Fig. 2

Experimental validated the stable expression of rice circRNAs a A rice circRNA (Chr9:10839626|10,840,970) exemplifies the validation strategy. According to the genomic loci, Chr9:10839626|10,840,970 derived from LOC_Os09g17730 gene. Red arrows represent divergent primers, which were designed to confirmed head-to-tail backsplicing by RT-PCR and sanger sequencing. b Divergent primers successfully amplify circRNAs in cDNA but not genomic DNA (gDNA). Convergent primers amplify circRNAs both in cDNA and gDNA. OsActin1 as a linear control. n = 3 replicates. c RT-PCR of RNase R (−) and RNase R (+) treated total RNA shows that circRNAs are stable transcripts. d qRT-PCR shows that seven circRNAs are more RNase R-resistant than linear mRNA. Data in (d) are the means ± standard deviation of three replicates. Asterisks indicate a significant difference as determined by Student’s t-test (*P < 0.05; **P < 0.01).