Molecular Biotechnology

, Volume 50, Issue 1, pp 49–56 | Cite as

Identification of Suitable Reference Genes for qRT-PCR Analysis of Circulating microRNAs in Hepatitis B Virus-Infected Patients

  • Hai-Tao Zhu
  • Qiong-Zhu Dong
  • Guan Wang
  • Hai-Jun Zhou
  • Ning Ren
  • Hu-Liang Jia
  • Qing-Hai Ye
  • Lun-Xiu Qin
Original Paper


Circulating microRNAs (miRNAs) were found to exist in serum/plasma in a highly stable, cell-free form, and aberrantly expressed in many human diseases. Currently, the expression levels of circulating miRNAs are estimated by quantitative real-time polymerase chain reaction. However, no study has systematically evaluated reference genes for evaluating circulating microRNA expression. This study describes the identification and characterization of an appropriate reference gene for the normalization of circulating miRNA levels in hepatitis B virus (HBV)-infected patients and healthy people. Ten miRNAs that resemble the mean expression of the TaqMan low density array together with U6, RNU6B, and miR-16 were validated with two algorithms, geNorm, and NormFinder, after ensuring their equivalent expression between the two study groups. The combination of miR-26a, miR-221, and miR-22* is recommended as the most stable set of reference genes for circulating miRNA evaluation in HBV patients and healthy people.


Hepatitis B virus Circulating microRNA Quantitative real-time PCR Reference genes Normalization 



Cycle threshold


Hepatitis B virus


Hepatitis C virus


Quantitative reverse-transcription polymerase chain reaction



This article was supported by grants from the National Key Projects for Infectious Disease of China (2008ZX10002-021), the Program of Shanghai Chief Scientist (08XD1400800), the National Natural Science Foundation of China (30700991), and the Research Fund for New Teachers of Fudan University.

Supplementary material

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12033_2011_9414_MOESM7_ESM.tif (12 mb)
Fig. S1 a Real-time PCR amplification plots for the miR-22* assay using a dilution series of known input amounts of synthetic miR-22* (Invitrogen, China; from left to right: 1.0E + 12 to 1.0E + 4 copies). Each graph represents amplification [presented on the y-axis as Rn: the fluorescence emission intensity of the reporter dye (FAM) normalized to the passive reference dye (ROX)] plotted against cycle number (presented on the x-axis as the cycle at which fluorescence was detected above an automatically determined threshold) for miR-22*. The curves represent technical replicates (in triplicate or duplicate) of real-time PCR. b Standard curve for miR-22* TaqMan qRT-PCR assays. Standard curves were generated for the miR-22* assay by using a dilution series of known input amounts of synthetic miR-22* corresponding to the target of the assay (Table 1). The dilution series samples were run using common RT and PCR enzyme master mixes and on the same plate as experimental samples. The standard curve revealed that the Ct values between 14 and 37 were reliable, stable in replicate, and quantitative in this assay. (TIFF 12276 kb)


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Hai-Tao Zhu
    • 1
    • 2
  • Qiong-Zhu Dong
    • 1
    • 2
  • Guan Wang
    • 1
    • 2
  • Hai-Jun Zhou
    • 1
    • 2
  • Ning Ren
    • 1
    • 2
  • Hu-Liang Jia
    • 1
    • 2
  • Qing-Hai Ye
    • 1
    • 2
  • Lun-Xiu Qin
    • 1
    • 2
  1. 1.Live Cancer Institute and Zhongshan Hospital, Institutes of Biomedical SciencesFudan UniversityShanghaiChina
  2. 2.Key Laboratory of Carcinogenesis and Cancer InvasionMinistry of EducationShanghaiChina

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