Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes death within a mean of 2–3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP) can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses revealed similar pathways differentially regulated in disease compared to controls.
Highlights
Amyotrophic lateral sclerosis (ALS) is a devastating and fatal disease that preferentially affects the motor system
Gene expression profiling (GEP) with blood is a feasible approach for biomarker discovery in neurological disease
The present study primarily highlights the importance of reducing the high levels of globin present when using blood for GEP profiling in neurological disease [7]
Summary
Amyotrophic lateral sclerosis (ALS) is a devastating and fatal disease that preferentially affects the motor system. The identification of biomarkers that may detect the early signs of ALS, assess disease progression, monitor the effects of treatment, or even help identify the cause of the disease is of great importance. Gene expression profiling (GEP) is a powerful tool to help identify potential diagnostic and therapeutic targets in neurological diseases [3,4,5]. GEP of whole blood is informative in studying the mechanisms and pathogenesis of a number of diseases, including neurological disorders, the high proportion of globin mRNA present in red blood cells masks potentially important genetic information, and increases noise, thereby reducing sensitivity [6,7]
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