Selection of reliable reference genes for qPCR studies on chondroprotective action

Background  

Chondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected step in the analysis process. Therefore, the present study aimed to determine the expression stability of common reference genes (ACTB, Beta actin; GAPDH, Glyceraldehyde-3-phosphate; B2M, Beta-2-microglobulin; HPRT1, Hypoxanthine phosphoribosyl-transferase I; SDHA, Succinate dehydrogenase complex, subunit A; YWHAZ, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) under the influence of glucosamine, curcumin and diacerein in the IL-1β-stimulated C-28/I2 chondrocyte model, using the geNorm software tool.     

Husbandry and growth rates of neonate epaulette sharks,Hemiscyllium ocellatum in captivity

In this report, I describe husbandry techniques that are used to successfully hatch and raise epaulette sharks, Hemiscyllium ocellatum, in captivity. Egg cases and hatchlings were held in floating baskets within a 1,306‐L tank, at water temperatures of 24.4–25.5°C. From 56 egg cases, 27 neonates successfully hatched, and 22 survived. Based on seven tagged egg cases, the average gestation time was 140.3 ± 4.6 days with a range of 134 and 148 days. Upon hatching, neonate length was 16.2 ± 0.522 cm, with a range of 15.24–17.30 cm, and weight was 19.9 ± 2.06 g, with a range of 16.50–24.5 g. Growth rates of four individuals were recorded over time. Finely chopped capelin, Mallotus villosus, fillets, supplemented with a multivitamin were the primary food items accepted by neonates. The success in rearing H. ocellatum can likely be attributed to a low stress environment, limited disruption, an easy to consume diet, multivitamin supplementation, and excellent water quality. Zoo Biol 31:718‐724, 2012. © 2011 Wiley Periodicals, Inc.     

RNA-Seq is not required to determine stable reference genes for qPCR normalization

Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization. In this study, however, we demonstrate that the statistical approach to determine the best reference genes from commonly used conventional candidates is more important than the preselection of ‘stable’ candidates from RNA-Seq data. Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using conventional reference genes render the same results as stable reference genes selected from RNA-Seq data. We validated these observations in two distinct cross-sectional experimental conditions involving human iPSC derived microglial cells and mouse sciatic nerves. These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays.     

Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia

The majority of vertebrates are not tolerant to hypoxia but epaulette sharks (Hemiscyllium ocellatum) living on shallow reef platforms appear to tolerate hypoxic periods during tidal fluctuations. The effects of progressive hypoxia on the metabolic and ventilatory responses of these elasmobranchs were examined in a closed respirometer. In order to determine whether repeated exposure to hypoxia primes these sharks to alter their metabolism, one group of sharks was exposed to repeated sub-lethal hypoxia, at 5% of air saturation, prior to respirometry. In response to falling oxygen concentration [O(2)], the epaulette shark increased its ventilatory rate and maintained its O(2) consumption rate (VO(2)) down to 2.2 mg O(2) l(-1) at 25 degrees C. This is the lowest critical [O(2)] ([O(2)](crit)) ever measured for any elasmobranch. After reaching the [O(2)](crit), the shark remained in the respirometer for a further 4-5 h of progressive hypoxia. Only after the [O(2)] fell to 1.0 mg l(-1) was there a decrease in the ventilatory rate followed by a rise in blood lactate levels, indicating that the epaulette shark responds to severe hypoxia by entering a phase of metabolic and ventilatory depression. Interestingly, hypoxia tolerance was dynamic because hypoxic pre-conditioning lowered the VO(2) of the epaulette shark by 29%, which resulted in a significantly reduced [O(2)](crit) (1.7 mg O(2) l(-1)), revealing that hypoxic pre-conditioning elicits an enhanced physiological response to hypoxia.     

Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies

ABSTRACT: BACKGROUND: Quantitative PCR (qPCR) is a widely used technique for gene expression analysis. A common normalization method for accurate qPCR data analysis involves stable reference genes to determine relative gene expression. Despite extensive research in the forest tree species Populus, there is not a resource for reference genes that meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards for qPCR techniques and analysis. Since Populus is a woody perennial species, studies of seasonal changes in gene expression are important towards advancing knowledge of this important developmental and physiological trait. The objective of this study was to evaluate reference gene expression stability in various tissues and growth conditions in two important Populus genotypes (P. trichocarpa "Nisqually 1" and P. tremula X P. alba 717 1-B4) following MIQE guidelines. RESULTS: We evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from P. trichocarpa and P. tremula. x P. alba grown under long-day (LD), short-day (SD) or SD plus low-temperatures conditions. Gene expression data were analyzed for stable reference genes among 18S rRNA, ACT2, CDC2, CYC063, TIP4-like, UBQ7, PT1 and ANT using two software packages, geNormPLUS and BestKeeper. GeNormPLUS ranked TIP4-like and PT1 among the most stable genes in most genotype/tissue combinations while BestKeeper ranked CDC2 and ACT2 among the most stable genes. CONCLUSIONS: This is the first comprehensive evaluation of reference genes in two important Populus genotypes and the only study in Populus that meets MIQE standards. Both analysis programs identified stable reference genes in both genotypes and all tissues grown under different photoperiods. This set of reference genes was found to be suitable for either genotype considered here and may potentially be suitable for other Populus species and genotypes. These results provide a valuable resource for the Populus research community.     

