Search

Studying the molecular mechanisms of the pathogenesis of Parkinson's disease (PD) is critical to improve PD treatment. We used OpenArray technology to assess gene expression in the substantia nigra (SN) cells of mice in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD and in controls. Among the 11 housekeeping genes tested, Rps27a was taken as the reference gene due to its most stable expression in normal and experimental conditions. From 101 genes encoding functionally significant proteins of nigrostriatal dopaminergic neurons, 57 highly expressed genes were selected to assess their expressions in the PD model and in the controls. The expressions of Th, Ddc, Maoa, Comt, Slc6a3, Slc18a2, Drd2, and Nr4a2 decreased in the experiment compared to the control, indicating decreases in the synthesis, degradation, and transport of dopamine and the impaired autoregulation of dopaminergic neurons. The expressions of Tubb3, Map2, Syn1, Syt1, Rab7, Sod1, Cib1, Gpx1, Psmd4, Ubb, Usp47, and Ctsb genes were also decreased in the MPTP-treated mice, indicating impairments of axonal and vesicular transport and abnormal functioning of the antioxidant and ubiquitin-proteasome systems in the SN. The detected decreases in the expressions of Snca, Nsf, Dnm1l, and Keap1 may serve to reduce pathological protein aggregation, increase dopamine release in the striatum, prevent mitophagy, and restore the redox status of SN cells. [ABSTRACT FROM AUTHOR]

Sepsis is a dysregulated immune response to infections that frequently precipitates multiple organ dysfunction and death despite intensive supportive therapy. The aim of the present study was to identify sepsis-induced alterations in the signaling transcriptome of peripheral blood leukocytes that might shed light on the elusive transition from proinflammatory to anti-inflammatory responses and underlie long-term post-sepsis immunosuppression. Peripheral blood leukocytes were collected from subjects (i) with systemic inflammation, (ii) with sepsis in the acute phase and (iii) 6 months after recovery from sepsis, corresponding to progressive stages of the disease. Transcriptomic analysis was performed with the QuantStudio 12K Flex OpenArray Human Signal Transduction Panel analyzing transcripts of 573 genes playing a significant role in signaling. Of them, 145 genes exhibited differential expression in sepsis as compared to systemic inflammation. Pathway analysis revealed enhanced expression levels of genes involved in primary immune responses (proinflammatory cytokines, neutrophil and macrophage activation markers) and signatures characteristic of immunosuppression (increased expression of anti-inflammatory cytokines and proapoptotic genes; diminished expression of T and B cell receptor dependent activating and survival pathways). Importantly, sepsis-induced expression patterns of 39 genes were not normalized by the end of the 6-month follow-up period, indicating expression aberrations persisting long after clinical recovery. Functional analysis of these transcripts revealed downregulation of the antiapoptotic Wnt and mTOR signaling pathways that might explain the post-sepsis immunosuppression commonly seen in sepsis survivors. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a neurodegenerative disorder caused by the selective destruction of dopaminergic neurons (DA-nergic). Clinically, PD is diagnosed based on developing signs and symptoms. A neurological and physical examination and sometimes medical and family history also help in the diagnosis of PD. However, most of these features are visible when more than 80% of the dopaminergic neurons have degenerated. An understanding of the selective degeneration process at the cellular and molecular level and the development of new biomarkers are required for effective PD management. Several studies have been carried out using a selected set of miRNAs/ mRNAs and proteins to develop biomarkers of PD; however, an unbiased and combined miRNA–protein profiling study was required to identify the markers of progressive and selected degeneration of dopaminergic neurons in PD patients. In the present study, we have carried out global protein profiling through LC–MS/MS and miRNA profiling by using a "brain-specific" miRNA array panel of 112 miRNAs in PD patients and healthy controls to find the unprejudiced group of proteins and miRNAs that are deregulating in PD. In the whole blood samples of PD patients compared to healthy controls, the expression of 23 miRNAs and 289 proteins was significantly increased, whereas the expression of 4 miRNAs and 132 proteins was considerably downregulated. Network analysis, functional enrichment, annotation, and analysis of miRNA–protein interactions were also performed as part of the bioinformatics investigation of the discovered miRNAs and proteins revealing several pathways that lead to PD development and pathogenesis. Based on the analysis of miRNA and protein profiling, we have identified four miRNAs (hsa-miR-186-5p, miR-29b, miR-139 & has-miR-150-5p) and four proteins (YWHAZ, PSMA4, HYOU1, & SERPINA1), which can be targeted for the development of new biomarkers of PD. In vitro studies have identified the role of miR-186-5p in regulating the levels of the YWHAZ/YWHAB & CALM2 gene, which has shown maximum downregulation in PD patients and is known for its role in neuroprotection from apoptotic cell death & calcium regulation. In conclusion, our research has identified a group of miRNA–proteins that can be developed as PD biomarkers; however, future studies on the release of these miRNAs and proteins in extracellular vesicles circulating in the blood of PD patients can further validate these as specific biomarkers of PD. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the age-related neurological disorder characterized by the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). PD is based on motor deficits which start to appear when up to 80% of the DA neurons of SNpc have been lost. Effective management of PD requires the development of novel biomarkers. Therefore, the present study aimed to characterize biomarkers of PD using miRNomics, proteomics, and bioinformatics approaches. Rats exposed to rotenone (2.5 mg/kg b.wt) for 2 months were used as an animal model to identify the unbiased set of miRNAs and proteins deregulated in blood samples. OpenArray, a real-time PCR-based array, is used for high-throughput profiling of miRNAs, and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to carry out the global protein profiling. Systematic bioinformatics analysis of miRNAs and proteins was also performed, including annotation, functional classification and functional enrichment, network analysis, and miRNA–protein interaction analysis. Expression of 19 miRNAs and 96 proteins was significantly upregulated in the blood, while 22 proteins were significantly downregulated in blood samples of rotenone-exposed rats. In silico pathway analysis of deregulated proteins and miRNAs in rotenone-exposed rats has identified multiple pathways leading to PD. In summary, we have identified a set of miRNAs (miR-144, miR-96, and miR-29a) and proteins (PLP1, TUBB4A, and TUBA1C), which can be used as a potential biomarker of PD, while further validation required large human population studies. [ABSTRACT FROM AUTHOR]

