Comparison of blood RNA extraction methods used for gene expression profiling in amyotrophic lateral sclerosis.
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.
Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom.
Nadhim
N
Bayatti
Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom.
Johnathan
J
Cooper-Knock
Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom.
Joanna J
JJ
Bury
Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom.
Matthew
M
Wyles
Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom.
Paul R
PR
Heath
Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom.
Janine
J
Kirby
Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom.
Pamela J
PJ
Shaw
D000690:Amyotrophic Lateral Sclerosis / Q000175:diagnosis* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D063990:Gene Ontology; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods; D012313:RNA / Q000097:blood / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction
D017931:DNA Primers; D012313:RNA