RPAD (RNase R treatment, polyadenylation, and poly(A)+ RNA depletion) method to isolate highly pure circular RNA

Abstract

Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with deep-sequencing is widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently non-polyadenylated and highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial RNase R treatment followed by Polyadenylation and poly(A)⁺ RNA Depletion. These joint interventions drastically depleted linear RNAs leading to isolation of highly pure circRNAs from total RNA pools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.

Keywords: Backsplice sequence; CircRNAs; Linear RNA depletion; Polyadenylation; RNase R; RT-qPCR.

Figure 1.. Schematic of the RPAD method.

Methodology discussed in this report. Step 1, total RNA is isolated from cultured cells.Step 2, linear RNAs are depleted by RNase R digestion. Step 3, RNA left after RNase R digestion is polyadenylated and the poly(A)+ RNA is depleted using oligo(dT) beads, yielding a pool of RNA highly enriched in circRNAs. Step 4, RNAs isolated from above steps are used in RT-qPCR for validation of linear RNA depletion.

Figure 2.. Schematic of cDNA synthesis for small RNAs.

In RPAD, the cDNA from small RNAs are prepared using the Mir-X™ miRNA First Strand Synthesis Kit. cDNA is synthesized in a single-step reaction where the RNAs are polyadenylated using poly(A) polymerase followed by the hybridization of the modified oligo-dT primer and cDNA synthesis by using reverse transcriptase. Quantitative (q)PCR analysis is performed using the specific primer pairs (Table 1).

Figure 3.. Depletion of linear RNA by RNase R digestion.

RT-qPCR analysis of subsets of linear rRNA, mRNAs and circRNAs (A), as well as snRNAs and 5S rRNA (B) in HeLa cell RNA samples left untreated or treated with RNase R. In (A) since the circRNA levels were not affected by RNase R treatment, mRNAs left after RNase R treatment were normalized to the levels of the corresponding circRNAs. Data represent the means ± SEM from 4 or more experiments.

Figure 4.. Validation of RPAD.

Subsets of rRNAs, circRNAs and mRNAs (A), as well as snRNAs and 5S rRNA (B) from HeLa cell RNAs were quantified by RT-qPCR analysis following RPAD. In (A) since circRNAs lack poly(A) tails, mRNAs left after RPAD treatment were normalized to the levels of the corresponding circRNAs. The data represent the means ± SEM from 3 or more experiments.