Background: Pharmacogenomics has the potential to improve patient outcomes through predicting drug response. We designed and evaluated the analytical performance of a custom OpenArray® pharmacogenomics panel targeting 478 single-nucleotide variants (SNVs). Methods: Forty Coriell Institute cell line (CCL) DNA samples and DNA isolated from 28 whole-blood samples were used for accuracy evaluation. Genotyping calls were compared to at least 1 reference method: next-generation sequencing, Sequenom MassARRAY®, or Sanger sequencing. For precision evaluation, 23 CCL samples were analyzed 3 times and reproducibility of the assays was assessed. For sensitivity evaluation, 6 CCL samples and 5 whole-blood DNA samples were analyzed at DNA concentrations of 10 ng/µL and 50 ng/µL, and their reproducibility and genotyping call rates were compared. Results: For 443 variants, all samples assayed had concordant calls with at least 1 reference genotype and also demonstrated reproducibility. However, 6 of these 443 variants showed an unsatisfactory performance, such as low PCR amplification or insufficient separation of genotypes in scatter plots. Call rates were comparable between 50 ng/µL DNA (99.6%) and 10 ng/µL (99.2%). Use of 10 ng/µL DNA resulted in an incorrect call for a single sample for a single variant. Thus, as recommended by the manufacturer, 50 ng/µL is the preferred concentration for patient genotyping. Conclusions: We evaluated a custom-designed pharmacogenomics panel and found that it reliably interrogated 437 variants. Clinically actionable results from selected variants on this panel are currently used in clinical studies employing pharmacogenomics for clinical decision-making. [ABSTRACT FROM AUTHOR]

