Robust RT-qPCR Data Normalization: Validation and Selection of Internal Reference Genes during Post-Experimental Data Analysis

Reverse transcription and real-time PCR (RT-qPCR) has been widely used for rapid quantification of relative gene expression. To offset technical confounding variations, stably-expressed internal reference genes are measured simultaneously along with target genes for data normalization. Statistic methods have been developed for reference validation; however normalization of RT-qPCR data still remains arbitrary due to pre-experimental determination of particular reference genes. To establish a method for determination of the most stable normalizing factor (NF) across samples for robust data normalization, we measured the expression of 20 candidate reference genes and 7 target genes in 15 Drosophila head cDNA samples using RT-qPCR. The 20 reference genes exhibit sample-specific variation in their expression stability. Unexpectedly the NF variation across samples does not exhibit a continuous decrease with pairwise inclusion of more reference genes, suggesting that either too few or too many reference genes may detriment the robustness of data normalization. The optimal number of reference genes predicted by the minimal and most stable NF variation differs greatly from 1 to more than 10 based on particular sample sets. We also found that GstD1, InR and Hsp70 expression exhibits an age-dependent increase in fly heads; however their relative expression levels are significantly affected by NF using different numbers of reference genes. Due to highly dependent on actual data, RT-qPCR reference genes thus have to be validated and selected at post-experimental data analysis stage rather than by pre-experimental determination

CRF-Like Diuretic Hormone Negatively Affects Both Feeding and Reproduction in the Desert Locust, Schistocerca gregaria

Diuretic hormones (DH) related to the vertebrate Corticotropin Releasing Factor (CRF) have been identified in diverse insect species. In the migratory locust, Locusta migratoria, the CRF-like DH (CRF/DH) is localized in the same neurosecretory cells as the Ovary Maturating Parsin (OMP), a neurohormone that stimulates oocyte growth, vitellogenesis and hemolymph ecdysteroid levels in adult female locusts. In this study, we investigated whether CRF-like DH can influence feeding and reproduction in the desert locust, Schistocerca gregaria. We identified two highly similar S. gregaria CRF-like DH precursor cDNAs, each of which also encodes an OMP isoform. Alignment with other insect CRF-like DH precursors shows relatively high conservation of the CRF/DH sequence while the precursor region corresponding to OMP is not well conserved. Quantitative real-time RT-PCR revealed that the precursor transcripts mainly occur in the central nervous system and their highest expression level was observed in the brain. Injection of locust CRF/DH caused a significantly reduced food intake, while RNAi knockdown stimulated food intake. Therefore, our data indicate that CRF-like DH induces satiety. Furthermore, injection of CRF/DH in adult females retarded oocyte growth and caused lower ecdysteroid titers in hemolymph and ovaries, while RNAi knockdown resulted in opposite effects. The observed effects of CRF/DH may be part of a wider repertoire of neurohormonal activities, constituting an integrating control system that affects food intake and excretion, as well as anabolic processes like oocyte growth and ecdysteroidogenesis, following a meal. Our discussion about the functional relationship between CRF/DH and OMP led to the hypothesis that OMP may possibly act as a monitoring peptide that can elicit negative feedback effects

RNA-Seq reveals large quantitative differences between the transcriptomes of outbreak and non-outbreak locusts

Outbreaks of locust populations repeatedly devastate economies and ecosystems in large parts of the world. The consequent behavioural shift from solitarious to gregarious and the concomitant changes in the locusts’ biology are of relevant scientific interest. Yet, research on the main locust species has not benefitted from recent advances in genomics. In this first RNA-Seq study on Schistocerca gregaria, we report two transcriptomes, including many novel genes, as well as differential gene expression results. In line with the large biological differences between solitarious and gregarious locusts, almost half of the transcripts are differentially expressed between their central nervous systems. Most of these transcripts are over-expressed in the gregarious locusts, suggesting positive correlations between the levels of activity at the population, individual, tissue and gene expression levels. We group these differentially expressed transcripts by gene function and highlight those that are most likely to be associated with locusts’ phase change either in a species-specific or general manner. Finally, we discuss our findings in the context of population-level and physiological events leading to gregariousness.M. Bakkali wishes to thank the Spanish Ministerio de Ciencia y Tecnología for the for the Ramón y Cajal fellowship and for the BFU2010-16438 grant that supported both this research and the FPI studentship to Rubén Martín Blázquez. We thank Mrs. Pernille Lavgesen for revision of the English language writing of this manuscript. We also thank the editor for the valuable comments on the manuscript

Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust

<p>Abstract</p> <p>Background</p> <p>The Australian plague locust, <it>Chortoicetes terminifera</it>, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust.</p> <p>Results</p> <p>Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5^thinstar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca²⁺binding glycoprotein, <it>SPARC</it>, which was previously found to be up-regulated in brains of gregarious desert locusts, <it>Schistocerca gregaria</it>. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike <it>S. gregaria</it>, there was no effect of any crowding treatment in the Australian plague locust.</p> <p>Conclusion</p> <p>Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that <it>C. terminifera </it>crowding did not change the neuronal expression of the <it>SPARC </it>gene, a gregarious phase-specific gene identified in brains of the desert locust, <it>S. gregaria</it>. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.</p

Transcriptomics of the Bed Bug (Cimex lectularius)

BACKGROUND: Bed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance. METHODOLOGY AND PRINCIPAL FINDINGS: Using 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database. CONCLUSIONS: To our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies

Transcriptome Analysis of the Desert Locust Central Nervous System: Production and Annotation of a Schistocerca gregaria EST Database

) displays a fascinating type of phenotypic plasticity, designated as ‘phase polyphenism’. Depending on environmental conditions, one genome can be translated into two highly divergent phenotypes, termed the solitarious and gregarious (swarming) phase. Although many of the underlying molecular events remain elusive, the central nervous system (CNS) is expected to play a crucial role in the phase transition process. Locusts have also proven to be interesting model organisms in a physiological and neurobiological research context. However, molecular studies in locusts are hampered by the fact that genome/transcriptome sequence information available for this branch of insects is still limited. EST information is highly complementary to the existing orthopteran transcriptomic data. Since many novel transcripts encode neuronal signaling and signal transduction components, this paper includes an overview of these sequences. Furthermore, several transcripts being differentially represented in solitarious and gregarious locusts were retrieved from this EST database. The findings highlight the involvement of the CNS in the phase transition process and indicate that this novel annotated database may also add to the emerging knowledge of concomitant neuronal signaling and neuroplasticity events. EST data constitute an important new source of information that will be instrumental in further unraveling the molecular principles of phase polyphenism, in further establishing locusts as valuable research model organisms and in molecular evolutionary and comparative entomology

Insect neuropeptide and peptide hormone receptors: Current knowledge and future directions

Peptides form a very versatile class of extracellular messenger molecules that function as chemical communication signals between the cells of an organism. Molecular diversity is created at different levels of the peptide synthesis scheme. Peptide messengers exert their biological functions via specific signal-transducing membrane receptors. The evolutionary origin of several peptide precursor and receptor gene families precedes the divergence of the important animal Phyla. In this chapter, current knowledge is reviewed with respect to the analysis of peptide receptors from insects, incorporating many recent data that result from the sequencing of different insect genomes. Therefore, detailed information is provided on six different peptide receptor families belonging to two distinct receptor categories (i.e., the heptahelical and the single transmembrane receptors). In addition, the remaining problems, the emerging concepts, and the future prospects in this area of research are discussed.status: publishe