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

Background

Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. The chosen experimental system was the correction of a hprt-point mutation in a hamster cell line, the generation of an additional nucleotide exchange at a variable distance from the first exchange position and the investigation of the rate of simultaneous nucleotide exchanges.

Results

The smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position.

Conclusion

We suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.     

Male-biased sexual size dimorphism in sharks: the narrowmouth catshark Schroederichthys bivius as case study

Hydrobiologia - In shark populations, variation in sexual size dimorphism (SSD) appears to be related with the reproductive mode. Here, we aimed to investigate the reproductive biology of the...     

Reproductive and feeding ecology of parasitic gnathiid isopods of epaulette sharks (Hemiscyllium ocellatum) with consideration of their role in the transmission of a haemogregarine

Epaulette sharks Hemiscyllium ocellatum were surveyed on Heron Island, Great Barrier Reef, Australia for gnathiid isopods and protozoan (haemogregarine) parasites to determine the prevalence and intensity of infection and to investigate the potential role of gnathiids as vectors of these haemogregarines, the first such study carried out on elasmobranchs. Juvenile gnathiids were collected and quantified using a novel non-invasive and chemical-free technique and gnathiid squashes were examined for haemogregarine developmental stages. The feeding and reproductive ecology of the Gnathia spp. was investigated to better understand the relationship between gnathiids and haemogregarines. Gnathiids were found on all sharks and intensities ranged between two and 66. Only third-stage gnathiid juveniles were found, which fell into two size groups (A and B). These juveniles remained attached to H. ocellatum for up to 17 days, the longest period of attachment yet recorded for gnathiids. Group A female gnathiids produced broods of 45-187 (median =120) first stage juveniles from between 54 and 82 days (median=63 days) after detachment. First stage juveniles survived for an average of 15.8+/-0.1 (SEM) days without feeding. The prevalence (6.7%) and parasitaemia (usually <0.1% infected erythrocytes) of infections of the haemogregarine Haemogregarina hemiscyllii were relatively low and most stages were immature gamonts. Two undescribed Gnathia spp. were identified by examining adult male gnathiids that metamorphosed from juveniles from each of the two size groups. Our hypothesis that Gnathia spp. transmit H. hemiscyllii is neither supported or refuted, as although intact H. hemiscyllii gamonts were detected in squashes of gnathiids that had engorged on haemogregarine-positive H. ocellatum 24-57 days previously, no further developmental stages were detected.     

Getting real with real-time qPCR: a case study of reference gene selection for morphological variation in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> wings

Accurate estimation of gene expression differences during development requires sensitive techniques combined with gold-standard normalization procedures. This is particularly true in the case of quantitative traits, where expression changes might be small. Nevertheless, systematic selection and validation of reference genes has been overlooked, even in Drosophila studies. Here, we tested the stability of six traditional reference genes across samples of imaginal wing disks from morphologically divergent strains of Drosophila melanogaster, in a two-class comparison: quantitative or qualitative variation in wing morphology. Overall, we identified and validated a pair of genes (RpL32 and Tbp) as being stably expressed in both experimental comparisons. These genes might be considered as a bona fide pair of reference genes for gene expression analyses of morphological divergence in D. melanogaster wings. They might also be taken as good candidates for experimental identification of stable reference genes in other morphological comparisons using Drosophila or other insect species. Besides, we found that some genes traditionally used as reference in qPCR experiments were not stably expressed in wing disks from the different fly strains. In fact, a significant bias was observed when the expression of three genes of interest, which are involved in the regulation of growth and patterning during imaginal wing development, was normalized with such putative reference genes. Our results demonstrate how inaccurate findings and opposite conclusions might be drawn if traditional reference genes are arbitrarily used for internal normalization without proper validation in the given experimental condition, a practice still common in qPCR experiments.     

Sustainability of threatened species displayed in public aquaria,with a case study of Australian sharks and rays

Zoos and public aquaria exhibit numerous threatened species globally, and in the modern context of these institutions as conservation hubs, it is crucial that displays are ecologically sustainable. Elasmobranchs (sharks and rays) are of particular conservation concern and a higher proportion of threatened species are exhibited than any other assessed vertebrate group. Many of these lack sustainable captive populations, so comprehensive assessments of sustainability may be needed to support the management of future harvests and safeguard wild populations. We propose an approach to identify species that require an assessment of sustainability. Species at risk of extinction in the wild were considered to be those assessed as threatened (CR, EN or VU) on the IUCN Red List of Threatened Species, or data deficient species that may be at an elevated risk of extinction due to life history traits and habitat associations. We defined sustainable captive populations as self-maintaining, or from a source population that can sustain harvest levels without risk of population declines below sustainable levels. The captive breeding and wild harvest records of at risk species displayed by Australian aquaria were examined as a case study. Two species, largetooth sawfish Pristis pristis and grey nurse shark Carcharias taurus, were found to have unsustainable captive populations and were identified as high priorities for comprehensive sustainability assessments. This review highlights the need for changes in permitting practices and zoo and aquarium record management systems to improve conservation outcomes for captive elasmobranchs.