Circular RNAs do not have a poly(A) tail. Random primers, instead of oligo(dT), are used in the first cDNA strand synthesis. To distinguish circular RNA from the linear counterpart in qPCR, the forward and reverse primers are situated in the two separate exons that are joined together at the circular junction site to form circular RNA (e.g. exon 4 and exon 2 in Fig. 1), such that the amplicon spans the circular junction. The directions of the primers face toward each other for the circular RNA sequence but away from each other with the linear RNA, also referred to as “outward facing” or “divergent” primers [1-2]. The linear housekeeping genes, such as GAPDH, ACTB, or B2M, can continue to be used normally as the qPCR normalization reference. The differential expression of circular RNA can be calculated by the “??Ct” method.
Optionally, circular RNA enrichment and circular/linear RNA ratio change, with and without RNase R treatment, can be compared in the parallel qPCR reactions using the primers for the circular RNA only and primers for both linear and circular RNA.
Figure 1. qPCR validation of differentially expressed circRNAs. The concordance between qPCR and microarray for the differentially expressed circRNA is related to the magnitude of change (FC), p-value, as well as the abundance level.
|RNA sample source
||Total RNA, the same prep as used in the microarray profiling for best concordance. |
|Total RNA amount
||1.5 ug |
|Sample RNA quality
||OD260/280 ratio ~ 2 OD260/230 ratio ~ 2 |
||GAPDH, unless otherwise requested |
||Reverse transcription with random primers |
||SYBR Green |
||384-well plate |
1. Salzman et al. "Cell-type specific features of circular RNA expression", PLOS Genet.(2013) [PMID: 24039610]
2. Jeck et al. "Circular RNAs are abundant, conserved, and associated with ALU repeats", RNA (2013) [PMID: 23249747]