158.5
作者：
van de Vijver, MJ;He, YD;Bernards, R
通讯作者：
Bernards, R
影响因子：
14.9
作者：
Schouten, JP;McElgunn, CJ;Pals, G
通讯作者：
Pals, G
影响因子：
6
作者：
Specht, K;Richter, T;Höfler, H
通讯作者：
Höfler, H
影响因子：
51.1
作者：
Albain, Kathy S.;Barlow, William E.;Shak, Steven;Hortobagyi, Gabriel N.;Livingston, Robert B.;Yeh, I-Tien;Ravdin, Peter;Bugarini, Roberto;Boehner, Frederick L.;Davidson, Nancy E.;Sledge, George W.;Winer, Eric P.;Hudis, Clifford;Ingle, James N.;Perez, Edith A.;Pritchard, Kathleen I.;Shepherd, Lois;Gralow, Julie R.;Yoshizawa, Carl;Allred, D. Craig;Osborne, C. Kent;Hayes, Daniel F.
通讯作者：
Hayes, Daniel F.