RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells.
RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells.
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DOI：
10.1371/journal.pone.0197517
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Saiani A
中科院分区：
文献类型：
--
作者：
Burgess KA;Workman VL;Elsawy MA;Miller AF;Oceandy D;Saiani A
自组装肽水凝胶为细胞培养和组织工程中的许多应用提供了新的三维平台，但由于RNA和生物材料之间的相互作用，其与当前的RNA分离方法不兼容。本研究探讨了基于两种不同的基本提取原理的两种技术的使用：基于溶液的提取和RNA的直接固态结合，以从包封在四个β-折叠形成具有不同净正电荷的自组装肽水凝胶中的细胞中提取RNA。RNA-肽原纤维相互作用，而不是RNA-肽分子络合，被发现干扰提取过程，导致低产率。依赖于RNA特异性结合的基于柱的方法被证明更适合于从这些肽水凝胶中提取具有更高纯度的RNA，这是由于其依赖于与RNA-肽原纤维相互作用直接竞争的强特异性RNA结合相互作用。为了减少存在的原纤维的量并提高RNA产率，在RNA提取之前使用广谱酶溶液-链霉蛋白酶来部分消化水凝胶。这种预处理显示出显著增加提取的RNA的产率，允许进行下游RT-qPCR。
Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution—pronase—was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed.
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