Biotechnology Letters

, Volume 41, Issue 10, pp 1111–1119 | Cite as

Identification of internal control genes for circular RNAs

  • Shanliang Zhong
  • Siying Zhou
  • Sujin Yang
  • Xinnian Yu
  • Hanzi Xu
  • Jinyan Wang
  • Qian Zhang
  • Mengmeng Lv
  • Jifeng FengEmail author
Original Research Paper



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.


Circular 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).

Supporting information

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.

Supplementary material

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  1. Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250CrossRefGoogle Scholar
  2. Ashwal-Fluss R, Meyer M, Pamudurti NR, Ivanov A, Bartok O, Hanan M, Evantal N, Memczak S, Rajewsky N, Kadener S (2014) circRNA biogenesis competes with pre-mRNA splicing. Mol Cell 56:55–66CrossRefGoogle Scholar
  3. Chen L-L (2016) The biogenesis and emerging roles of circular RNAs. Nat Rev Mol Cell Biol 17:205CrossRefGoogle Scholar
  4. Chuang TJ, Chen YJ, Chen CY, Mai TL, Wang YD, Yeh CS, Yang MY, Hsiao YT, Chang TH, Kuo TC, Cho HH, Shen CN, Kuo HC, Lu MY, Chen YH, Hsieh SC, Chiang TW (2018) Integrative transcriptome sequencing reveals extensive alternative trans-splicing and cis-backsplicing in human cells. Nucleic Acids Res 46:3671–3691CrossRefGoogle Scholar
  5. Du WW, Yang W, Liu E, Yang Z, Dhaliwal P, Yang BB (2016) Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Res 44:2846–2858CrossRefGoogle Scholar
  6. Gao Y, Wang J, Zhao F (2015) CIRI: an efficient and unbiased algorithm for de novo circular RNA identification. Genome Biol 16:4CrossRefGoogle Scholar
  7. Glazar P, Papavasileiou P, Rajewsky N (2014) circBase: a database for circular RNAs. RNA 20:1666–1670CrossRefGoogle Scholar
  8. Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Finsen B, Damgaard CK, Kjems J (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384–388CrossRefGoogle Scholar
  9. Hansen TB, Veno MT, Damgaard CK, Kjems J (2016) Comparison of circular RNA prediction tools. Nucleic Acids Res 44:e58CrossRefGoogle Scholar
  10. Hoffmann S, Otto C, Doose G, Tanzer A, Langenberger D, Christ S, Kunz M, Holdt LM, Teupser D, Hackermuller J, Stadler PF (2014) A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection. Genome Biol 15:R34CrossRefGoogle Scholar
  11. Jeck WR, Sharpless NE (2014) Detecting and characterizing circular RNAs. Nat Biotechnol 32:453–461CrossRefGoogle Scholar
  12. Kong Q, Yuan J, Gao L, Zhao S, Jiang W, Huang Y, Bie Z (2014) Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon. PLoS ONE 9:e90612CrossRefGoogle Scholar
  13. Lasda E, Parker R (2016) Circular RNAs co-precipitate with extracellular vesicles: a possible mechanism for circrna clearance. PLoS ONE 11:e0148407CrossRefGoogle Scholar
  14. Legnini I, Di Timoteo G, Rossi F, Morlando M, Briganti F, Sthandier O, Fatica A, Santini T, Andronache A, Wade M, Laneve P, Rajewsky N, Bozzoni I (2017) Circ-ZNF609 Is a circular RNA that can be translated and functions in myogenesis. Mol Cell 66:22–37.e29CrossRefGoogle Scholar
  15. Li P, Chen H, Chen S, Mo X, Li T, Xiao B, Yu R, Guo J (2017) Circular RNA 0000096 affects cell growth and migration in gastric cancer. Br J Cancer 116:626–633CrossRefGoogle Scholar
  16. Li X, Yang L, Chen L-L (2018) The biogenesis, functions, and challenges of circular RNAs. Mol Cell 71:428–442CrossRefGoogle Scholar
  17. Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak SD, Gregersen LH, Munschauer M, Loewer A, Ziebold U, Landthaler M, Kocks C, le Noble F, Rajewsky N (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338CrossRefGoogle Scholar
  18. Ning L, Long B, Zhang W, Yu M, Wang S, Cao D, Yang J, Shen K, Huang Y, Lang J (2018) Circular RNA profiling reveals circEXOC6B and circN4BP2L2 as novel prognostic biomarkers in epithelial ovarian cancer. Int J Oncol 53:2637–2646Google Scholar