Abstract Objective A simple, non-invasive sample collection method is key for the integration of pharmacogenetics into clinical practice. The aim of this study was to gain samples for pharmacogenetic testing and evaluate the variation between dry-flocked and sponge-tipped buccal swabs in yield and quality of DNA isolated. Results Thirty-one participants collected samples using dry-flocked swabs and sponge-tipped swabs. Samples were assessed for DNA yield, quality and genotyping performance on a qPCR OpenArray platform of 28 pharmacogenetic SNPs and a CYP2D6 TaqMan copy number variant. DNA from sponge-tipped swabs had a significantly greater yield compared to DNA collected with dry-flocked swabs (p = 4.4 × 10−7). Moreover, highest genotyping call rates across all assays and highest CNV confidence scores were observed in DNA samples collected from sponge-tipped swabs (97% vs. 54% dry-flocked swabs; 0.99 vs. 0.88 dry-flocked swabs, respectively). Sample collection using sponge-tipped swabs provides a DNA source of sufficient quantity and quality for pharmacogenetic variant detection using qPCR.

Nurgul Sikhayeva,1,2 Yerkebulan Talzhanov,1 Aisha Iskakova,1 Jarkyn Dzharmukhanov,1 Raushan Nugmanova,1 Elena Zholdybaeva,1 Erlan Ramanculov1–3 1National Scientific Laboratory of Biotechnology, National Center for Biotechnology, Astana, Kazakhstan; 2Faculty of Natural Sciences, L.N. Gumilyov Eurasian National University, Astana, Kazakhstan; 3School of Science and Technology, Nazarbayev University, Astana, Kazakhstan Background: Ethnic differences exist in the frequencies of genetic variations that contribute to the risk of common disease. This study aimed to analyse the distribution of several genes, previously associated with susceptibility to type 2 diabetes and obesity-related phenotypes, in a Kazakh population.Methods: A total of 966 individuals belonging to the Kazakh ethnicity were recruited from an outpatient clinic. We genotyped 41 common single nucleotide polymorphisms (SNPs) previously associated with type 2 diabetes in other ethnic groups and 31 of these were in Hardy–Weinberg equilibrium. The obtained allele frequencies were further compared to publicly available data from other ethnic populations. Allele frequencies for other (compared) populations were pooled from the haplotype map (HapMap) database. Principal component analysis (PCA), cluster analysis, and multidimensional scaling (MDS) were used for the analysis of genetic relationship between the populations.Results: Comparative analysis of allele frequencies of the studied SNPs showed significant differentiation among the studied populations. The Kazakh population was grouped with Asian populations according to the cluster analysis and with the Caucasian populations according to PCA. According to MDS, results of the current study show that the Kazakh population holds an intermediate position between Caucasian and Asian populations.Conclusion: A high percentage of population differentiation was observed between Kazakh and world populations. The Kazakh population was clustered with Caucasian populations, and this result may indicate a significant Caucasian component in the Kazakh gene pool. Keywords: single-nucleotide polymorphism, Kazakh population, type 2 diabetes, OpenArray

Background: The study of circulating miRNAs in CSF has gained tremendous attention during the last years, as these molecules might be promising candidates to be used as biomarkers and provide new insights into the disease pathology of neurological disorders. Objective: The main aim of this study was to describe an OpenArray panel of CSF-enriched miRNAs to offer a suitable tool to identify and characterize new molecular signatures in different neurological diseases. Methods: Two hundred and fifteen human miRNAs were selected to be included in the panel, and their expression and abundance in CSF samples were analyzed. In addition, their stability was studied in order to propose suitable endogenous controls for CSF miRNA studies. Results: miR-143-3p and miR-23a-3p were detected in all CSF samples, while another 80 miRNAs were detected in at least 70% of samples. miR-770-5p was the most abundant miRNA in CSF, presenting the lowest mean Cq value. In addition, miR-26b-5p, miR-335-5p and miR-92b-3p were the most stable miRNAs and could be suitable endogenous normalizers for CSF miRNA studies. Conclusions: These OpenArray plates might be a suitable and efficient tool to identify and characterize new molecular signatures in different neurological diseases and would improve the yield of miRNA detection in CSF.

