使用红外激光烧蚀从组织切片中采集 RNA。
RNA sampling from tissue sections using infrared laser ablation.
机构信息
Department of Chemistry, Louisiana State University, Baton Rouge, LA, 70803, United States.
Genomics Facility, College of Science, Louisiana State University, Baton Rouge, LA, 70803, United States.
出版信息
Anal Chim Acta. 2019 Jul 31;1063:91-98. doi: 10.1016/j.aca.2019.02.054. Epub 2019 Mar 7.
RNA was obtained from discrete locations of frozen rat brain tissue sections through infrared (IR) laser ablation using a 3-μm wavelength in transmission geometry. The ablated plume was captured in a microcentrifuge tube containing RNAse-free buffer and processed using a commercial RNA purification kit. RNA transfer efficiency and integrity were evaluated based on automated electrophoresis in microfluidic chips. Reproducible IR-laser ablation of intact RNA was demonstrated with purified RNA at laser fluences of 3-5 kJ/m (72 ± 12% transfer efficiency) and with tissue sections at a laser fluence of 13 kJ/m (79 ± 14% transfer efficiency); laser energies were attenuated ∼20% by the soda-lime glass slides used to support the samples. RNA integrity from tissue ablation was >90% of its original RIN value (∼7) and the purified RNA was sufficiently intact for conversion to cDNA and subsequent qPCR assay.
通过使用 3μm 波长的透射几何结构对冷冻大鼠脑组织切片的离散位置进行红外（IR）激光烧蚀，获得 RNA。将烧蚀的羽流收集在含有无 RNA 酶缓冲液的微量离心管中，并使用商业 RNA 纯化试剂盒进行处理。基于微流控芯片中的自动电泳评估 RNA 转移效率和完整性。用 3-5 kJ/m 的激光强度（72±12%的转移效率）对纯化的 RNA 以及用 13 kJ/m 的激光强度（79±14%的转移效率）对组织切片进行了可重复的 IR 激光烧蚀；用于支撑样品的钠钙玻璃载玻片将激光能量衰减约 20%。来自组织烧蚀的 RNA 完整性>原始 RIN 值（约 7）的 90%，并且纯化的 RNA 足够完整，可用于转换为 cDNA 并随后进行 qPCR 分析。
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