Evaluation of Reference Genes for Real-Time Quantitative PCR Analysis in Tissues from Bumble Bees (Bombus Terrestris) of Different Lines

Abstract

Bumble bees are important alternative pollinators and model insects due to their highly developed sociality and colony management. In order to better understand their molecular mechanisms, studies focusing on the genetic and molecular aspects of their development and behavior are needed. Although quantitative real-time polymerase chain reaction (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different lines and tissues) must first be identified to ensure the accurate normalization of target genes. In order to contribute to molecular studies on bumble bees, we used Bombus terrestris to determine the expression stability of eight reference genes (β-actin (ACT), Arginine Kinase (AK), Phospholipase A2 (PLA2), Elongation factor 1 alpha (EF-1), Ribosomal proteins (S5, S18, S28) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in five different lines and several tissues (ovary, thorax, fat body, and head) using RT-qPCR procedures and four analysis programs (RefFinder, NormFinder, BestKeeper, and geNorm). In general, the S28, S5, and S18 ribosomal protein genes and the PLA2 and EF-1 genes showed the highest stability and were therefore identified as suitable reference genes for the bumble bee species and their defined lines and tissues. Our results also emphasized the need to evaluate the stability of candidate reference genes for any differently designed lines and tissue conditions in bumble bee species.

Seasonal Stability Assessment of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalization in Bombus terrestris

“…However, EF1, S18 and S28 were identified as the most stable reference genes based on the outcomes of the other four algorithms (Tables 1 and 2). Moreover, a study on a related insect did not find such striking differences between lab-raised and wild-collected insect tissues [22]. In contrast to these geNorm methods (Tables 1 and 2), seasonal changes significantly alter the gene expression levels in bumblebees, increasing the diversity in reference gene expression.…”

“…All primers were designed using Primer-BLAST software (NCBI) in accordance with MIQE requirements. Primers from a previous study [22] were used for seven reference genes, and three primers for were newly designed (Table S1). After qRT-PCR validation of the generated primer pairs, the most suitable gene was selected for subsequent experiments.…”

Selection of Reference Genes for Gene Expression Analysis in Acacia melanoxylon under Different Conditions

“…Generally, housekeeping genes encode proteins that are necessary to maintain fundamental biological processes, such as actin (ACT), ubiquitin (UBI), elongation factor (EF), protein phosphatase 2a (PP2a), and tubulin (TUB), which are commonly utilized as internal reference genes [19]. However, increasing experimental evidence has shown that housekeeping genes are not as stable as previously believed in different conditions [20,21]. Several studies have reported that some novel characterized genes exhibit more excellent stability than transitional housekeeping genes under certain conditions.…”