Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects
- 238 Downloads
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.
KeywordsGene 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.
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.
- de Kok JB et al (2005) Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab Invest 15:159–185Google Scholar
- Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechnology 37:112–119Google Scholar
- Pabinger S, Rödiger S, Kriegner A, Vierlinger K, Weinhäusel A (2014) A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomol Det Quant 1:23–33Google Scholar
- Pfaffl MW (2004) Quantification strategies in real-time PCR. In: Bustin SA (ed) AZ of quantitative PCR. International University Line (IUL), La Jolla, pp 87–212Google Scholar
- Rhinn H, Marchand-Leroux C, Croci N, Plotkine M, Scherman D, Escriou V (2008) Housekeeping while brain’s storming validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury. BMC Mol Biol. https://doi.org/10.1186/1471-2199-9-62 PubMedPubMedCentralGoogle Scholar
- Scharlaken B, de Graaf DC, Goossens K, Brunain M, Peelman LJ, Jacobs FJ (2008) Reference gene selection for insect expression studies using quantitative real-time PCR: The head of the honeybee, Apis mellifera, after a bacterial challenge. J Ins Sci. https://doi.org/10.1673/031.008.3301 Google Scholar
- Suzuki T, Higgins P, Crawford D (2000) Control selection for RNA quantitation. Biotechnology 29:332–337Google Scholar
- van Doorn R, Szemes M, Bonants P, Kowalchuk GA, Salles JF, Ortenberg E, Schoen CD (2007) Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™. BMC Genom. https://doi.org/10.1186/1471-2164-8-276 Google Scholar
- Vandesompele J, Kubista M, Pfaffl MW (2009) Reference gene validation software for improved normalization. In: Logan J, Edwards K, Saunders N (eds) Real-time PCR: current technology and applications. Caister Academic Press, London, pp 47–64Google Scholar