Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be ...relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.
qPCR is more complex than perceived by many scientists.
The production of an amplification curve and an associated quantitative cycle value does not necessarily mean interpretable data.
The MIQE guidelines and associated methodology articles published thereafter, underline the ongoing drive to help scientists produce reproducible data from qPCR, culminating in a simple, stepwise methodology to ensure high-quality, reproducible data from qPCR experiments.
The concept of data normalization has led to the ongoing publication of articles solely focused on this subject for various sample types and experimental parameters.
The analysis of qPCR data can be challenging, especially as experiments grow in sample number and complexity of biological groups. A defined approach to qPCR data analysis is necessary to clarify gene expression analysis.
We developed a practical and easy two-step multiplex PCR assay to aid in serotyping of Streptococcus suis. The assay accurately typed almost all of the serotype reference strains and field isolates ...of various serotypes and also identified the genotypes of capsular polysaccharide synthesis gene clusters of some serologically nontypeable strains.
The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine pathogens and often serves as an entry door for other viral or bacterial pathogens, of which ...Streptococcus suis is one of the most common. Pre-infection with PRRSV leads to exacerbated disease caused by S. suis infection. Very few studies have assessed the immunological mechanisms underlying this higher susceptibility. Since antigen presenting cells play a major role in the initiation of the immune response, the in vitro transcriptional response of bone marrow-derived dendritic cells (BMDCs) and monocytes in the context of PRRSV and S. suis co-infection was investigated. BMDCs were found to be more permissive than monocytes to PRRSV infection; S. suis phagocytosis by PRRSV-infected BMDCs was found to be impaired, whereas no effect was found on bacterial intracellular survival. Transcription profile analysis, with a major focus on inflammatory genes, following S. suis infection, with and without pre-infection with PRRSV, was then performed. While PRRSV pre-infection had little effect on monocytes response to S. suis infection, a significant expression of several pro-inflammatory molecules was observed in BMDCs pre-infected with PRRSV after a subsequent infection with S. suis. While an additive effect could be observed for CCL4, CCL14, CCL20, and IL-15, a distinct synergistic up-regulatory effect was observed for IL-6, CCL5 and TNF-α after co-infection. This increased pro-inflammatory response by DCs could participate in the exacerbation of the disease observed during PRRSV and S. suis co-infection.
Streptococcus suis is an emerging zoonotic agent causing meningitis and septicemia. Outbreaks in humans in China with atypical cases of streptococcal toxic shock-like syndrome have been described to ...be caused by a clonal epidemic S. suis strain characterized as sequence type (ST) 7 by multilocus sequence typing, different from the classical ST1 usually isolated in Europe. Previous in vitro studies showed that Toll-like receptor (TLR) 2 plays a major role in S. suis ST1 interactions with host cells. In the present study, the in vivo role of TLR2 in systemic infections caused by S. suis ST1 or ST7 strains using TLR2 deficient (TLR2(-/-)) mice was evaluated. TLR2-mediated recognition significantly contributes to the acute disease caused by the highly virulent S. suis ST1 strain, since the TLR2(-/-) mice remained unaffected when compared to wild type (WT) mice. The lack of mortality could not be associated with a lower bacterial burden; however, a significant decrease in the induction of pro-inflammatory mediators, as evaluated by microarray, real-time PCR and protein assays, was observed. On the other hand, TLR2(-/-) mice infected with the epidemic ST7 strain presented no significant differences regarding survival and expression of pro-inflammatory mediators when compared to the WT mice. Together, these results show a TLR2-independent host innate immune response to S. suis that depends on the strain.
Streptococcus suis is a major swine pathogen and important zoonotic agent causing mainly septicemia and meningitis. However, the mechanisms involved in host innate and adaptive immune responses ...toward S. suis as well as the mechanisms used by S. suis to subvert these responses are unknown. Here, and for the first time, the ability of S. suis to interact with bone marrow-derived swine dendritic cells (DCs) was evaluated. In addition, the role of S. suis capsular polysaccharide in modulation of DC functions was also assessed. Well encapsulated S. suis was relatively resistant to phagocytosis, but it increased the relative expression of Toll-like receptors 2 and 6 and triggered the release of several cytokines by DCs, including IL-1β, IL-6, IL-8, IL-12p40 and TNF-α. The capsular polysaccharide was shown to interfere with DC phagocytosis; however, once internalized, S. suis was readily destroyed by DCs independently of the presence of the capsular polysaccharide. Cell wall components were mainly responsible for DC activation, since the capsular polysaccharide-negative mutant induced higher cytokine levels than the wild-type strain. The capsular polysaccharide also interfered with the expression of the co-stimulatory molecules CD80/86 and MHC-II on DCs. To conclude, our results show for the first time that S. suis interacts with swine origin DCs and suggest that these cells might play a role in the development of host innate and adaptive immunity during an infection with S. suis serotype 2.
The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These ...new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.
We previously identified Fragile X-related protein 1 (FXR1) as an RNA-binding protein involved in the post-transcriptional control of TNF and other cytokines in macrophages. Macrophages derived from ...FXR1-KO mice overexpress several inflammatory cytokines including TNF. Recently, we showed that fenretinide (4HPR) is able to inhibit several inflammatory cytokines in the lungs of cystic fibrosis mice, which also have abnormal immune responses. Therefore, we hypothesized that 4HPR might also be able to downregulate excessive inflammation even in macrophages with ablated FXR1. Indeed, our results demonstrate that 4HPR inhibited the excessive production of inflammatory mediators, including TNF, IL-6, CCL2 and CCL-5 in LPS-stimulated FXR1-KO macrophages, by selectively inhibiting phosphorylation of ERK1/2, which is naturally more phosphorylated in FXR1-KO cells. We also found that LPS stimulation of FXR1-KO macrophages led to significantly higher ratio of arachidonic acid/docosahexaenoic acid than observed in FXR1-WT macrophages. Interestingly, treatment with 4HPR was associated with the normalization of arachidonic acid/docosahexaenoic acid ratio in macrophages, which we found to impact phosphorylation of ERK1/2. Overall, this study shows for the first time that 4HPR modulates inflammatory cytokine expression in macrophages by correcting a phospholipid-bound fatty acid imbalance that impacts the phosphorylation of ERK1/2.
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