Table 1.

Comparison of RNA analysis techniques. In comparing the most common RNA analysis procedures with one another for use in single-cell transcriptomics, each procedure has advantages and disadvantages. The quantitation of signal with ISH is compromised by the permeability of the tissue while quantitation of qPCR is difficult because of the need to quantitate in the limited linear range of PCR amplification. Only RNA-seq does not require choice of probe for analysis and hence is the only procedure that is unbiased in the data that are generated. False-positives (calling a RNA present when it is not) arise in part from difficulty in controlling for specificity of detection methodology and only ISH can be selectively controlled so that no false-positives arise. Microarray gives rise to the most false-positives as sequence-specific hybridization hotspots are difficult to eliminate and control for. False-negatives (calling a RNA absent when it is not) arise most dramatically with ISH and qPCR, as specific sequences are needed to generate a positive signal, and if those sequences are incapable of binding to the probe (secondary structure, etc.), then no signal will be generated. False-positives and negatives for RNA-seq are negligible when performing paired-end 100 base reads but increase if doing shorter sequencing reactions (e.g. single-end 50 base reads).

	ISH	qPCR	microarray	RNA-seq
single-cell resolution	yes	yes	yes	yes
high-throughput	no	no	yes	yes
quantitative	with difficulty	with difficulty	yes	yes
unbiased	no	no	no	yes
cost	$	$$	$$$	$$$
ease of use	+++	+++	++	+
amount of data	+	++	+++	++++
false-positives	−	+	+++	+
false-negatives	+++	+++	++	+