Gould Barbara R, Damgaard Tina, Nielsen Boye Schnack
Exiqon Inc., 12 Gill St. Suite 1650, Woburn, MA, 01801, USA.
Exiqon A/S, Vedbæk, Denmark.
Methods Mol Biol. 2017;1641:399-412. doi: 10.1007/978-1-4939-7172-5_22.
Disease research and treatment development have turned to the impact and utility of microRNA. The dynamic and highly specific expression of these molecular regulators can be used to predict and monitor disease progression as well as therapeutic treatment efficacy and safety, thus aiding decisions in patient care. In situ hybridization (ISH) of biopsy material has become a routine clinical pathology procedure for monitoring gene structure, expression, and sample characterization. For ribonucleic acid (RNA), determining cell source and level of expression of these biomarkers gives insight into the cellular function and physiopathology. Identification and monitoring of microRNA biomarkers are made possible through locked nucleic acid (LNA)™-based detection probes. LNA™ enhances the sensitivity and specificity of target binding, most profoundly so for the short, highly similar, microRNA sequences. We present a robust 1-day ISH protocol for formalin-fixed, paraffin-embedded (FFPE) tissue sections based on microRNA-specific LNA™ detection probes which can be labeled with digoxigenin (DIG) or 6-carboxyfluorescein (FAM) and detected through enzyme-linked specific antibodies that catalyze substrates into deposited chromogen products at the target RNA site. The variety of haptens and detection reagents in combination with LNA™ chemistry offer flexibility and ease to multiple target assessment of therapeutic response.
疾病研究与治疗发展已转向微小RNA的影响及效用。这些分子调节因子动态且高度特异性的表达可用于预测和监测疾病进展以及治疗效果与安全性，从而辅助临床护理决策。活检材料的原位杂交（ISH）已成为监测基因结构、表达及样本特征的常规临床病理程序。对于核糖核酸（RNA）而言，确定这些生物标志物的细胞来源及表达水平有助于深入了解细胞功能和生理病理学。基于锁核酸（LNA）™的检测探针使得微小RNA生物标志物的识别与监测成为可能。LNA™增强了靶标结合的灵敏度和特异性，对于短的、高度相似的微小RNA序列影响尤为显著。我们基于微小RNA特异性LNA™检测探针，针对福尔马林固定、石蜡包埋（FFPE）组织切片，提出了一种稳健的1天ISH方案，该探针可用地高辛（DIG）或6-羧基荧光素（FAM）标记，并通过酶联特异性抗体进行检测，这些抗体可催化底物在靶RNA位点形成沉积的显色产物。多种半抗原和检测试剂与LNA™化学相结合，为治疗反应的多靶点评估提供了灵活性和便利性。
