Quantitative Polymerase Chain Reaction Platform Research Articles

The molecular and functional characterization of ferritins in the hard tick Hyalomma rufipes

BackgroundThe protein ferritin, which plays an important role in the maintenance of iron homeostasis, is indispensable for iron detoxification, resistance to oxidative stress and innate immunity. Ticks, which are obligate blood-sucking ectoparasites, have to deal with a large amount of iron when they take a blood meal.MethodsSequence analysis was undertaken using bioinformatics. A recombinant (r) expression vector, rferritin, was constructed for a prokaryotic expression system. A quantitative polymerase chain reaction platform was used to detect the spatial and temporal expression patterns of target genes and their responses to a low temperature environment. Knockdown of the ferritin genes through RNA interference was used to analyze their effects on physiological parameters of ticks.ResultsTwo ferritin genes, HrFer1 and HrFer2, were cloned from the tick Hyalomma rufipes. Their open reading frames are 519 base pairs (bp) and 573 bp in length, and number of coding amino acids 170 and 190, respectively. The phylogenetic tree showed that HrFer1 and HrFer2 have a close evolutionary relationship with the H subunit of ferritin. In vitro experiments showed that rHrFer1 and rHrFer2 had concentration-dependent iron chelating activity. The relative expression of the two ferritin genes was higher in the ovary and midgut of H. rufipes. RNA interference results demonstrated that HrFer1 and HrFer2 expression had a significant effect on engorged body weight, number of eggs laid, and mortality of H. rufipes, and that HrFer2 also had a significant effect on feeding duration. Furthermore, the relative expression of ferritin decreased significantly in a low temperature environment, suggesting that HrFer1 and HrFer2 play a regulatory role in the cold stress response of H. rufipes.ConclusionsThe results of the present study improve our understanding of the involvement of ferritins in tick blood-feeding.Graphical

A multiplexed electrochemical quantitative polymerase chain reaction platform for single-base mutation analysis

Detection of single-based mutation (SbM), which is of ultra-low abundance against wild-type alleles, are typically constrained by the level of multiplexing, sensitivity for single-base resolution and quantification accuracy. In this work, an electrochemical quantitative polymerase chain reaction (E-PCR) platform was developed for multiplexed and quantitative SbM analysis in limited and precious samples with single-nucleotide discrimination. A locked nucleic acid (LNA)-mediated multiplexed PCR system in a single, closed tube setup was firstly constructed to selectively amplify the SbM genes while suppressing the wild-type alleles. The amplicons were detected simultaneously through hybridization with the sequence-specific hairpin probes anchored on a reduced graphene oxide-gold nanoparticles functionalized electrode surface. With the inclusion of an LNA-mediated PCR step upstream of the electrochemical detection, we improved the limit of detection (LOD) by 2 orders of magnitude, down to an ultralow-level of 5 copies μL−1. The platform achieved an ultra-sensitive and specific detection with 0.05% against a background of 10, 000 copies of wild-type alleles. It is highly adaptive and has the potential to enable expanded multiplexed detection in parallel, thus providing a universal tool for multiplexed SbM identification.

A host-based two-gene model for the identification of bacterial infection in general clinical settings

ObjectivesIn this study, we aimed to develop a simple gene model to identify bacterial infection, which can be implemented in general clinical settings. MethodsWe used a clinically availablereal-time quantitative polymerase chain reaction platform to conduct focused gene expression assays on clinical blood samples. Samples were collected from 2 tertiary hospitals. ResultsWe found that the 8 candidate genes for bacterial infection were significantly dysregulated in bacterial infection and displayed good performance in group classification, whereas the 2 genes for viral infection displayed poor performance. A two-gene model (S100A12 and CD177) displayed 93.0% sensitivity and 93.7% specificity in the modeling stage. In the independent validation stage, 87.8% sensitivity and 96.6% specificity were achieved in one set of case-control groups, and 93.6% sensitivity and 97.1% specificity in another set. ConclusionsWe have validated the signature genes for bacterial infection and developed a two-gene model to identify bacterial infection in general clinical settings.

Bartonella rochalimae Detection by a Sensitive and Specific PCR Platform.

