RNA sampling from tissue sections using infrared laser ablation
RNA sampling from tissue sections using infrared laser ablation
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DOI：
10.1016/j.aca.2019.02.054
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发表时间：
2019-07-31
影响因子：
6.2
通讯作者：
Murray，Kermit K.
中科院分区：
文献类型：
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作者：
Wang，Kelin;Donnarumma，Fabrizio;Murray，Kermit K.
用3 μm波长的红外激光从冰冻的大鼠脑组织切片中分离出RNA。将烧蚀的羽流捕获在含有无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，评价RNA转移效率和完整性。用纯化的RNA在3-5 kJ/m2的激光能量密度（72 ± 12%转移效率）下和用13 kJ/m2的激光能量密度（79 ± 14%转移效率）下的组织切片证明了完整RNA的可再现IR激光消融;用于支持样品的钠钙载玻片使激光能量衰减约20%。来自组织消融的RNA完整性>其原始RIN值的90%（RIN 7），并且纯化的RNA对于转化为cDNA和随后的qPCR测定是足够完整的。
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/m2(72 ± 12% transfer efficiency) and with tissue sections at a laser fluence of 13 kJ/m2(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.