The goal of this project is to develop a localized sampling approach for analysis and quantification of DNA and RNA in tissue. Mid-IR laser at a wavelength of 3 µm was used to ablate specific regions from tissue samples. Thin tissue sections are mounted on microscope slides and selected regions are irradiated in transmission geometry with an IR laser while the ablated material is captured in microcentrifuge tubes. The transfer efficiency and the molecular integrity of the captured DNA was evaluated using Sanger sequencing, gel electrophoresis and fluorimetric analysis. It was found that a 3200-bp double-stranded plasmid DNA template was reproducibly transferred with an efficiency of 59±3% at laser fluences between 10 and 20 kJ/m<sup>2</sup>. IR laser ablation was used to ablate and capture DNA from rat tissue sections and the extracted DNA was amplified by polymerase chain reaction (PCR). All samples produced PCR products of the expected size. In the case of RNA sampling, human kidney total RNA was used to demonstrate IR laser ablation sample transfer of intact biomolecules. The transfer efficiency and the molecular integrity of the captured RNA was evaluated using electrophoresis. The laser energy range in which intact RNA transfer could be achieved at high efficiency was determined. RNA from rat brain tissue sections is ablated, captured and amplified using quantitative PCR. Four primers targeting myelin basic protein gene were used to measure the RNA copy number and the quantitative results were consistent with previously reported distribution of the protein. The long-term goal of this project is to develop a comprehensive approach for localized biomolecule analysis on heterogeneous tissue.

Infrared laser ablation of tissue for localized genomics analysis

255th National Meeting (2018)

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