Several lines of epidemiological and biochemical evidence support the association of type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC). T2DM has been shown to impinge on the transcriptome of colon tumor cells, promoting their proliferation and invasion. In order to gain insight into diabetes-specific modulation of colon cancer signaling, we analyzed gene expression patterns of more than five hundred genes encoding signaling proteins on TaqMan OpenArray panels from colonoscopic colorectal tumor samples of type 2 diabetic and non-diabetic patients. In total, 48 transcripts were found to be differentially expressed in tumors of T2DM patients as compared to healthy colon samples. Enrichment analysis with the g:GOSt (Gene Ontology Statistics) functional profiling tool revealed that the underlying genes can be classified into five signaling pathways (in decreasing order of significance: Wnt (wingless-type)/β-catenin; Hippo; TNF (tumor necrosis factor); PI3K/Akt (phosphoinositide-3 kinase/protein kinase B), and platelet activation), implying that targeted downregulation of these signaling cascades might help combat CRC in diabetic patients. Transcript levels of some of the differentially expressed genes were also measured from surgically removed diabetic and non-diabetic CRC specimens by individual qPCR (quantitative real-time PCR) assays using the adjacent normal tissue mRNA levels as an internal control. The most significantly altered genes in diabetic tumor samples were largely different from those in non-diabetic ones, implying that T2DM profoundly alters the expression of signaling genes and presumably the biological characteristics of CRC.

Background: Studies of genes involved in the absorption, distribution, metabolism, and excretion (ADME) of drugs are crucial to the development of therapeutics in clinical medicine. Such data provide information that may improve our understanding of individual differences in sensitivity or resistance to certain drugs, thereby helping to avoid adverse drug reactions (ADRs) in patients and improve the quality of therapies. Here, we aimed to analyse single nucleotide polymorphisms (SNPs) involved in the ADME of multiple drugs in Kazakhs from Kazakhstan. Results: A total of 158 SNPs involved in the ADME of various drugs were studied. We analysed 320 Kazakh DNA samples using OpenArray genotyping. Of the 158 SNPs, 75 were not found in heterozygous or homozygous variants. Comparative analysis among Kazakhs and world populations showed a fairly high percentage of population differentiation. Conclusion: These results provide further information for pharmacogenetic databases and may contribute to the development of personalized approaches and safer therapies for the Kazakh population. Moreover, these data provide insights into the different racial groups that may have contributed to the Kazakh population. [ABSTRACT FROM AUTHOR]

To date, most research on larval lipid nutrition has been centered on essential fatty acid requirements. However, less is known about effects of dietary fatty acid (FA) composition on lipid absorption and metabolism, as addressed in this study. Senegalese sole were fed live preys enriched with different oils: cod liver oil (CLO), linseed oil (LSO), soybean oil (SBO), and olive oil (OO), that are rich in long-chain polyunsaturated FAs (LC-PUFA), n-3 PUFA, n-6 PUFA or monounsaturated FAs (MUFA), respectively. Larvae reared on CLO showed significantly improved growth and survival, faster onset of metamorphosis and maturation of the intestine, lower lipid accumulation in liver after metamorphosis and an up-regulation of genes involved in lipid transport and phospholipid metabolism, while key lipogenesis genes were down-regulated. From the remaining treatments, the LSO diet induced the closest performance to CLO, with larvae completing metamorphosis at a similar time and having the second best growth and survival by the end of the experiment. They were also grouped closer to the CLO treatment than the remaining vegetable oil treatments, based on the patterns of gene expression. These results showed that oil sources rich in LC-PUFA and n-3 PUFA were superior to those having high n-6 PUFA or MUFA levels in the larval nutrition of Senegalese sole and indicate that this effect might be at least partly explained by an up-regulation of phospholipid metabolism and apolipoprotein synthesis, likely leading to enhanced lipid transport and mobilization, as well as tissue growth and remodeling. Statement of relevance The nutritional and physiological roles of dietary C18 PUFA in the absence of LC-PUFA have rarely been investigated in fish, particularly in larvae, as it is unfeasible to culture most marine species in such extreme dietary conditions. Senegalese sole (Solea senegalensis), a marine species of high commercial aquaculture importance in southern Europe, has relatively low LC-PUFA requirements during the larval stage compared to other marine finfish, making it a good model species to investigate this subject. A holistic approach was implemented to determine the effects these FAs may have on fish performance and metabolism, by analyzing growth, development, intestinal maturation, lipid and FA composition, hepatic and intestinal lipid accumulation, and expression of genes involved in lipid metabolism, absorption and transport (qPCR). [ABSTRACT FROM AUTHOR]

