RNA sampling from tissue sections using infrared laser ablation.

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 ±â€¯12% transfer efficiency) and with tissue sections at a laser fluence of 13 kJ/m2 (79 ±â€¯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.

Infrared laser ablation sample transfer of tissue DNA for genomic analysis.

Infrared (IR) laser ablation was used to remove material from tissue sections mounted on microscope slides, with subsequent capture in a solvent-containing microcentrifuge tube. Experiments conducted with a 3200-bp double-stranded plasmid DNA template demonstrated IR-laser ablation transfer of intact DNA. The transfer efficiency and the molecular integrity of the captured DNA were evaluated using Sanger sequencing, gel electrophoresis, and fluorimetric analysis. The plasmid DNA was reproducibly transferred with an efficiency of 59 ± 3% at laser fluences of between 10 and 20 kJ/m2 at a wavelength of 3 µm. IR laser ablation sample transfer was then used to ablate and capture DNA from 50-µm-thick rat brain and kidney tissue sections. DNA was extracted from the captured material using five commercial DNA extraction kits that employed significantly divergent methodologies, with all kits recovering sufficient DNA for successful amplification by polymerase chain reaction (PCR). Four sets of primers were employed, targeting one region of the CYP 11b2 gene (376 bp) and three different regions of the Snn1g gene (298, 168, and 281 bp). The PCR results were not consistently reliable when using unpurified ablation samples; however, after extraction, all samples produced PCR products of the expected size. This work expands the sampling capabilities of IR laser ablation, demonstrating that DNA can be isolated from tissue samples for genomic assays. Due to the small size of the ablation regions (1 mm2), this technique will be useful for sampling discrete cell populations from tissue sections. Graphical abstract Infrared laser ablation transfer of intact DNA from a tissue section.