RNA sampling from tissue sections using infrared laser ablation

Highlights: • Localized RNA microsampling from tissue using IR laser ablation capture. • RNA sampling efficiency 75%. • RNA integrity >90% after laser ablation. • Captured RNA used for cDNA synthesis and mRNA quantification. -- Abstract: 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.