Bartonella rochalimae is an emerging zoonotic pathogen present in the United States, South America, and Europe. The molecular detection of B. rochalimae frequently relies on polymerase chain reaction (PCR) assays that target the genus Bartonella coupled with DNA sequencing for species determination. However, the presence of other Bartonella spp. in the sample being tested may result in false-negative results for B. rochalimae, especially when Sanger sequencing is used. We developed a sensitive and specific quantitative PCR platform for B. rochalimae by targeting the intergenic transcribed spacer, gltA, and rpoB genes, which are recommended for subtyping characterization. This PCR platform achieved the limit of detection between five and 10 genomic equivalents per reaction and did not amplify DNA from other Bartonella species or selected hosts. This PCR platform is a fast and cost-effective option to be used in epidemiological evaluations of reservoirs and vectors and in detecting and quantifying B. rochalimae infection in humans.

Role of regulatory T cells and CD8+ T lymphocytes in the dissemination of circulating tumor cells in primary invasive breast cancer.

Circulating tumor cells (CTCs) dissemination is involved in tumor metastasis and is an independent prognostic factor in patients with primary and metastatic breast cancer. Regulatory T cells (Tregs) and cluster of differentiation (CD)8+ T lymphocytes are the main types of immune cells in the tumor microenvironment and exert opposite roles on the progression and outcome of breast cancer. The aim of the present study was to evaluate the associations between CTCs and intratumoral/peritumoral Tregs and CD8+ T lymphocytes in breast cancer. Peripheral CTCs were detected by a multi-marker quantitative polymerase chain reaction platform in 167 patients with invasive breast cancer. Intratumoral/peritumoral Tregs and CD8+ T lymphocytes were assessed by immunohistochemistry in 167 patients with invasive breast cancer to establish an association between these cell types and detection of peripheral CTCs. CTCs were detected in 55% of the patients with breast cancer. The prevalence of CTCs was positively associated with the number of intratumoral (P=0.002) and peritumoral Tregs (P=0.045), and the primary tumor size (P=0.012). This result was verified by analyzing intratumoral Tregs (P=0.044) and primary tumor size (P=0.044) with multivariate analysis, which indicated that the CTC-positive rate increased with an increasing number of intratumoral Tregs and a larger tumor size In the multivariate analysis, other variables including menopause, tumor-node-metastasis stage and peritumoral Tregs were not associated with the prevalence of CTCs. The prevalence of CTCs was inversely and weakly associated with the number of peritumoral CD8+ T lymphocytes using the univariate analysis, however this result was not statistically significant (P=0.470). In conclusion, regulatory T cells and CD8+ T lymphocytes may be involved, at least in part, in the CTCs dissemination of breast cancer, whereby Tregs appear to exert the dominant effect. Furthermore, the role of Tregs in the progression of breast cancer may be mediated by suppressing the dissemination of CTCs, which is primarily determined by intratumoral Tregs.

Pigs are useful for the molecular study of bone inflammation and regeneration in humans.

Pigs are used with increased frequency to model different kinds of orthopedic surgical conditions. In order to show the full potential of porcine models in orthopedic research, it is therefore required to examine the expression of bone regulatory genes in pigs affected by orthopedic surgery and compare it to the expression in humans and mice as mice, are one of the most applied animal species in orthopedics today. In the present study, the local molecular response to drilling of a tibial implant cavity, and the subsequent insertion of a steel implant was examined in a porcine model. Pigs were euthanized five days after drilling of the bone. The molecular response of 73 different genes was analyzed using a high-throughput quantitative polymerase chain reaction platform and compared to histopathology. Histologically, it was found that bone remodeling was initiated on day 5 after surgery and was associated with upregulation of several genes involved in bone degradation and formation ( CTSK, ACP5, IBSP, RANK, RANKL and COL1A1). Interleukin-6 and several acute-phase proteins (C3, SAA and ITIH4) were significantly upregulated, indicating their importance in the initial process of healing and osseointegration. All tested bone morphogenic proteins (BMP2, -4 and -7) including their inhibitor noggin were also significantly upregulated. Surprisingly, vascular endothelial growth factor A was not found to be regulated five days after surgery while several other vascular growth factors (ANGPT1, ANGPT2 and PTN) were upregulated. The pig was found to be a useful model for elucidation of bone regulatory genes in humans.

Technical Validation of a Reverse-Transcription Quantitative Polymerase Chain Reaction In Vitro Diagnostic Test for the Determination of MiR-31-3p Expression Levels in Formalin-Fixed Paraffin-Embedded Metastatic Colorectal Cancer Tumor Specimens.

