Comparison of blood RNA extraction methods used for gene expression profiling in amyotrophic lateral sclerosis.
Comparison of blood RNA extraction methods used for gene expression profiling in amyotrophic lateral sclerosis.
复制标题
DOI：
10.1371/journal.pone.0087508
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Shaw PJ
中科院分区：
文献类型：
--
作者：
Bayatti N;Cooper-Knock J;Bury JJ;Wyles M;Heath PR;Kirby J;Shaw PJ
肌萎缩性侧索硬化症（ALS）是一种神经退行性疾病，从症状开始平均2-3年内导致死亡。没有诊断测试，从症状出现到诊断平均延迟12个月。识别ALS的预后和诊断性生物标志物将有助于早期诊断和更快地监测治疗。基因表达谱（GEP）可以帮助识别这些标记以及神经系统疾病的治疗靶点。ALS患者GEP遗传物质的一个来源是外周血，通常从患者身上获取。然而，血液中高比例的珠蛋白mRNA可以掩盖重要的遗传信息。许多方法允许安全收集，储存和运输血液以及RNA稳定，包括用于收集全血的PAXGENE和TEMPUS系统以及丰富白细胞群的LEUKOLOCK。在这里，我们比较了这三种系统，并评估了它们对ALS患者GEP的适用性。我们采集了8例散发性ALS患者和7例对照者的血液。PAXGENE和TEMPUS RNA提取的样品还使用GlobinClear去除珠蛋白。将RNA扩增并杂交到Affymetrix U133 Plus 2.0阵列上。利用R包PUMA建立每种方法在ALS患者和对照组中的差异调控基因列表，并对所选基因进行RT-PCR验证。TEMPUS/GlobinClear和LEUKOLOCK生产出高质量的RNA，产量充足，阵列表达谱一致。PAXGENE/GlobinClear产量和质量较低。PAXGENE和TEMPUS的珠蛋白耗尽发现，与未耗尽样品相比，存在超过60%的转录本。TEMPUS/GlobinClear和LEUKOLOCK基因列表分别包含3619个和3047个患者与对照组差异表达的基因。实时PCR验证显示，这两种方法之间的可靠性相似，基因本体分析显示，与对照组相比，疾病中存在相似的差异调节途径。
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.
登录
查看更多内容
影响因子：
3.5
作者：
Kudo, Lili C.;Parfenova, Liubov;Karsten, Stanislav L.
通讯作者：
Karsten, Stanislav L.
影响因子：
4.1
作者：
Debey-Pascher, Svenja;Hofmann, Andrea;Staratschek-Jox, Andrea
通讯作者：
Staratschek-Jox, Andrea
影响因子：
2.7
作者：
Prezeau, N;Silvy, M;Picard, C
通讯作者：
Picard, C
差异基因表达谱取决于外周血收集和RNA分离的方法。
影响因子：
4.4
作者：
Asare, Adam L.;Kolchinsky, Svetlana A.;Gao, Zhong;Wang, Richard;Raddassi, Khadir;Bourcier, Katarzyna;Seyfert-Margolis, Vicki
通讯作者：
Seyfert-Margolis, Vicki
影响因子：
1.1
作者：
Oster, Marina;Pollinger, John P.;Wayne, Robert K.
通讯作者：
Wayne, Robert K.