Li Jinghuan, Smyth Paul, Flavin Richard, Cahill Susanne, Denning Karen, Aherne Sinead, Guenther Simone M, O'Leary John J, Sheils Orla
Department of Histopathology, University of Dublin, Trinity College, Dublin, Ireland.
BMC Biotechnol. 2007 Jun 29;7:36. doi: 10.1186/1472-6750-7-36.
Archival formalin-fixed paraffin-embedded (FFPE) tissues have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully detected in archival specimens.
Our results show that miRNA extracted from FFPE blocks was successfully amplified using Q-RT-PCR. The levels of expression of miRNA detected in total RNA extracted from FFPE were higher than that extracted from snap frozen cells when the quantity of total RNA was identical. This phenomenon is most likely explained by the fact that larger numbers of FFPE cells were required to generate equivalent quantities of total RNA than their snap frozen counterparts.
We hypothesise that methylol cross-links between RNA and protein which occur during tissue processing inhibit the yield of total RNA. However, small RNA molecules appear to be less affected by this process and are recovered more easily in the extraction process. In general miRNAs demonstrated reliable expression levels in FFPE compared with snap frozen paired samples, suggesting these molecules might prove to be robust targets amenable to detection in archival material in the molecular pathology setting.
由于在固定和处理过程中mRNA会发生降解和修饰，存档的福尔马林固定石蜡包埋（FFPE）组织在涉及基因表达分析的应用中效用有限。本研究分析了配对的速冻细胞和FFPE细胞中的160种微小RNA（miRNA），以研究是否能在存档标本中成功检测到miRNA。
我们的结果表明，使用Q-RT-PCR成功扩增了从FFPE组织块中提取的miRNA。当总RNA量相同时，从FFPE提取的总RNA中检测到的miRNA表达水平高于从速冻细胞中提取的。这种现象很可能是由于与速冻细胞相比，需要更多数量的FFPE细胞才能产生等量的总RNA。
我们推测，组织处理过程中RNA与蛋白质之间形成的羟甲基交联会抑制总RNA的产量。然而，小RNA分子似乎受此过程的影响较小，并且在提取过程中更容易回收。总体而言，与配对的速冻样本相比，miRNA在FFPE中表现出可靠的表达水平，这表明这些分子可能是在分子病理学环境中存档材料中易于检测的可靠靶点。