  19. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515CrossRefGoogle Scholar
  20. Qu S, Yang X, Li X, Wang J, Gao Y, Shang R, Sun W, Dou K, Li H (2015) Circular RNA: a new star of noncoding RNAs. Cancer Lett 365:141–148CrossRefGoogle Scholar
  21. Rong D, Tang W, Li Z, Zhou J, Shi J, Wang H, Cao H (2017) Novel insights into circular RNAs in clinical application of carcinomas. OncoTargets Ther 10:2183–2188CrossRefGoogle Scholar
  22. Tu C, Du T, Shao C, Liu Z, Li L, Shen Y (2018) Evaluating the potential of housekeeping genes, rRNAs, snRNAs, microRNAs and circRNAs as reference genes for the estimation of PMI. Forensic Sci Med Pathol 14:194–201CrossRefGoogle Scholar
  23. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034CrossRefGoogle Scholar
  24. Wan L, Zhang L, Fan K, Cheng ZX, Sun QC, Wang JJ (2016) Circular RNA-ITCH suppresses lung cancer proliferation via inhibiting the Wnt/beta-catenin pathway. Biomed Res Int 2016:1579490Google Scholar
  25. Wang K, Singh D, Zeng Z, Coleman SJ, Huang Y, Savich GL, He X, Mieczkowski P, Grimm SA, Perou CM, MacLeod JN, Chiang DY, Prins JF, Liu J (2010) MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res 38:e178CrossRefGoogle Scholar
  26. Westholm JO, Miura P, Olson S, Shenker S, Joseph B, Sanfilippo P, Celniker SE, Graveley BR, Lai EC (2014) Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation. Cell Rep 9:1966–1980CrossRefGoogle Scholar
  27. Xiao-Long M, Kun-Peng Z, Chun-Lin Z (2018) Circular RNA circ_HIPK3 is down-regulated and suppresses cell proliferation, migration and invasion in osteosarcoma. J Cancer 9:1856–1862CrossRefGoogle Scholar
  28. Yang Y, Fan X, Mao M, Song X, Wu P, Zhang Y, Jin Y, Yang Y, Chen LL, Wang Y, Wong CC, Xiao X, Wang Z (2017) Extensive translation of circular RNAs driven by N(6)-methyladenosine. Cell Res 27:626–641CrossRefGoogle Scholar
  29. Zhang Y, Zhang XO, Chen T, Xiang JF, Yin QF, Xing YH, Zhu S, Yang L, Chen LL (2013) Circular intronic long noncoding RNAs. Mol Cell 51:792–806CrossRefGoogle Scholar
  30. Zhang XO, Wang HB, Zhang Y, Lu X, Chen LL, Yang L (2014) Complementary sequence-mediated exon circularization. Cell 159:134–147CrossRefGoogle Scholar
  31. Zheng Q, Bao C, Guo W, Li S, Chen J, Chen B, Luo Y, Lyu D, Li Y, Shi G, Liang L, Gu J, He X, Huang S (2016) Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat Commun 7:11215CrossRefGoogle Scholar
  32. Zheng J, Liu X, Xue Y, Gong W, Ma J, Xi Z, Que Z, Liu Y (2017) TTBK2 circular RNA promotes glioma malignancy by regulating miR-217/HNF1beta/Derlin-1 pathway. J Hematol Oncol 10:52CrossRefGoogle Scholar
  33. Zhong S, Wang J, Hou J, Zhang Q, Xu H, Hu J, Zhao J, Feng J (2018a) Circular RNA hsa_circ_0000993 inhibits metastasis of gastric cancer cells. Epigenomics 10:1301–1313CrossRefGoogle Scholar
  34. Zhong S, Wang J, Zhang Q, Xu H, Feng J (2018b) CircPrimer: a software for annotating circRNAs and determining the specificity of circRNA primers. BMC Bioinform 19:292CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Center of Clinical Laboratory ScienceThe Affiliated Cancer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer ResearchNanjingChina
  2. 2.The First Clinical Medical CollegeNanjing University of Chinese MedicineNanjingChina
  3. 3.Department of General SurgeryThe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina
  4. 4.Department of Medical OncologyThe Affiliated Cancer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer ResearchNanjingChina
  5. 5.Department of Radiation OncologyThe Affiliated Cancer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer ResearchNanjingChina
  6. 6.Department of Gynecologic OncologyThe Affiliated Cancer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer ResearchNanjingChina

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