RNase R



RNase R (Ribonuclease R) is a 3 '-5' exonuclease derived from the Escherichia coli RNR superfamily that progressively cuts RNA into dinucleotides and trinucleotides from the 3 '-5' direction. RNase R can digest almost all linear RNA molecules, but it is difficult to digest circular RNA, lasso structures, or double-stranded RNA molecules with fewer than seven nucleotides at the 3' protruding end.

RNase R is an essential tool enzyme for circRNA identification and enrichment experiments, digesting linear RNA to enrich for circular RNAs (circRNAs) or lasso-structured RNAs (lariat RNAs).

Fig. 1: Diagram of RNase R digesting RNA

Application

In Fig. 2, detection of mRNA with a band in RNase R(-) and no band in RNase R(+); detection of Lariat/Circular RNA with a band in both RNase R(-) and RNase R(+) samples suggests that the mRNA is digested, whereas the Lariat/Circular RNA is resistant to digestion (Suzuki H et al., 2006).

2. circRNA enrichment

Fig. 3: Schematic of RNase R(+) and RNase R(-) RNA sequencing (Jeck WR et al., 2014)

Some studies reported that sequencing showed a 5- to 10-fold enrichment of Junction Reads in the RNase R(+) group relative to the RNase R(-) samples, which allowed the identification of thousands to tens of thousands of circRNAs (Fig. 3).

3.circRNA purification

Elimination of interference from linear RNA residues is the key to the artificial synthesis of high-purity circRNA. Efficient RNase R is not only a key tool for the ligase method of circRNA synthesis, but also essential in the intron self-splicing circRNA synthesis.

Product Advantages

High specificity: specific digestion  of linear RNA;

Highly efficient: most linear RNA can be digested in 5-15min;

Buffer compatible: the digested product can be directly used in downstream experiments;

Easy to use: simple system, one-step reaction at 37℃.

Quality Control

The purity test of SDS-PAGE >95%; Total RNA digested by RNase R and then detected by RT-qPCR, the abundance of linear RNA was significantly reduced, while the abundance of circular RNA was basically unchanged.

Performance comparison

1.RNA electrophoresis detection

Add 10U RNase R to 2.5 μg Total RNA, incubate at 37°C for 15 min and then directly carry out electrophoretic detection, the results showed that the bands of RNase R(+) group became fainter (not visible), which indicated that the RNase R exerted a digestive effect on Total RNA. The effects of RNase R on Total RNA digestion by Geneseed and Company A or Company E were comparable.

2.RT-qPCR test

Add 10U RNase R to 2.5 μg total RNA, incubate at 37°C for 30 min and then perform RT-qPCR experiments, the results showed that the abundance of both β-actin and FGFR2 was significantly reduced in the RNase R+ group, which indicated that the RNase R could digest linear RNA. The RNase R of Geneseed and Company E had comparable effects on linear RNA digestion and were superior to the RNase R of Company A.

Add 10U RNase R to 2.5 μg total RNA, incubate at 37°C for 30 min and then perform RT-qPCR experiments, the results showed that the abundance of hsa_circFOXO3_002 and hsa_circMTO1_001 in the RNase R+ group was basically unchanged, indicating that circRNAs were resistant to RNase R digestion. The effects of RNase R were comparable between Geneseed and Company E or Company A.

Note: The data were calculated using the "RNase R+Geneseed" group as a control, and its relative abundance value was set to 1.

  Product Specification