MiR-31-3p expression has been shown to be a predictive biomarker for response to anti-epithelial growth factor receptor therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). To aid in the quantification of miR-31-3p expression in formalin-fixed paraffin-embedded (FFPE) primary tumor samples from patients with mCRC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed and validated. Assay development included the identification of a microRNA reference standard and the determination of an appropriate relative quantification cutoff for differentiating low versus high miR-31-3p expression. Sample specimens for the validation studies included both FFPE slides and shavings. Polymerase chain reaction (PCR) efficiency and linearity, analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy were demonstrated across a number of test conditions and differing quantitative PCR platforms. The data from this study provide evidence as to the feasibility of quantifying the expression of miR-31-3p from FFPE tumor tissue using a standardized RT-qPCR assay.

Metallothioneins as dynamic markers for brain disease in lysosomal disorders

ObjectiveTo facilitate development of novel disease-modifying therapies for lysosomal storage disorder (LSDs) characterized by nervous system involvement such as metachromatic leukodystrophy (MLD), molecular markers for monitoring disease progression and therapeutic response are needed. To this end, we sought to identify blood transcripts associated with the progression of MLD.MethodsGenome-wide expression analysis was performed in primary T lymphocytes of 24 patients with MLD compared to 24 age- and sex-matched healthy controls. Genes associated with MLD were identified, confirmed on a quantitative polymerase chain reaction platform, and replicated in an independent patient cohort. mRNA and protein expression of the prioritized gene family of metallothioneins was evaluated in postmortem patient brains and in mouse models representing 6 other LSDs. Metallothionein expression during disease progression and in response to specific treatment was evaluated in 1 of the tested LSD mouse models. Finally, a set of in vitro studies was planned to dissect the biological functions exerted by this class of molecules.ResultsMetallothionein genes were significantly overexpressed in T lymphocytes and brain of patients with MLD and generally marked nervous tissue damage in the LSDs here evaluated. Overexpression of metallothioneins correlated with measures of disease progression in mice and patients, whereas their levels decreased in mice upon therapeutic treatment. In vitro studies indicated that metallothionein expression is regulated in response to oxidative stress and inflammation, which are biochemical hallmarks of lysosomal storage diseases.InterpretationMetallothioneins are potential markers of neurologic disease processes and treatment response in LSDs.

Rapid Temporal Changes in the Expression of a Set of Neuromodulatory Genes During Alcohol Withdrawal in the Dorsal Vagal Complex: Molecular Evidence of Homeostatic Disturbance

Chronic alcohol exposure produces neuroadaptation, which increases the risk of cellular excitotoxicity and autonomic dysfunction during withdrawal. The temporal progression and regulation of the gene expression that contributes to this physiologic and behavioral phenotype is poorly understood early in the withdrawal period. Further, it is unexplored in the dorsal vagal complex (DVC), a brainstem autonomic regulatory structure. We use a quantitative polymerase chain reaction platform to precisely and simultaneously measure the expression of 145 neuromodulatory genes in more than 100 rat DVC samples from control, chronically alcohol-exposed, and withdrawn rats. To gain insight into the dynamic progression and regulation of withdrawal, we focus on the expression of a subset of functionally relevant genes during the first 48hours, when behavioral symptoms are most severe. In the DVC, expression of this gene subset is essentially normal in chronically alcohol-exposed rats. However, withdrawal results in rapid, large-magnitude expression changes in this group. We observed differential regulation in 86 of the 145 genes measured (59%), some as early as 4hours into withdrawal. Time series measurements (4, 8, 18, 32, and 48hours after alcohol removal) revealed dynamic expression responses in immediate early genes, γ-aminobutyric acid type A, ionotropic glutamate, and G-protein coupled receptors and the Ras/Raf signaling pathway. Together, these changes elucidate a complex, temporally coordinated response that involves correlated expression of many functionally related groups. In particular, the expression patterns of Gabra1, Grin2a, Grin3a, and Grik3 were tightly correlated. These receptor subunits share overrepresented transcription factor binding sites for Pax-8 and other transcription factors, suggesting a common regulatory mechanism and a role for these transcription factors in the regulation of neurotransmission within the first 48hours of alcohol withdrawal. Expression in this gene set is essentially normal in the alcohol-adapted DVC, but withdrawal results in immediate, large-magnitude, and dynamic changes. These data support both increased research focus on the biological ramifications of alcohol withdrawal and enable novel insights into the dynamic withdrawal expression response in this understudied homeostatic control center.

