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Comparative Study
. 2015 Jul;48(7):388-94.
doi: 10.5483/bmbrep.2015.48.7.218.

Cell type-specific gene expression profiling in brain tissue: comparison between TRAP, LCM and RNA-seq

TaeHyun Kim  1 , Chae-Seok Lim  1 , Bong-Kiun Kaang  1
Affiliations
Comparative Study

Cell type-specific gene expression profiling in brain tissue: comparison between TRAP, LCM and RNA-seq

TaeHyun Kim et al. BMB Rep. 2015 Jul.

Abstract

The brain is an organ that consists of various cell types. As our knowledge of the structure and function of the brain progresses, cell type-specific research is gaining importance. Together with advances in sequencing technology and bioinformatics, cell type-specific transcriptome studies are providing important insights into brain cell function. In this review, we discuss 3 different cell type-specific transcriptome analyses i.e., Laser Capture Microdissection (LCM), Translating Ribosome Affinity Purification (TRAP)/RiboTag, and single cell RNA-Seq, that are widely used in the field of neuroscience.

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Figures

Fig. 1.
Fig. 1.. Sampling transcripts from target cells using LCM or TRAP/ RiboTag (A) Sampling targeted cell population with laser capture microdissection (LCM). A transparent polymer membrane is attached to the brain slice prepared on a slide glass. When the desired cell’s border is defined using the software linked to the LCM machine, a UV laser is emitted onto the target region. The UV laser cuts the target cell out from the tissue slice. The target cell population is pulled from the tissue slice and then transcriptome of the specific cell population can be determined. (B) Sampling polysome- bound mRNAs from target cell type with antigen-tagged ribosome. Antigen (HA or GFP)-tagged ribosomal subunit is expressed in the desired cell type using a cell type-specific promoter or Cre recombinase-driven gene expression. After lysing the tissue sample, mRNAs bound to antigen-tagged ribosomes are pulled down using antigen-binding antibodies that are bound to beads. mRNAs are eluted from the beads and then analyzed further.
Fig. 2.
Fig. 2.. Single-cell transcriptome analysis. (A) Gathering single cell transcriptome via patch pipette aspiration. A target cell is selected from a tissue slice or neuron culture and patch pipette is attached to the cell membrane. Electrophysiological properties can be measured from the whole-cell preparation. Cell soma components can be acquired by aspiration, and then the transcriptome can be purified from the acquired cell. (B) Capturing target RNAs with activated TIVA-tag. When the tissue is incubated with solution containing TIVA-tag, TIVA-tag can penetrate into the cell by the attached cell penetrating peptide (CPP). UV laser is emitted on desired cell population. The UV laser cleaves the photocleavable linker connecting the masking RNA with the tag, and masking RNA is released from the probe RNA segment, thus the tag is ‘activated’. RNAs in the cell containing complementary sequence with the probe is captured by the probe, and the tag is immunoprecipitated with streptavidin beads. Tag-bound RNAs can be eluted and then further analyzed.
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