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使用Illumina测序平台制备mRNA 5'端文库的四种方法。

Four methods of preparing mRNA 5' end libraries using the Illumina sequencing platform.

作者信息

Machida Ryuji J, Lin Ya-Ying

机构信息

Biodiversity Research Centre, Academia Sinica, Nankang, Taipei, Taiwan.

出版信息

PLoS One. 2014 Jul 8;9(7):e101812. doi: 10.1371/journal.pone.0101812. eCollection 2014.

DOI:10.1371/journal.pone.0101812
PMID:25003736
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4086933/
Abstract

BACKGROUND

The 5' untranslated regions of mRNA play an important role in their translation.

RESULTS

Here, we describe the development of four methods of profiling mRNA 5' ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5' end of RNA Transcript) technology, while the second involves replacing the 5' cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA molecules with (nuclear transcripts) and without (mitochondrial transcripts) 5' end cap structures, respectively. Libraries prepared using SMART technology gave more reproducible results, but the ligation method was advantageous in that it only sequenced mRNAs with a cap structure at the 5' end.

CONCLUSIONS

These methods are suitable for global mapping of mRNA 5' ends, both with and without cap structures, at a single molecule resolution. In addition, comparison of the present results obtained using different methods revealed the presence of abundant messenger RNAs without a cap structure.

摘要

背景

mRNA的5'非翻译区在其翻译过程中起着重要作用。

结果

在此，我们描述了使用Illumina测序平台分析mRNA 5'末端的四种方法的开发；第一种方法利用SMART（RNA转录本5'末端的切换机制）技术，而第二种方法涉及通过连接用RNA寡聚物替换5'帽结构。第三种和第四种方法是对SMART的改进，分别涉及富集具有（核转录本）和不具有（线粒体转录本）5'末端帽结构的mRNA分子。使用SMART技术制备的文库给出了更可重复的结果，但连接方法的优势在于它只对5'末端具有帽结构的mRNA进行测序。

结论

这些方法适用于在单分子分辨率下对具有和不具有帽结构的mRNA 5'末端进行全局定位。此外，对使用不同方法获得的当前结果的比较揭示了存在大量无帽结构的信使RNA。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/68b53b2e7c2c/pone.0101812.g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/4858e39dd3f5/pone.0101812.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/1814e4ad84f1/pone.0101812.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/177a8e390a2e/pone.0101812.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/678abaedd046/pone.0101812.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/d4d2c75696e1/pone.0101812.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/703d88da17a5/pone.0101812.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/5954e5b9701a/pone.0101812.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/5aa73d2170ec/pone.0101812.g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/68b53b2e7c2c/pone.0101812.g009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/4858e39dd3f5/pone.0101812.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/1814e4ad84f1/pone.0101812.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/177a8e390a2e/pone.0101812.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/678abaedd046/pone.0101812.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/d4d2c75696e1/pone.0101812.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/703d88da17a5/pone.0101812.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/5954e5b9701a/pone.0101812.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/5aa73d2170ec/pone.0101812.g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8247/4086933/68b53b2e7c2c/pone.0101812.g009.jpg

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