Acquiring genome-wide gene expression profiles in Guthrie card blood spots using microarrays

Standard Guthrie cards have been widely used to collect blood samples from essentially all USA and Japanese neonates for newborn screening programs. Thus, archival blood spot samples are a unique and comprehensive resource for molecular pathology studies. However, the challenge in using these samples is the presumed low quantity and degraded quality of nucleic acids that can be isolated from these samples, particularly the RNA. Here, we report a new assay using Agilent 4x44K microarrays for acquiring genome-wide gene expression profiles from blood spots on Guthrie cards. Due to the small amount of RNA obtained from each sample, major modifications, such as concentrating and amplifying the RNA and using a different labeling procedure, were performed. Approximately 9000 expressed genes can be detected after normalization of data, an increment of 260% in detection power compared with previously reported cDNA microarrays made in-house with standard procedures. The correlation coefficients in technical and biological replicates were 0.92 and 0.85, respectively, confirming the reproducibility of this study. This new and comprehensive assay will add value to the utility of archival Guthrie cards (e.g. neonatal blood spot cards) and open new opportunities to molecular epidemiology, pathology, genomic, and diagnostic studies of perinatal diseases.     

一种进行基因表达研究的有效技术--小鼠全胚胎RNA原位杂交

目的　建立小鼠整体胚胎水平研究基因表达的方法。　方法　采用地高辛配基标记的造血相关基因 Runx1和神经发生相关基因 Runx3RNA探针 ,对 10 .5～ 14天小鼠全胚胎进行 RNA原位杂交 ,通过检测胚胎组织中 m RNA的存在状况来观察基因的表达。结果　在小鼠胚胎观察到 Runx1和 Runx3基因在不同组织中的清晰的杂交信号 ;不同探针和不同大小的胚胎需要不同的最适蛋白酶 K处理条件。　结论　小鼠全胚胎 RNA原位杂交技术是一种有效的研究基因表达的方法 ,可以从整体水平反映基因表达的全貌 ,在功能基因组学时代将具有很大的应用潜力 ,为基因表达研究提供了一种可与 L ac Z染色和免疫组织化学媲美的选择。蛋白酶 K处理条件是否适当是小鼠全胚胎 RNA原位杂交成功的关键因素。     

Impact of acute psychosocial stress on peripheral blood gene expression pathways in healthy men

We investigated peripheral blood mononuclear cell gene expression responses to acute psychosocial stress to identify molecular pathways relevant to the stress response. Blood samples were obtained from 10 healthy male subjects before, during and after (at 0, 30, and 60 min) a standardized psychosocial laboratory stressor. Ribonucleic acid (RNA) was extracted and gene expression measured by hybridization to a 20,000-gene microarray. Gene Set Expression Comparisons (GSEC) using defined pathways were used for the analysis. Forty-nine pathways were significantly changed from baseline to immediately after the stressor (p < 0.05), implicating cell cycle, cell signaling, adhesion and immune responses. The comparison between stress and recovery (measured 30 min later) identified 36 pathways, several involving stress-responsive signaling cascades and cellular defense mechanisms. These results have relevance for understanding molecular mechanisms of the physiological stress response, and might be used to further study adverse health outcomes of psychosocial stress.     

多发性硬化外周全血mRNA差异表达的来源及其特异性分析

目的 探究多发性硬化外周全血mRNA差异表达的来源及其特异性.方法 利用外周全血mRNA表达谱数据,分别筛选多发性硬化和炎症性疾病(感染性休克、肺结核)患者相对正常人群的差异表达基因并进行比较.筛选正常外周血中髓系相对淋巴系细胞的差异表达基因,并与多发性硬化患者相对正常人群的差异表达基因作比较.结果 比较多发性硬化患者与两种炎症性疾病(感染性休克、肺结核)患者相对正常人群的差异表达基因,发现交叠基因上/下调方向的一致性分别为91.32%(P<0.05),93.23%(P<0.05).比较多发性硬化患者相对正常人群的差异表达基因及正常人群髓系相对淋巴系细胞的差异表达基因,发现交叠基因上/下调方向的一致性为96.82%(P<0.05).结论 多发性硬化患者相对于正常人群的差异表达基因与炎症性疾病患者相对正常人群的差异表达基因高度一致,同时与髓系相对淋巴系细胞的差异表达基因同样高度一致.上述结果表明多发性硬化患者外周全血中观察到的基因表达的改变可能是由于髓系/淋巴系细胞构成比例改变所致,是非特异的炎症性改变.     

