Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini)

Abstract

Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studies focusing on genetic and molecular aspects of their development and behavior. The most common method for looking at gene expression is real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR) of the mRNA of interest. This method requires the identification of reliable reference genes to correctly estimate fluctuations in transcript levels. To contribute to molecular studies on stingless bees, we used Frieseomelitta varia, Melipona quadrifasciata, and Scaptotrigona bipunctata species to test the expression stability of eight reference genes (act, ef1-α, gapdh, rpl32, rps5, rps18, tbp, and tbp-af) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the rpl32, rps5 and rps18 ribosomal protein genes and tpb-af gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species.

Screening and Validation of Reference Genes for RT-qPCR Under Different Honey Bee Viral Infections and dsRNA Treatment

“…Previous studies have shown that some ribosome-associated genes have been used as stable internal reference genes for quantitative analysis ( Wang et al, 2019 ). For instance, Freitas et al (2019) found that rpl 32, rps 5, and rps 18 were identified as suitable reference genes for the different tissues of stingless bees. Similarly, rpl23 was identified the reliable reference gene in bumblebees challenged by IAPV ( Niu et al, 2014 ; Table 3 ).…”

Longitudinal survey reveals delayed effects of forager gene expression on stingless bee colony health

“…Primers based on each respective gene sequence were designed with Primer3 from Geneious R11 [20] (electronic supplementary material, table S1). Actin ( act ) and 40S ribosomal protein S5 ( rps5 ) were used as references for gene expression normalization [21–23]. StepOnePlus Real-Time PCR System (Applied Biosystems, USA) was used for the RT-qPCR assays, and primer amplification efficiency was calculated with qBASE+ software [24] from the slope of a five-point 1:10 serial dilution of calibrator cDNA samples.…”

Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea

“…The commonality and discrepancies displayed here confirm the notion that no universal reference genes exist for all contexts and reference gene selection and validation is crucial for accurate quantification of gene expression under specific experimental conditions. Without these studies, single un-validated endogenous controls can have profound impacts on data analysis and lead to questionable interpretation 16,18,19,45,46 . In this study, the expression of Hex-1 was significantly underestimated in the 1st nymphs when the least stable instead of the most stable and recommended reference genes was used to normalize target gene expression.…”