The article looks at a study related to intraethnic variation in prostate cancer patients with steroid-5-alpha-reductase polymorphisms. It states that steroid-5-alpha-reductase inhibitors are used for treatment of male alopecia and benign prostatic enlargement. It mentions that genotype distribution within different populations of the same ethnicity could be responsible for the failure of treatments based on 5α-RI.

Background: Besides serum levels of PSA, there is a lack of prostate cancer specific biomarkers. It is need to develop new biological markers associated with the tumor behavior which would be valuable to better individualize treatment. The aim of this study was to elucidate the relationship between single nucleotide polymorphisms (SNPs) in genes involved in DNA repair and prostate cancer progression. Methods: A total of 494 prostate cancer patients from a Spanish multicenter study were genotyped for 10 SNPs in XRCC1, ERCC2, ERCC1, LIG4, ATM and TP53 genes. The SNP genotyping was made in a Biotrove OpenArray? NT Cycler. Clinical t umor stage, diagnostic PSA serum levels, and Gleason score at diagnosis were obtained for all participants. Genotypic and allelic frequencies were determined using the web-based environment SNPator. Results: SNPs rs11615 (ERCC1) and rs17503908 (ATM) appeared as risk factors for prostate cancer aggressiveness. Patients wild homozygous for these SNPs (AA and TT, respectively) were at higher risk for developing cT2b - cT4 (OR = 2.21 (confidence interval (CI) 95% 1.47 - 3.31), p < 0.001) and Gleason scores = 7 (OR = 2.22 (CI95% 1.38 - 3.57), p < 0.001), respectively. Moreover, those patients wild homozygous for both SNPs had the greatest risk of presenting D'Amico high-risk tumors (OR = 2.57 (CI95% 1.28 - 5.16)). Conclusions: Genetic variants at DNA repair genes are associated with prostate cancer progression, and would be taken into account when assessing the malignancy of prostate cancer. [ABSTRACT FROM AUTHOR]

Abstract: Background and aim: Single nucleotide polymorphisms (SNPs) are substitutions of one base for another in the gene sequence and conforms the basis for pharmacogenetics and the development of personalized medicine. Many methods have been developed for SNP genotyping. The aim of the present study was to validate the use of a novel high-throughput genotyping system. Methods: Five SNPs (rs25487, rs25489, rs1799782, rs13181, and rs11615) were genotyped in 118 cancer patients using the classical method PCR restriction fragment length polymorphism (RFLP) and the high-throughput, automated assay Biotrove OpenArray® NT Cycler, trying to explore the feasibility and reproducibility of the OpenArray system in the context of oncology. Results: The call rates obtained ranged from 95.7 to 100% for both techniques. The percentage of overlapping ranged from 96.2 to 100% among both assays, showing a high reproducibility between the techniques. Conclusion: These findings, together with the low-cost and the simple and fast work flow, suggest that the OpenArray system is a robust and easy methodology for genotyping in the field of oncology. [Copyright &y& Elsevier]

Abstract: Enterohemorrhagic Escherichia coli (EHEC), including O157 and non-O157 serotypes are significant foodborne pathogens that require sensitive and discriminatory methods for detection and characterization. There are numerous PCR-based methods for the detection of EHEC virulence factors, but the time and cost involved with large-scale screening efforts and population level analyses have limited the size and scope of studies. Recent technological advancements have combined the high-throughput performance of the microarray with the specificity and sensitivity of real-time qPCR to make large-scale screening efforts both time- and cost-effective. This study identified and evaluated a panel of 28 genetic markers including known virulence and regulatory genes, O-antigen genes, and select prophage regions of O157 and non-O157 EHEC that can be used with high-throughput PCR to virulotype, serotype, and preliminarily subtype large numbers of isolates. The PCR assays for the target genes were shown to be robust using multiple extraction methods and PCR platforms. Preliminary quantitative PCR showed that an EHEC concentration of 104 CFU/ml or lower could be detected, with a linear range of detection over five to six orders of magnitude. The panel of 28 target genes has the potential to become an integral tool in outbreak, environmental, and genetic investigations of EHEC. [Copyright &y& Elsevier]