Tumour necrosis factor (TNF) gene polymorphism influences TNF-α production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans

TNF-α is involved in infectious and immuno-inflammatory diseases. Different individuals may have different capacities for TNF-α production. This might determine a predisposition to develop some complications or phenotypes of these diseases. The aims of our study were to assess the inter-individual variability of TNF-α production and to correlate this variability to a single base pair polymorphism located at position ?308 in TNF gene. We studied 62 healthy individuals. TNF-α production after LPS stimulation was evaluated using a whole blood cell culture model. The TNF gene polymorphism was studied by an allele-specific polymerase chain reaction. Other cytokines produced in the culture, soluble CD14 concentrations and expression of CD14 on blood cells were also measured. Among the 62 individuals, 57 were successfully genotyped. There were 41 TNF1 homozygotes and 16 TNF1/TNF2 heterozygotes. TNF-α production after LPS stimulation of whole blood cell culture was higher among TNF2 carriers than among TNF1 homozygotes (929 pg/ml (480–1473 pg/ml) versus 521 pg/ml (178–1307 pg/ml); P < 0.05). This difference was even more significant after correction of TNF-α production for CD14 expression on blood cells. In conclusion, the single base pair polymorphism at position ?308 in the TNF gene may influence TNF-α production in healthy individuals.     

Tandem repeat sequence variation as causative cis-eQTLs for protein-coding gene expression variation: the case of CSTB

Association studies have revealed expression quantitative trait loci (eQTLs) for a large number of genes. However, the causative variants that regulate gene expression levels are generally unknown. We hypothesized that copy-number variation of sequence repeats contribute to the expression variation of some genes. Our laboratory has previously identified that the rare expansion of a repeat c.-174CGGGGCGGGGCG in the promoter region of the CSTB gene causes a silencing of the gene, resulting in progressive myoclonus epilepsy. Here, we genotyped the repeat length and quantified CSTB expression by quantitative real-time polymerase chain reaction in 173 lymphoblastoid cell lines (LCLs) and fibroblast samples from the GenCord collection. The majority of alleles contain either two or three copies of this repeat. Independent analysis revealed that the c.-174CGGGGCGGGGCG repeat length is strongly associated with CSTB expression (P = 3.14 × 10(-11)) in LCLs only. Examination of both genotyped and imputed single-nucleotide polymorphisms (SNPs) within 2 Mb of CSTB revealed that the dodecamer repeat represents the strongest cis-eQTL for CSTB in LCLs. We conclude that the common two or three copy variation is likely the causative cis-eQTL for CSTB expression variation. More broadly, we propose that polymorphic tandem repeats may represent the causative variation of a fraction of cis-eQTLs in the genome.     

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti-cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single-cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8⁻ T cells, IL-2 and interferon-gamma (IFN-γ) were optimally induced after 10 h stimulation with phorbol 12-myristate acetate (PMA)/ionomycin, and in CD8⁺ T cells IL-2 was optimally induced after 10 h and IFN-γ after 6 h. The levels of IL-2 and IFN-γ in CD8⁺ and CD8⁻ T cells in four healthy individuals were consistent on four occasions over a 3-month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL-2⁺ (range 9.7–41.3) and CD3/IFN-γ⁺ cells (10.1–44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3⁺/CD8⁺ peripheral blood T cells expressing IFN-γ and an increased percentage of CD3⁺/CD8⁻ T cells expressing IL-4 compared with non-atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.     

Microarray analysis of differential gene expression profile in peripheral blood cells of patients with human essential hypertension

The polygenic nature of essential hypertension and its dependence on environmental factors pose a challenge for biomedical research. We hypothesized that the analysis of gene expression profiles from peripheral blood cells would distinguish patients with hypertension from normotensives. In order to test this, total RNA from peripheral blood cells was isolated. RNA was reversed-transcribed and labeled and gene expression analyzed using significance Analysis Microarrays (Stanford University, CA, USA). Briefly, Significance Analysis Microarrays (SAM) thresholding identified 31 up-regulated and 18 down-regulated genes with fold changes of ≥2 or ≤0.5 and q-value≤5% in expression. Statistically significantly gene ontology (GO) function and biological process differentially expressed in essential hypertension were MHC class II receptor activity and immune response respectively. Biological pathway analysis identified several related pathways which are associated with immune/inflammatory responses. Quantitative Real-Time RT-PCR results were consistent with the microarray results. The levels of C-reactive protein were higher in hypertensive patients than normotensives and inflammation-related genes were increased as well. In conclusion, genes enriched for "immune/inflammatory responses" may be associated with essential hypertension. In addition, there is a correlation between systemic inflammation and hypertension. It is anticipated that these findings may provide accurate and efficient strategies for prevention, diagnosis and control of this disorder.     

