Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP)

Abstract

Cellular diversity and architectural complexity create barriers to understanding the function of the mammalian CNS at a molecular level. To address this problem, we have recently developed a methodology that provides the ability to profile the entire translated mRNA complement of any genetically defined cell population. This methodology, which we termed translating ribosome affinity purification, or TRAP, combines cell type-specific transgene expression with affinity purification of translating ribosomes. TRAP can be used to study the cell type-specific mRNA profiles of any genetically defined cell type, and it has been used in organisms ranging from Drosophila melanogaster to mice and human cultured cells. Unlike other methodologies that rely on microdissection, cell panning or cell sorting, the TRAP methodology bypasses the need for tissue fixation or single-cell suspensions (and the potential artifacts that these treatments introduce) and reports on mRNAs in the entire cell body. This protocol provides a step-by-step guide to implement the TRAP methodology, which takes 2 d to complete once all materials are in hand.

Figure 1. The Translating Ribosome Affinity Purification (TRAP) strategy

(A) The cell type of interest is targeted with appropriate genetic elements to express the EGFP-L10a transgene. Translating polyribosomes (polysomes) originating from non-targeted cells (grey cells, B) do not have an EGFP tag on their ribosomes, while those originating from targeted cells (green cells, C) do. Lysis of all cells releases both tagged and non-tagged polysomes. Only the latter are captured on an anti-GFP affinity matrix (D), which can be used for purification of the cell-type-specific mRNA associated with tagged polysomes.

Figure 2. Representative polyribosome profiles from fresh and frozen striatal tissue

Post-mitochondrial supernatants (prepared as described in this protocol) were prepared from freshly dissected striatal tissue (A) or tissue that had been flash-frozen and stored for four months at −80°C (B), subjected to sucrose-gradient zonal centrifugation as previously described and gradient fractions were collected while UV absorbance at 254 nm was monitored. An increase in 254 nm absorbance indicates an increase in RNA content in the fraction. Both fresh and frozen samples were prepared from young adult TRAP mice. Small ribosomal subunit (40S), large ribosomal subunit (60S), monosome (1) and polysome (2, 3, 4, 5, 6+) peaks are indicated. Red ticks indicate gradient fractions. Animal experiments were reviewed and approved by the Rockefeller University Institutional Animal Care and Use Committee.