The management of neuropsychiatric disorders involves different pharmacological treatments. In order to perform efficacious drug treatments, the metabolism of CYP genes can help to foresee potential drug–drug interactions. The NeuroPGx software is an open-source web-based tool for genotype/diplotype/phenotype interpretation for neuropharmacogenomic purposes. The software provides information about: (i) the genotypes of evaluated SNPs (single nucleotide polymorphisms); (ii) the main diplotypes in CYP genes and corresponding metabolization phenotypes; (iii) the list of neuropsychiatric drugs with recommended dosage adjustment (according to CPIC and DPWG guidelines); (iv) the list of possible (rare) diplotypes and corresponding metabolization phenotypes. The combined application of NeuroPGx software to the OpenArray technology results in an easy, quick, and highly automated device ready to be used in routine clinical practice. [ABSTRACT FROM AUTHOR]

Background: The study of circulating miRNAs in CSF has gained tremendous attention during the last years, as these molecules might be promising candidates to be used as biomarkers and provide new insights into the disease pathology of neurological disorders. Objective: The main aim of this study was to describe an OpenArray panel of CSF-enriched miRNAs to offer a suitable tool to identify and characterize new molecular signatures in different neurological diseases. Methods: Two hundred and fifteen human miRNAs were selected to be included in the panel, and their expression and abundance in CSF samples were analyzed. In addition, their stability was studied in order to propose suitable endogenous controls for CSF miRNA studies. Results: miR-143-3p and miR-23a-3p were detected in all CSF samples, while another 80 miRNAs were detected in at least 70% of samples. miR-770-5p was the most abundant miRNA in CSF, presenting the lowest mean Cq value. In addition, miR-26b-5p, miR-335-5p and miR-92b-3p were the most stable miRNAs and could be suitable endogenous normalizers for CSF miRNA studies. Conclusions: These OpenArray plates might be a suitable and efficient tool to identify and characterize new molecular signatures in different neurological diseases and would improve the yield of miRNA detection in CSF. [ABSTRACT FROM AUTHOR]

Several lines of epidemiological and biochemical evidence support the association of type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC). T2DM has been shown to impinge on the transcriptome of colon tumor cells, promoting their proliferation and invasion. In order to gain insight into diabetes-specific modulation of colon cancer signaling, we analyzed gene expression patterns of more than five hundred genes encoding signaling proteins on TaqMan OpenArray panels from colonoscopic colorectal tumor samples of type 2 diabetic and non-diabetic patients. In total, 48 transcripts were found to be differentially expressed in tumors of T2DM patients as compared to healthy colon samples. Enrichment analysis with the g:GOSt (Gene Ontology Statistics) functional profiling tool revealed that the underlying genes can be classified into five signaling pathways (in decreasing order of significance: Wnt (wingless-type)/β-catenin; Hippo; TNF (tumor necrosis factor); PI3K/Akt (phosphoinositide-3 kinase/protein kinase B), and platelet activation), implying that targeted downregulation of these signaling cascades might help combat CRC in diabetic patients. Transcript levels of some of the differentially expressed genes were also measured from surgically removed diabetic and non-diabetic CRC specimens by individual qPCR (quantitative real-time PCR) assays using the adjacent normal tissue mRNA levels as an internal control. The most significantly altered genes in diabetic tumor samples were largely different from those in non-diabetic ones, implying that T2DM profoundly alters the expression of signaling genes and presumably the biological characteristics of CRC. [ABSTRACT FROM AUTHOR]

OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material., (© 2013-2021 The Journal of Biological Methods, All rights reserved.)

