Transcriptome analysis of prion disease animal models

Prion diseases are incurable and fatal neurodegenerative disorders that affect both humans and animals. The causative agent is an infectious protein called prion (PrPSc), which is the pathological form of a normal protein (PrPC) present on the cell membrane. The molecular mechanisms underlying prion replication and subsequent degeneration of the Central Nervous System (CNS) are still poorly understood and therefore innovative approaches are needed to build diagnostic, therapeutic, taxonomic, and disease surveillance tools. We are going to adopt an unbiased genomic approach and conduct whole transcriptome analyses using microarray gene expression methods in brain and/or blood of infected animals versus healthy controls. We hope to identify a set of genes that can be used for early diagnosis and/or as targets for therapeutic strategies. Within the Trans2Care project we intend to promote collaboration and exchange of knowledge to facilitate all partners’ research objectives, and possibly find a common way to accelerate the process aimed at improving our healthcare system

Novel markers for neurodegeneration

Prion diseases are incurable and fatal neurodegenerative disorders that affect both humans and animals. The causative agent is an infectious protein called prion (PrPSc), which is the pathological form of a normal protein (PrPC) present on the cell membrane. The molecular mechanisms underlying prion replication and subsequent degeneration of the Central Nervous System (CNS) are still poorly understood and therefore innovative approaches are needed to build diagnostic, therapeutic, taxonomic, and disease surveillance tools. We adopted an unbiased genomic approach and conducted whole transcriptome analyses using microarray and RT-qPCR gene expression methods in brain of infected macaques versus healthy controls. We identified a set of genes that could become novel biomarkers for early diagnosis and/or therapeutic strategies for prion diseases and other neurodegenerative disorders

Real-time quantification of microRNAs by stem–loop RT–PCR

A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency

Whole blood gene expression profiling in preclinical and clinical cattle infected with atypical bovine spongiform encephalopathy

Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named Hand L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSEinfected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions. \ua9 2016 Xerxa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Assessment of DNA Extracted from Forensic Samples Prior to Genotyping

"The field of forensic DNA analysis has grown immensely in the past two decades and genotyping of biological samples is now routinely performed in human identification (HID) laboratories. Application areas include paternity testing, forensic casework, family lineage studies, identification of human remains, and DNA databasing. Forensic DNA Analysis: Current Practices and Emerging Technologies explores the fundamental principles and the application of technologies for each aspect of forensic DNA analysis. The book begins by discussing the value of DNA evidence and how to properly recognize, document, collect, and store it. The remaining chapters examine: - The most widely adopted methods and the best practices for DNA isolation from forensic biological samples and human remains - Studies carried out on the use of both messenger RNA and small (micro) RNA profiling - Real-time polymerase chain reaction (PCR) methods for quantification and assessment of human DNA prior to genotyping - Capillary electrophoresis (CE) as a tool for forensic DNA analysis - Next-generation short tandem repeat (STR) genotyping kits for forensic applications, the biological nature of STR loci, and Y-chromosome STRs (Y-STRs) - Mitochondrial DNA (mtDNA) sequence analysis - Single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (indels) in typing highly degraded DNA - Deep-sequencing technologies - The current state of integrated systems in forensic DNA analysis. The book concludes by discussing various aspects of sample-processing training and the entities that provide such training programs. This volume is an essential resource for students, researchers, teaching faculties, and other professionals interested in human identification/forensic DNA analysis.

Whole transcriptome analysis in brains from BSE-infected macaques

Prion diseases are neurodegenerative disorders that affect both humans and animals. The molecular mechanisms underlying prion replication and subsequent degeneration of the central nervous system are still poorly understood. In an attempt to identify molecules that are putatively involved in the etiology of these diseases, we conducted a whole transcriptome analysis with brain tissue from BSE-infected and uninfected cynomolgus macaques (M. fascicularis). Total RNA from the gyrus frontalis region of seven BSE-infected and five noninfected control macaques was isolated. The integrity of the RNA was assessed using an Agilent 2100 Bioanalyzer. The RNA was reverse-transcribed, labeled and analyzed on an GeneChip\uae Rhesus Macaque Genome Array (Affymetrix) containing 52,024 Macaca mulatta probe sets to monitor the gene expression of approximately 47,000 transcripts. Bioinformatic analysis revealed that about 100 transcripts were significantly up-or down-regulated more than twofold. Beside others, we found up-regulation of \u3b11-antichymotrypsin, which has also been described in scrapie-infected mice and Alzheimer\u2019s disease. We are currently validating the most interesting candidates using quantitative RT-PCR. Our approach may help to identify genes that are crucial to prion disease processes and may become potential targets for diagnostic and therapeutic strategies

Functional classification of differentially expressed genes in blood of infected cattle versus control group^a.

<p>Functional classification of differentially expressed genes in blood of infected cattle versus control group<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153425#t002fn001" target="_blank">^a</a>.</p

Validation of microarray data by RT-qPCR.

<p>Differential expression of selected genes in blood from preclinical (A) and clinical (B) atypical BSE-infected cattle. Ganulysin (GNLY), X-inactive specific transcript (XIST), pyruvate dehydrogenase kinase 4 (PDK4), CD40 ligand (CD40L), haemoglobin, alpha 2 (HBA2) and Sel-1 Suppressor Of Lin-12-Like 3 protein (SEL1L3). Gene expression (ratio) values are represented as relative to RNA levels in control animals. Ns = not significant; *<i>P</i> value ≤ 0.05.</p