Lunaphore Technologies SA, EPFL Innovation Park Building C, CH-1015, Lausanne, Switzerland.
Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Tomtebodavägen 23a, SE 171 65, Stockholm, Sweden.
Sci Rep. 2019 Mar 5;9(1):3542. doi: 10.1038/s41598-019-40026-6.
Advancements in multiplexed in situ RNA profiling techniques have given unprecedented insight into spatial organization of tissues by enabling single-molecule quantification and sub-micron localization of dozens to thousands of RNA species simultaneously in cells and entire tissue sections. However, the lack of automation of the associated complex experimental procedures represents a potential hurdle towards their routine use in laboratories. Here, we demonstrate an approach towards automated generation and sequencing of barcoded mRNA amplicons in situ, directly in fixed cells. This is achieved through adaptation of a microfluidic tool compatible with standard microscope slides and cover glasses. The adapted tool combines a programmable reagent delivery system with temperature controller and flow cell to perform established in situ sequencing protocols, comprising hybridization and ligation of gene-specific padlock probes, rolling circle amplification of the probes yielding barcoded amplicons and identification of amplicons through barcode sequencing. By adapting assay parameters (e.g. enzyme concentration and temperature), we achieve a near-identical performance in identifying mouse beta-actin transcripts, in comparison with the conventional manual protocol. The technically adapted assay features i) higher detection efficiency, ii) shorter protocol time, iii) lower consumption of oligonucleotide reagents but slightly more enzyme. Such an automated microfluidic tissue processor for in situ sequencing studies would greatly enhance its research potentials especially for cancer diagnostics, thus paving way to rapid and effective therapies.
多重原位 RNA 分析技术的进步使我们能够对组织的空间结构进行前所未有的深入了解，能够同时对细胞和整个组织切片中的数十到数千种 RNA 进行单分子定量和亚微米级定位。然而，相关复杂实验程序缺乏自动化，这可能成为其在实验室常规应用的一个障碍。在这里，我们展示了一种在固定细胞中直接进行原位生成和测序的方法。这是通过对与标准显微镜载玻片和盖玻片兼容的微流控工具进行改造来实现的。改造后的工具结合了可编程试剂输送系统、温度控制器和流控室，以执行已建立的原位测序方案，包括基因特异性锁式探针的杂交和连接、探针的滚环扩增产生带条形码的扩增子以及通过条形码测序对扩增子进行鉴定。通过调整测定参数（例如酶浓度和温度），我们实现了与传统手动方案在鉴定小鼠β-肌动蛋白转录本方面几乎相同的性能。经过技术改造的测定具有以下特点：i）更高的检测效率；ii）更短的方案时间；iii）寡核苷酸试剂的消耗更少，但酶的用量略多。这种用于原位测序研究的自动化微流控组织处理器将极大地提高其研究潜力，特别是在癌症诊断方面，从而为快速有效的治疗铺平道路。
