Biotechnology Letters

, Volume 40, Issue 2, pp 227–236 | Cite as

Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects

  • Muhammad Shakeel
  • Alicia Rodriguez
  • Urfa Bin Tahir
  • Fengliang Jin


Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.


Gene expression Insects Internal controls Reference genes RT-qPCR 



This work is supported by the National Natural Science Foundation of China (31572069, 31371989). We thank the reviewers for their constructive comments to improve our manuscript. We apologize to those scientists whose work was not cited in this manuscript owing to space limitations.

Supporting Information

Supplementary Table 1—List of reference genes used in insect studies

Supplementary Table 2—Traditional reference genes showing low stability under different biotic and abiotic conditions, specifically in insects, published in important scientific journals.

Supplementary Table 3—Reference genes (traditional and novel) showing high stability under different biotic conditions, specifically in insects, published in important scientific journals.

Supplementary Table 4—Reference genes (traditional and novel) showing high stability under different abiotic conditions, specifically in insects, published in important scientific journals.

Compliance with ethical standards

Conflict of interest

The authors report that there are no conflicts of interest.

Supplementary material

10529_2017_2465_MOESM1_ESM.docx (52 kb)
Supplementary material 1 (DOCX 51 kb)


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Muhammad Shakeel
    • 1
  • Alicia Rodriguez
    • 2
  • Urfa Bin Tahir
    • 3
  • Fengliang Jin
    • 1
  1. 1.Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, College of AgricultureSouth China Agricultural UniversityGuangzhouChina
  2. 2.Faculty of Veterinary Science, Food Hygiene and Safety, Meat and Meat Products Research InstituteUniversity of ExtremaduraCáceresSpain
  3. 3.Department of Aquatic Animal Medicine, College of FisheriesHuazhong Agricultural UniversityWuhanChina

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