Department of Chemical Engineering , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States.
HMS Initiative for RNA Medicine, Department of Pathology, Beth Israel Deaconess Medical Center , Harvard Medical School , 330 Brookline Avenue , Boston , Massachusetts 02215 , United States.
Anal Chem. 2018 Sep 4;90(17):10279-10285. doi: 10.1021/acs.analchem.8b02010. Epub 2018 Aug 14.
MicroRNAs (miRNA) are short, noncoding RNAs that have been implicated in many diseases, including cancers. Because miRNAs are dysregulated in disease, miRNAs show promise as highly stable biomarkers. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable sample type to assay for biomolecules because it is a convenient storage method and is often used by pathologists for histological staining. However, extracting biomolecules from FFPE tissue is challenging because of the presence of cellular and extracellular proteins, formaldehyde cross-links, and paraffin. Moreover, most protocols to measure miRNA in FFPE tissue are time-consuming and laborious. Here, we report a simple protocol to directly measure miRNA from formalin-fixed cells, FFPE tissue sections after paraffin is removed, and FFPE tissue sections using encoded hydrogel microparticles fabricated using stop flow lithography. Measurements by these particles show agreement between formalin-fixed cells and fresh cells, and measurement of FFPE tissue with paraffin is 10% less than FFPE tissue when paraffin is removed before the assay. When normal and tumor FFPE tissue are compared using this microparticle assay, we observe differential miRNA signal for oncogenic miRNAs and tumor suppressing miRNAs. This approach reduces assay times, reduces the use of hazardous chemicals to remove paraffin, and provides a sensitive, quantitative, and multiplexed measurement of miRNA in FFPE tissue.
MicroRNAs (miRNA) 是短的非编码 RNA，与许多疾病有关，包括癌症。由于 miRNA 在疾病中失调，因此作为高度稳定的生物标志物具有很大的应用前景。福尔马林固定、石蜡包埋 (FFPE) 组织是一种有价值的用于检测生物分子的样本类型，因为它是一种方便的储存方法，并且经常被病理学家用于组织学染色。然而，由于存在细胞和细胞外蛋白质、甲醛交联和石蜡，从 FFPE 组织中提取生物分子具有挑战性。此外，大多数用于测量 FFPE 组织中 miRNA 的方案都很耗时且费力。在这里，我们报告了一种简单的方案，可以直接从福尔马林固定的细胞、去除石蜡的 FFPE 组织切片以及使用停流光刻技术制造的编码水凝胶微珠中测量 miRNA。这些颗粒的测量结果表明福尔马林固定细胞与新鲜细胞之间存在一致性，并且在进行测定之前去除石蜡时，FFPE 组织的测量值比含有石蜡的 FFPE 组织低 10%。当使用这种微珠测定法比较正常和肿瘤 FFPE 组织时，我们观察到致癌 miRNA 和肿瘤抑制 miRNA 的 miRNA 信号存在差异。这种方法缩短了测定时间，减少了去除石蜡时危险化学品的使用，并提供了对 FFPE 组织中 miRNA 的敏感、定量和多重测量。
