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Honey bee viruses are one of the most important pathogens that have contributed to the decrease in honey bee colony health. To analyze the infection dynamics of honey bee viruses, quantification of viral gene expression by RT-qPCR is necessary. However, suitable reference genes have not been reported from viral and RNAi studies of honey bee. Here, we evaluated the expression of 11 common reference genes (ache2, rps18, β-actin, tbp, tif, rpl32, gadph, ubc, α-tubulin, rpl14, and rpsa) from Apis mellifera (Am) and Apis cerana (Ac) under Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV), and Chinese sacbrood virus (CSBV) infection as well as dsRNA-PGRP-SA treatment, and we confirmed their validation by evaluating the levels of the defensin 1 and prophenoloxidase (ppo) genes during viral infection. Our results showed that the expression of selected genes varied under different viral infections. ache2, rps18, β-actin, tbp, and tif can be used to normalize expression levels in Apis mellifera under IAPV infection, while the combination of actin and tif is suitable for CBPV-infected experiments. The combination of rpl14, tif, rpsa, ubc, and ache2 as well as more reference genes is suitable for CSBV treatment in Apis cerana. Rpl14, tif, rps18, ubc, and α-tubulin were the most stable reference genes under dsRNA treatment in Apis mellifera. Furthermore, the geNorm and NormFinder algorithms showed that tif was the best suitable reference gene for these four treatments. This study screened and validated suitable reference genes for the quantification of viral levels in honey bee, as well as for RNAi experiments.
Pubmed ID: 32849362
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NIH genetic sequence database that provides annotated collection of all publicly available DNA sequences for almost 280 000 formally described species (Jan 2014) .These sequences are obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole-genome shotgun (WGS) and environmental sampling projects. Most submissions are made using web-based BankIt or standalone Sequin programs, and GenBank staff assigns accession numbers upon data receipt. It is part of International Nucleotide Sequence Database Collaboration and daily data exchange with European Nucleotide Archive (ENA) and DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through NCBI Entrez retrieval system, which integrates data from major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of GenBank database are available by FTP.
View all literature mentionsA tool to design target-specific primers for polymerase chain reaction (PCR). It uses Primer3 to design PCR primers and then uses BLAST and global alignment algorithm to screen primers against user-selected database in order to avoid primer pairs (all combinations including forward-reverse primer pair, forward-forward as well as reverse-reverse pairs) that can cause non-specific amplifications.
View all literature mentionsExcel-based tool using pair-wise correlations for determination of stable housekeeping genes, differentially regulated target genes and sample integrity. It determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points.
View all literature mentionsSoftware for identifying the optimal normalization gene among a set of candidates. It ranks the set of candidate normalization genes according to their expression stability in a given sample set and given experimental design. It can analyze expression data obtained through any quantitative method e.g. real time RT-PCR and microarray based expression analysis. NormFinder.xla adds the NormFinder functionality directly to Excel. A version for R is also available.
View all literature mentionsSoftware to determine most stable reference (housekeeping) genes from set of tested candidate reference genes in given sample panel. From this, gene expression normalization factor can be calculated for each sample based geometric mean of user-defined number of reference genes.
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