Table 1.

Advantages and disadvantages of different RNA production methods to aid, in combination with Fig. 1, in the selection of the most suitable method

Method		Advantages	Disadvantages
Recombinant overexpression	tRNA scaffolds	Cost-effective, high yields	Cloning steps required, insert must not interfere with scaffold folding, extensive downstream purification
	Circular RNAs	Increased stability toward exonucleases	Not fully developed, extensive downstream purification
	Alternative hosts	Fast and easy purification from growth medium	Not fully developed, lower yield than Escherichia coli
	T7 in vitro transcription	Well-established method, fast, easy, and reproducible	Lower size limitation (construct >10 nt), yields and purity are construct dependent
Enzymatic methods	Ribozyme cleavage and T4 ligation	Allows segmental labeling, production of small constructs (<10 nt)	Multiple enzymatic and purification steps required, low yield
	PLOR	Allows segmental labeling, fast protocol	New technology, relies on a noncommercial robotic platform, low yield for multiple labels/modifications
	PCT	Exponential amplification of DNA template, cost-efficient, fast, and can incorporate modified nucleotides	New method, 1 reaction always produces 2 RNA fragments, construct sizes are limited (12 and 25 nt)
Chemical synthesis	Solid-phase chemical synthesis	Modifications possible, easy purification, fast, no sequence-specific optimization	Expensive equipment required, length limited to ~100 nt, limited availability and high price of labeled or modified phosphoramidites

nt nucleotides, PCT polymerase chain transcription, PLOR position-selective labeling of RNA, tRNA transfer RNA