在cDNA文库构建中探究癌症病人血浆外泌体miRNA的特异性扩增

Specific amplification of plasma exosome miRNA in cancer patients for construction of cDNA library

摘要:

血浆外泌体微小核糖核酸（microRNAs，miRNAs）与癌症的发生、诊断和治疗密切相关，但其分子机制尚不明晰。本研究探讨了癌症病人血浆外泌体miRNAs在cDNA文库构建中非特异性扩增的解决方案。在酶切法中，采用核酸外切酶T （exonuclease T，EXOT）和phi29 DNA聚合酶降解引物；在磁珠法中，利用DNA结合磁珠分离模板和引物。随后，采取琼脂糖凝胶电泳和变性聚丙烯酰胺凝胶电泳检测磁珠分离情况，运用RT-qPCR检测癌症病人血浆外泌体miRNA和不同连接物的含量变化。结果显示，非特异性扩增来源于miRNA的连接物USR5SR；核酸外切酶T （EXOT）和phi29 DNA聚合酶虽可降解USR5SR，但模板链也会发生降解；磁珠分离法中以9% PEG沉淀引物片段、15% PEG沉淀模板链效果最佳。综上所述，磁珠分离法能够高效解决cDNA文库构建中的非特异性扩增，从而实现293T细胞和癌症病人血浆外泌体miRNA cDNA文库的成功构建。

Abstract:

Plasma exosome microRNAs (miRNAs) are closely related with the occurrence, diagnosis, and treatment of cancers. However, the underlying molecular mechanisms remain unclear. We herein investigated the solution for tackling the unspecific amplification of plasma exosome microRNAs from cancer patients during the construction of its cDNA library. For the restriction enzyme digesting method, the primers were degraded by exonuclease T (EXOT) and phi29 DNA polymerase. For the magnetic bead separation method, the templates and primers were separated through the DNA binding beads. The separation effects of magnetic beads were detected by agarose gel electrophoresis and modified polyacrylamide gel electrophoresis. The levels of plasma exosome miRNAs from cancer patients and various primers were assayed by RT-qPCR. The results indicated that the unspecific amplification stemmed from USR5SR. EXOT and phi29 DNA polymerase could degrade USR5SR, but the templates were also degraded simultaneously. Regarding the magnetic bead separation method, the best effect was achieved via precipitation of primer fragments by 9% PEG and precipitation of templates by 15% PEG. In conclusion, the magnetic bead separation method efficiently circumvented the unspecific amplification during the construction of cDNA library, and therefore led to the successful construction of cDNA library from plasma exosome miRNA of cancer patients and 293T cells.