Identification of internal control genes for circular RNAs
At present, no studies have established internal control genes for circular RNA (circRNA) analyses. We aimed to identify reference circRNAs for real-time quantitative PCR (RT-qPCR).
After analyzing the RNA-seq data, we obtained 50 circRNAs that were expressed in all samples. We ranked these 50 circRNAs according to their stability and obtained the six most stable circRNAs. We further evaluated the stability of the six circRNAs and three linear control genes (i.e., GAPDH, β-actin and 18S rRNA) in 22 cell lines. Our results indicated that hsa_circ_0000284 (circHIPK3) and hsa_circ_0000471 (circN4BP2L2) were the two most stable genes. After removing linear RNAs or including the cells treated with Adriamycin, NH4Cl and shikonin, the two most stable genes were hsa_circ_0000471 and hsa_circ_0000284. The amplification efficiency was 100% for hsa_circ_0000471 and 95% for hsa_circ_0000284.
In conclusion, since the stability of circRNAs is higher than that of linear RNAs, hsa_circ_0000284 and hsa_circ_0000471 may be used as reference genes not only for circRNAs but also for other kinds of RNAs. The findings in the present study fill the gap of lacking reference genes in the detection of circRNAs.
KeywordsCircular RNAs circRNAs circHIPK3 circN4BP2L2 Normalization genes Control genes
This study was funded by the National Natural Science Foundation of China (Grant Number 81602551 and 81702895) and the Young Talents Program of Jiangsu Cancer Hospital (Grant Number 2017YQL-10).
Supplementary Fig. S1—Specificity of the primers. (a) Specificity of the primers for GAPDH and β-actin was checked using circPrimer. (b) The melting curve analysis of hsa_circ_0000471 and hsa_circ_0000284. (c) The amplification specificity of the candidate internal control genes was determined using electrophoresis on an agarose gel. M, marker; 1, hsa_circ_0000471; 2, hsa_circ_0000284; 3, hsa_circ_0002484; 4, hsa_circ_0001445; 5, hsa_circ_0000944; 6, hsa_circ_0000567; 7, GAPDH; 8, 18S rRNA; 9, β-actin.
Supplementary Table S1—The 50 circRNAs expressed in all the specimens.
Supplementary Table S2—Cycle threshold values of the candidate genes in the cell lines with or without treatment of Rnase R.
Supplementary Table S3—The stability of candidate genes was assessed using 22 cell lines and cell lines treasted with different drugs*.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest.
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