mRNA的纯化方法对文库质量的影响

Effects of mRNA Purification Methods on Library Quality

高质量mRNA的分离和纯化是转录组文库构建和基因表达调控等生物学实验的前提。为了调取较完整、纯度较高的mRNA,本研究采用磁性分离技术调取mRNA,磁性复合微粒通过其表面的功能团与oligo(dT)_(25)相偶联,形成杂交体-磁珠复合体,外加磁场来实现mRNA的快速分离,并经过不同试剂纯化后得到高质量的mRNA,用于构建转录组文库。结果表明:mRNA被调取后,使用不同的缓冲液纯化时对mRNA质量影响较大,经多次试验验证:结合缓冲液为2.5 mol/L LiCl,洗脱缓冲液采用0.1 mol/L LiCl和1%Li DS相结合进行纯化时,检测结果显示文库条带位于400~600 bp之间,条带大小符合上机要求。高质量转录组文库的构建为高通量测序和基因克隆等实验提供帮助。

The isolation and purification of high-quality mRNA is the prerequisite for the construction of transcriptome libraries, gene expression regulation and other biological experiments. In order to obtain more complete and high purity mRNA, magnetic separation technology was used to get mRNA in this research. Magnetic composite particles conjugated with oligo（dT）_（25） through their surface functional groups and formed a hybrid-bead complex.The mRNA was rapidly separated by external magnetic field and purified by different reagents to obtain high quality mRNA for constructing transcriptome library. Results showed that different buffers had greatly influence on mRNA quality after the mRNA was taken. Through several experiments, we found that if the library was constructed when the buffer solution was 2.5 mol/L LiCl and the elution buffer was the combination of 0.1 mol/L LiCl and 1% Li DS, the band of the library was located between 400 and 600 bp, and the size of the band conformed to the requirements of the machine. Construction of high-quality transcriptome libraries could provide help for high-throughput sequencing experiment and gene cloning.