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从细胞/琼脂糖构建体中提取高完整性RNA的方法。

Methods of high integrity RNA extraction from cell/agarose construct.

作者信息

Ogura Takahiro, Tsuchiya Akihiro, Minas Tom, Mizuno Shuichi

机构信息

Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA, 02115, USA.

Funabashi Orthopaedic Hospital Sports Medicine Center, 1-833, Hasamacho, Funabashi, Chiba, 274-0822, Japan.

出版信息

BMC Res Notes. 2015 Nov 4;8:644. doi: 10.1186/s13104-015-1627-5.

DOI:10.1186/s13104-015-1627-5
PMID:26537242
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4632373/
Abstract

BACKGROUND

Agarose hydrogels are widely used for three-dimensional cell scaffolding in tissue engineering and cell biology. Recently, molecular profiles have been obtained with extraction of a minimal volume of RNA using fluorescent-tagged quantitative polymerase chain reaction (qPCR), which requires high integrity RNA. However, the agarose interferes considerably with the quantity and quality of the extracted RNA. Moreover, little is known about RNA integrity when the RNA is extracted from cell/agarose construct. Thus, in order to obtain RNA of sufficient integrity, we examined various extraction methods and addressed reproducible methodologies for RNA extraction from cell/agarose constructs using spectrophotometry and microfluidic capillary electrophoresis.

RESULTS

With various extraction methods using a mono-phasic solution of phenol and guanidine isothiocyanate, we evaluated quantity and quality of total RNA from cell/agarose construct. Extraction with solution of phenol and guanidine isothiocyanate followed by a silica based membrane filter column gave sufficient RNA integrity number, which allowed us to proceed to fluorescent-tagged qPCR for evaluating various cellular activities.

CONCLUSIONS

The RNA extraction methods using phenol and guanidine isothiocyanate solution and a silica membrane column can be useful for obtaining high integrity RNA from cell/agarose constructs rich in polysaccharide and extracellular matrix. Our study contributes to further investigation using agarose hydrogels and other materials rich in polysaccharide in the field of cellular and tissue engineering.

摘要

背景

琼脂糖水凝胶广泛应用于组织工程和细胞生物学中的三维细胞支架构建。最近，通过使用荧光标记定量聚合酶链反应（qPCR）提取最小体积的RNA获得了分子谱，这需要高完整性的RNA。然而，琼脂糖对提取的RNA的数量和质量有很大干扰。此外，从细胞/琼脂糖构建体中提取RNA时，对RNA完整性了解甚少。因此，为了获得足够完整性的RNA，我们研究了各种提取方法，并使用分光光度法和微流控毛细管电泳确定了从细胞/琼脂糖构建体中提取RNA的可重复方法。

结果

使用苯酚和异硫氰酸胍的单相溶液的各种提取方法，我们评估了细胞/琼脂糖构建体中总RNA的数量和质量。用苯酚和异硫氰酸胍溶液提取，然后通过基于硅胶的膜过滤柱，得到了足够的RNA完整性数值，这使我们能够进行荧光标记qPCR来评估各种细胞活性。

结论

使用苯酚和异硫氰酸胍溶液以及硅胶膜柱的RNA提取方法可用于从富含多糖和细胞外基质的细胞/琼脂糖构建体中获得高完整性的RNA。我们的研究有助于在细胞和组织工程领域进一步研究使用琼脂糖水凝胶和其他富含多糖的材料。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/3df3a0a98c81/13104_2015_1627_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/8375a4dcb368/13104_2015_1627_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/a935e81ec9de/13104_2015_1627_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/888c1e6ab700/13104_2015_1627_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/6a4bd37e52fd/13104_2015_1627_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/4eb7d845eba7/13104_2015_1627_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/fe1f09502d37/13104_2015_1627_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/3df3a0a98c81/13104_2015_1627_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/8375a4dcb368/13104_2015_1627_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/a935e81ec9de/13104_2015_1627_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/888c1e6ab700/13104_2015_1627_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/6a4bd37e52fd/13104_2015_1627_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/4eb7d845eba7/13104_2015_1627_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/fe1f09502d37/13104_2015_1627_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8e54/4632373/3df3a0a98c81/13104_2015_1627_Fig7_HTML.jpg

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