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

Abstract

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.

Selection of reference genes for tissue/organ samples of adults of Eucryptorrhynchus scrobiculatus

“…The delta Ct method analyzes the relative expression of gene pairs to identify suitable reference genes [33]. In our study, the delta Ct method results were similar to those of geNorm and NormFinder, and all stable reference genes were ribosomal protein genes, which in previous studies were identified as being highly stable between different insect tissues [35]. However, the rankings by Best-Keeper differed greatly from those of the other three algorithms, similar to the results of previous reports [44,49].…”

“…Many biological processes are closely related to the expression level of genes, and detection of gene expression levels is very important. Due to its high accuracy, sensitivity, specificity, and rapidity, RT-qPCR has been widely used for validating the results of gene expression profiles [35]. However, RT-qPCR requires normalization by reference genes to offset variation among samples processed from different tissues [36].…”

Blood Meal Induced Regulation of Gene Expression in the Maxillary Palps, a Chemosensory Organ of the Mosquito Aedes aegypti

“…Ninetynine percent of the CEGs were found at detectable levels in the maxillary palps, indicating that the sequencing depth was sufficient to reliably identify gene expression ( Figure 1; Supplementary File 2). The fold change in abundance for 99% and >92% of the CEGs transcripts, between physiological states and between replicates, was <2.0 and <1.5, respectively, as would be expected for housekeeping genes (Figure 1) (Dzaki et al, 2017;Shakeel et al, 2017).…”

Molecular identification, characterization, and expression analysis of a novel trypsin inhibitor-like cysteine-rich peptide from the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

“…The thermal reaction was as follows: denaturation for 30 s at 95°C, then 40 cycles for 5 s at 95°C, and 60°C for 10 s. Furthermore, a melting curve analysis was carried out for the amplified product. To analyze the relative expression, the 2 − ΔΔCT method (Livak and Schmittgen 2001) was employed with a ribosomal protein L28 (RPL28) used as an internal control for normalization (Chandra et al 2014;Shakeel et al 2015Shakeel et al , 2018. The list of primers for RT-qPCR is presented in Table 1.…”