Methods for small RNA preparation for digital gene expression profiling by next-generation sequencing.

Digital gene expression (DGE) profiling techniques are playing an eminent role in the detection, localization, and differential expression quantification of many small RNA species, including microRNAs (1-3). Procedures in small RNA library preparation techniques typically include adapter ligation by RNA ligase, followed by reverse transcription and amplification by PCR. This chapter describes three protocols that were successfully applied to generate small RNA sequencing SOLiD(TM) libraries. The Ambion SREK(TM)-adopted protocol can be readily used for multiplexing samples; the modban-based protocol is cost-efficient, but biased toward certain microRNAs; the poly(A)-based protocol is less biased, but less precise because of the A-tail that is introduced. In summary, each of these protocols has its advantages and disadvantages with respect to the ease of including barcodes, costs, and outcome.