Table 2.
An overview of cell type-specific mRNA purification methods reviewed from the literature
| Method | Preamplification yield of total RNA | Pros | Cons | References |
|---|---|---|---|---|
| Patch/aspirate | 5–10 pg (1 cell) | Allows direct comparison between electrophysiology and gene expression at the single-cell level | Lowest preamplification yield, potentially leading to false negatives and low reproducibility | (Subkhankulova et al., 2010; Toledo-Rodriguez et al., 2004) |
| LCM | 1–10 ng (100–700 cells) | Readily applied to preserved human postmortem tissue | Tissue fixation degrades mRNA | (Chung et al., 2005; Rossner et al., 2006; Pietersen et al., 2009) |
| Manual | 0.25–1 ng (30–100 cells) | Compatible with dim, sparsely labeled cell populations | Low preamplification yield | (Sugino et al., 2006; Hempel et al., 2007; Okaty et al., 2009) |
| FACS | 10–500 ng (103-105 cells) | Highest preamplification yield | Processing samples from mature animals requires optimization to overcome stress of procedure | (Arlotta et al., 2005; Lobo et al., 2006; Cahoy et al., 2008; Marsh et al., 2008; Molyneaux et al., 2009) |
| Immunopanning | 30–50 ng (103-105 cells) | Compatible with unlabeled cells | Cell type must be distinguishable by a surface protein | (Barres et al., 1988, 1992; Cahoy et al., 2008) |
| Procedure often requires multiple iterations of plating and antibody reaction steps which may result in stress effects | ||||
| TRAP/RiboTag | >15 ng (>103 cells) | Targets actively translated transcripts | Requires specific transgenic mouse lines | (Doyle et al., 2008; Heiman et al., 2008; Sanz et al., 2009; Dougherty et al., 2010) |
| Incompatible with detection of noncoding RNAs |
Preamplification yield of total RNA indicates the reported quantity of RNA that served as input to the two round in vitro transcription amplifications used by each method. Cell numbers were either given explicitly in the references or were estimated from the amount of RNA, using 10 pg per cell as an approximation (however, in general, the quantity of total RNA in a given cell varies by cell type). Pros and Cons are by no means complete, but are provided to illustrate some of the key differences between methods, particularly as they may relate to deciding which method is best suited to a given scientific question.