Obtaining high quality RNA from complex biological tissues, such as the brain, is needed for establishing high-fidelity cell-type specific transcriptomes. Although combining genetic labeling techniques with laser capture microdissection (LCM) is generally sufficient, concerns over RNA degradation and limited yields call into question results of many sequencing studies.
Researchers from the National Institute of Environmental Health Sciences set out to address both of these issues by: (1) developing a fluorescence-assisted LCM protocol that yields high quality RNA from fresh-frozen tissues; and (2) determining a suitable RNA-Seq library generation method for limited amounts of RNA (1-5 ng total RNA). The latter focused on comparing commercially available kits able to produce libraries of sufficient concentration and complexity while limiting PCR amplification biases. They found that high quality RNA (RNA integrity number, RIN, >9) of sufficient concentration can be isolated from laser-captured material from thinly-sectioned tissues when digestion time and temperature are minimized. Furthermore, they found that library generation approaches that retain ribosomal RNA (rRNA) through cDNA library generation required fewer cycles of PCR, minimizing bias in the resulting libraries. Lastly, end stage depletion of rRNA prior to sequencing enriches for target RNAs, thereby increasing read depth and level of gene detection while decreasing sequencing costs.
(A) Schematic of steps that were optimized in the current study (LCM instrument, RNA extraction and RNA-Seq library kit). (B) Representative images of (left) nissl stained mouse hippocampus from Allen Brain Atlas, (middle) Amigo2-EGFP mouse hippocampus showing the fluorescently labeled CA2 cells and projections prior to LCM and (right) post LCM. (C) Representative image of a glass slide with five dehydrated mouse brain sections post-LCM of hippocampal subregions CA1 and CA2. On the left is a magnified image of the dashed box. Scale bars: 500 μm (B,C left) and 4 mm (C right). CA, cornus ammonis; DG, dentate gyrus; LCM, laser capture microdissection; RIN, RNA Integrity Number.