期刊
EXPERIMENTAL AND MOLECULAR PATHOLOGY
卷 110, 期 -, 页码 -出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.yexmp.2019.104289
关键词
RNA extraction; Proteinase K; Formalin fixation; Paraffin embedding; Molecular analysis
类别
资金
- Brazilian Coordination of Superior Level Staff Improvement (CAPES)/National Council for Scientific and Technological Development (CNPq)
- German Academic Exchange Service (DAAD) [490235/2013-1]
- German Federal Ministry of Education and Research (BMBF) - Nationales Forschungsnetz zoonotische Infektionskrankheiten - ZooBoCo [01K11722E]
A suitable RNA extraction protocol was established to gain high quality RNA from formalin-fixed paraffin embedded tissues to perform reliable molecular assays either applicable for using FFPE tissue archives or tissues with harsh formalin-fixation. Eighteen FFPE samples from the central nervous system of horses, stored up to 11 years, were used as archive cases. To test the influence of the fixation period, brain, liver, kidney, and skeletal muscle tissue fragments from another horse, were treated either with water or tris-acetate-EDTA buffer after fixation under different timepoints with 10% unbuffered formalin. Two deparaffinization methods and three proteinase K-based lysis step were tested and translated into three protocols. After detailed statistical analysis it was determined that a longer period and increase in volume of proteinase K incubation provide higher yields and purity of RNA (P < 0.01) of archived samples. Alongside, amplification of equid-housekeeping gene up to 298 bp was successful with the protocol adaptations. For different formalin-fixation timepoints, it was demonstrated that the right choice for treatment and formalin-fixation period is organ-related (P <= 0.05). Essentially, little alterations to pre-existing extraction protocols unwound the RNA of up to 11-year-old samples, enabling the use of FFPE tissue archives or e.g. harshly fixed material needed in infection research under high biosafety levels for a variety of molecular analysis.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据