Small RNA expression profiling by high-throughput sequencing: implications of enzymatic manipulation

Abstract

Eukaryotic regulatory small RNAs (sRNAs) play significant roles in many fundamental cellular processes. As such, they have emerged as useful biomarkers for diseases and cell differentiation states. sRNA-based biomarkers outperform traditional messenger RNA-based biomarkers by testing fewer targets with greater accuracy and providing earlier detection for disease states. Therefore, expression profiling of sRNAs is fundamentally important to further advance the understanding of biological processes, as well as diagnosis and treatment of diseases. High-throughput sequencing (HTS) is a powerful approach for both sRNA discovery and expression profiling. Here, we discuss the general considerations for sRNA-based HTS profiling methods from RNA preparation to sequencing library construction, with a focus on the causes of systematic error. By examining the enzymatic manipulation steps of sRNA expression profiling, this paper aims to demystify current HTS-based sRNA profiling approaches and to aid researchers in the informed design and interpretation of profiling experiments.

Figure 1

Enzymatic manipulation of RNAs with modifications at their 5′- or 3′-ends. Black lines represent RNA with the left and right ends representing the 5′- and 3′-ends, respectively. One, two, or three grey circles represent mono-, di-, or triphosphate at the 5′-end. “A” and “mG” represent a 3′ to 5′ AMP and cap structure at RNA 5′-end. “2′-OH” or “2′, 3′-OH” represents RNAs with no modification at the 3′-end. “2′-OCH₃” and “2′, 3′-CP” represent 2′-O-methylation and 2′, 3′-cyclic phosphate at the 3′-end, respectively. Dashed lines represent degraded RNA. The nucleotide “N” in grey color represents the nucleotide removed during the β-elimination reaction. MthRnl, TAP, T4 PNK, and XRN1 are the abbreviations of Methanobacterium thermoautotrophicum RNA ligase, tobacco acid pyrophosphatase, T4 polynucleotide kinase, and a 5′-monophosphate-dependent 5′ to 3′exoribonuclease, respectively.

Figure 2

Small RNA high-throughput sequencing library construction methods. The 5′- and 3′-adapters are shown as blue and red lines, respectively. sRNAs are depicted as grey lines. After sRNAs are converted into DNA, the sequences are shown as black lines. The asterisks represent steps suitable for introducing barcodes in each method. The dashed lines with arrows illustrate cDNA synthesis. At the bottom of each schematic diagram, RNA 5′-end requirement and sensitivity to 2′-O-methyl modification at the 3′-end for each method are noted.