Alzheimer's disease is the most common form of dementia and is characterized by a progressive loss of cognitive functions. As the result of predicted demographic changes over the next decades, Alzheimer's disease is expected to be one of the most pressing medical and social challenges facing our generation. Current treatment strategies remain symptomatic. However, new approaches have shown promise in clinical trials, particularly in patients with only mild or moderate symptoms. Early detection of Alzheimer's disease is therefore of critical importance. Currently available diagnostic approaches (such as protein analysis in cerebrospinal fluid or neuroimaging), however, are expensive and invasive and therefore unsuitable for the screening of a large population. Consequently, Alzheimer's disease is generally diagnosed too late for effective intervention. MicroRNAs-readily measurable in biofluids and resistant to freeze-thaw and pH changes, have shown encouraging diagnostic potential in Alzheimer's disease. Several studies have attempted to correlate changes of specific microRNAs to disease progression using different approaches and profiling platforms including micro-arrays, RNA sequencing, and qPCR-based systems. In the present book chapter, we will describe the different steps involved in how to determine the microRNA profile in plasma samples from patients using the OpenArray platform.

Background Prostate cancer (PCa) is an androgen-dependent disease. Nonetheless, the role of single nucleotide polymorphisms (SNPs) in genes encoding androgen metabolism remains an unexplored area. Purpose To investigate the role of germline variations in cytochrome P450 17A1 ( CYP17A1 ) and steroid-5α-reductase, α-polypeptides 1 and 2 ( SRD5A1 and SRD5A2 ) genes in PCa. Patients and methods In total, 494 consecutive Spanish patients diagnosed with nonmetastatic localized PCa were included in this multicenter study and were genotyped for 32 SNPs in SRD5A1, SRD5A2, and CYP17A1 genes using a Biotrove OpenArray NT Cycler. Clinical data were available. Genotypic and allelic frequencies, as well as haplotype analyses, were determined using the web-based environment SNPator. All additional statistical analyses comparing clinical data and SNPs were performed using PASW Statistics 15. Results The call rate obtained (determined as the percentage of successful determinations) was 97.3% of detection. A total of 2 SNPs in SRD5A1 —rs3822430 and rs1691053—were associated with prostate-specific antigen level at diagnosis. Moreover, G carriers for both SNPs were at higher risk of presenting initial prostate-specific antigen levels>20 ng/ml (Exp( B ) = 2.812, 95% CI: 1.397–5.657, P = 0.004) than those who are AA-AA carriers. Haplotype analyses showed that patients with PCa nonhomozygous for the haplotype GCTTGTAGTA were at an elevated risk of presenting bigger clinical tumor size (Exp( B ) = 3.823, 95% CI: 1.280–11.416, P = 0.016), and higher Gleason score (Exp( B ) = 2.808, 95% CI: 1.134–6.953, P = 0.026). Conclusions SNPs in SRD5A1 seem to affect the clinical characteristics of Spanish patients with PCa. [ABSTRACT FROM AUTHOR]

Patient derived xenografts (PDXs) have become a vital, frequently used, component of anti-cancer drug development. PDXs can be serially passaged in vivo for years, and shared across laboratories. As a consequence, the potential for mis-identification and cross-contamination is possible, yet authentication of PDXs appears limited. We present a PDX Authentication System (PAS), by combining a commercially available OpenArray assay of single nucleotide polymorphisms (SNPs) with in-house R studio programs, to validate PDXs established in individual mice from acute lymphoblastic leukemia biopsies. The PAS is sufficiently robust to identify contamination at levels as low as 3%, similar to the gold standard of short tandem repeat (STR) profiling. We have surveyed a panel of PDXs established from 73 individual leukemia patients, and found that the PAS provided sufficient discriminatory power to identify each xenograft. The identified SNP-discrepant PDXs demonstrated distinct gene expression profiles, indicating a risk of contamination for PDXs at high passage number. The PAS also allows for the authentication of tumor cells with complex karyotypes from solid tumors including prostate cancer and Ewing's sarcoma. This study highlights the demands of authenticating PDXs for cancer research, and evaluates a reliable authentication platform that utilizes a commercially available and cost-effective system.