Systematic Evaluation of DNA-Based Quantitative-Polymerase Chain Reaction (Q-PCR) Assays to Track Treatment Response in Patients with JAK2-V617F Associated Myeloproliferative Neoplasms: A Joint European LeukemiaNet/ MPN&MPNr-EuroNet Study

Abstract▪2812▪This icon denotes a clinically relevant abstractSince the discovery of the JAK2-V617F mutation, the relative level of the mutant allele has been widely studied to gain further insights into the biology of myeloproliferative neoplasms and to establish utility in predicting clinical outcome. Mutant allele burden has been correlated with risk of thrombotic events in essential thrombocythemia, severity of the disease phenotype in polycythemia vera (PV) and survival in primary myelofibrosis (PMF). JAK2-V617F has also been quantified to assess disease response, with significant reductions in allele burden reported in PV patients following interferon (IFN) α-2b and pegylated IFN α-2a treatment. Moreover, serial DNA-based quantitative polymerase chain reaction (Q-PCR) assays have been used after allogeneic transplantation for myelofibrosis to predict outcome and guide donor lymphocyte infusion. With quantification of JAK2-V617F being considered an endpoint in a number of trials with novel agents including JAK2 inhibitors, it is important that assays are robust, mutant-specific and afford a suitable level of sensitivity. However, numerous JAK2-V617F Q-PCR assays have now been published, which based on experience of molecular monitoring in other hematological malignancies, are likely to vary markedly in their performance. Indeed, this concern was confirmed by the results of a quality control (QC) exercise involving 5 European LeukemiaNet (ELN) laboratories, each with their own established JAK2-V617F Q-PCR assay, who reported markedly differing levels of mutant allele percentage (ranging from 23–80%) in a QC sample distributed by UK NEQAS that comprised a 1 in 30 dilution of HEL cells (which harbor multiple copies of JAK2-V617F and lack the wild type [WT] JAK2 allele) in K562 cells (WT JAK2). To address this issue, a joint initiative was established within European LeukemiaNet between the Chronic Myeloproliferative Diseases and Minimal Residual Disease working groups (WP9 & WP12) to systematically evaluate a range of published and “in-house” JAK2-V617F assays, with the aim of identifying sensitive mutant-specific assays that perform similarly on different Q-PCR platforms – factors of fundamental importance for reliable tracking of disease response. The study involved 12 expert centers - in 6 European countries and included 1 US laboratory, which between them used Q-PCR platforms from 3 different manufacturers. The study involved central distribution of DNA from serial dilutions of JAK2-V617F mutant cell lines (HEL &/or UKE-1) in K562 and plasmid standards for JAK2 WT, JAK2-V617F and independent control genes (albumin, cyclophilin). Reagents for each assay were coded and also centrally distributed; reaction conditions for each coded assay were provided. Laboratories with established published or in-house Q-PCR assays were encouraged to perform these in parallel with the coded test assays on the distributed QC materials, to control for local variations in test conditions. Data from each QC round were submitted by participating laboratories for independent central analysis (Dept. of Genetics, King’s College London); conclusions were drawn concerning the performance of the various assays before breaking the code to reveal their identities. Overall, 7 successive QC rounds were conducted, evaluating 9 assays (6 published, 3 unpublished “in-house”). Six assays were eliminated, due to insufficient/inferior sensitivity (n=5) or inconsistent performance (n=1). Of the 3 assays with better performance profile, the assay published by Lippert et al (Blood 2006;108 :1865) reliably quantified JAK2-V617F across the diagnostic range, with consistent results obtained between laboratories irrespective of Q-PCR platform. The 2 remaining assays (Larsen et al, Br J Haematol. 2007;136 :745 & in-house assay from the Oppliger Leibundgut lab [Bern]), afforded better sensitivity, capable of detecting a 0.08–0.008 % level of JAK2-V617F in DNA preparations from HEL and K562 cells. The latter assays were tested on serial post-transplant samples from 2 patients allografted for JAK2-V617F mutant PMF, successfully identifying the presence of residual disease, complementing chimerism analysis to predict clinical outcome. In conclusion, this ELN study has identified robust assays suitable for quantifying JAK2-V617F in clinical trials and which merit further investigation as a tool to guide management of patients undergoing allogeneic transplant. Disclosures:No relevant conflicts of interest to declare.