Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays.
Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays.
复制标题
链特异性的转录组分析，在基因组平铺微阵列上直接标记RNA。
DOI：
10.1186/1471-2199-12-3
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发表时间：
2011-01-14
影响因子：
--
通讯作者：
Chen T
中科院分区：
文献类型：
--
作者：
Yu WH;Høvik H;Olsen I;Chen T
基因组平铺微阵列具有较低的制造成本、高点密度和灵活的探针设计，是综合转录组研究的理想选择。通常，使用微阵列进行转录组分析涉及逆转录，将 RNA 转化为 cDNA。然后 cDNA 被标记并与阵列上的探针杂交，从而间接检测 RNA 信号。众所周知，逆转录会产生人工 cDNA，特别是第二链 cDNA 的合成，从而导致反义 RNA 的错误发现。为了解决这个问题，我们开发了一种有效的方法，使用直接标记的 RNA，从而绕过 cDNA 的生成。本文描述了该方法及其在转录组图谱映射中的应用。从牙龈卟啉单胞菌实验室培养物中提取的 RNA 用烷基化试剂进行荧光标记，并直接与专门为这种牙周病原体设计的基因组平铺微阵列上的探针杂交。生成的转录组谱是链特异性的，并且在基因组的大多数反义区域中产生接近背景水平的信号。相反，当用 cDNA 进行杂交时，在反义区域检测到高水平的信号。用独立链特异性 RT-PCR 测试了五个反义区域，没有检测到可忽略的扩增，表明强反义 cDNA 信号是实验假象。开发了一种有效的方法来绘制细菌基因组两条编码链特异的转录组图谱。该方法对提取的 RNA 进行化学标记并直接用于微阵列杂交。生成的转录组图谱不含 cDNA 人为信号。此外，与传统的末端标记方法相比，该方法需要更少的处理步骤，并且由于每个 RNA 片段掺入了更多的荧光分子，因此在检测少量 RNA 时可能更灵敏。
With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment.
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