Circular RNA qPCR Service

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  • circRNA qPCR Assay Design
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Arraystar circRNA qPCR Service performs qPCR quantification of circRNAs, commonly used as an independent measure to validate selected circRNAs from circRNA microarray profiling data. The well established and optimized circRNA qPCR workflow includes primers designed specifically for the circular junction sites to achieve highly specific and accurate detection of circular RNAs, even in the presence of linear counterparts.

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Circular RNA qPCR Service

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.

Circular RNA qPCR assay design
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
Reference gene GAPDH, unless otherwise requested
cDNA conversion Reverse transcription with random primers
Reporter SYBR Green
Assay format 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]