Measurement of peripheral B cell subpopulations in common variable immunodeficiency (CVID) using a whole blood method

Recent reports have described reduced populations of CD27+ memory B cells and increased percentages of undifferentiated B cells in peripheral blood of patients with common variable immunodeficiency (CVID). This work has prompted two attempts to classify CVID based on rapid flow cytometric quantification of peripheral blood memory B cells and immature B cells. Evidence to support the hypothesis that such in vitro B cell classification systems correlate with clinical subtypes of CVID is being sought. For the classification to be useful in routine diagnosis, it is important that the flow cytometric method can be used without prior separation of peripheral blood mononuclear cells (PBMC). We have examined 23 CVID patients and 24 controls, using both PBMC and whole blood, and find an excellent correlation between these methods. The reproducibility of the method was excellent. We classified the CVID patients by all three of the existing classifications, including secretion of immunoglobulin by B cells in vitro as described by Bryant, as well as the more recent flow cytometric classification methods. Only one patient changed classification as a result of using whole blood.     

人脑胶质瘤基因表达谱芯片的构建和初步验证

目的：构建人脑胶质瘤特异的基因表达谱芯片,促进大规模胶质瘤表达谱的研究。方法：采用389条基因探针,优化基因芯片制作的各个条件：片基、点样液、点样大小、紫外交联、采集信号强度等。使用自制芯片检测20例胶质瘤标本的基因表达谱,并与以前报道的实验结果对比,验证芯片的特异性和灵敏度。结果：构建了针对人脑胶质瘤的基因表达谱芯片,并用于脑胶质瘤的基因表达谱分析,证实本产品可以有效地高通量检测相关基因的表达水平。结论：构建的胶质瘤芯片有望用于胶质瘤基因表达谱分析。     

应用微阵列初步分析髓母细胞瘤的基因表达谱

目的　应用微阵列技术研究髓母细胞瘤的分子发病机理。方法　收集新鲜髓母细胞瘤 4例及正常脑组织 1份的组织标本 ,提取总 RNA,逆转录成 32 P标记的 c DNA探针 ,与 Atlas人肿瘤芯片杂交 ,通过放射自显影获得基因谱 ,应用 Atlas Image TM1.0 1a分析。结果　与正常脑组织相比 ,髓母细胞瘤下调基因 6个 ,上调基因 35个 ;逆转录 -聚合酶链反应技术验证结果与芯片检测结果相符。除少数基因外 ,大部分基因的表达趋势与肿瘤生物学特性相符。结论　髓母细胞瘤是与星形细胞起源胶质瘤具有不同分子发病机理的多基因病变 ,不同基因之间可能存在复杂的相互作用和联系 ,值得进一步研究。     

应用微阵列初步研究少支胶质细胞瘤的基因表达谱

目的：应用微阵列研究少支胶质细胞瘤的基因表达谱。方法：从2例新鲜少支胶质细胞瘤及1份正常脑组织标本中提取总RNA，逆转录为^32P标记的cDNA、与Atlas微阵列杂交，洗膜、放射自显影，应用专用软件分析所得杂交图。结果：与正常脑组织相比，2例少支胶质细胞瘤共有63个基因表达上调，4个基因表达下调。2例肿瘤间基因表达量有显著差异。部分基因的表达趋与已知基因信息不同。结论：Atlas微阵列可高效显示少支胶质瘤的基因表达谱，并为进一步肿瘤分子发病机理提供新信息。     

Analysis of the inhibitory effect of whole human red blood cells on plasma heparin activity

The accelerating effect of whole human red blood cells (RBC) on the thrombin time of plasma (deprothrombinized, platelet-deprived) is manifested regardless of the presence or absence of calcium ions or of preincubation of the RBC with plasma and it is not accompanied by a fall of the free heparin level in the plasma. Acceleration of the transformation of the plasma fibrinogen into fibrin under the influence of whole RBC is unconnected with inhibition of endogenous heparin but reflects the fibrinoplastic effect of the cell. The experimental results do not support the view expressed in the literature that whole erythrocytes have a regulating effect on the blood level of endogenous heparin.Laboratory of Experimental and Clinical Hematology, I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. (Presented by Academician V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 11, pp. 526–528, November, 1977.     

Review of whole blood use in trauma

Traumatic haemorrhage is the leading cause of preventable death in both military and civilian settings. Haemostatic resuscitation with red cells, plasma, and platelets helps to address the problems caused by haemorrhagic shock and coagulopathy associated with trauma to improve survival. Whole blood (WB) is once again the primary recommended resuscitative blood product to be transfused in the military setting, and successes here have led to renewed interest in WB use in civilian trauma. This review provides a brief history of WB in trauma, highlights recent WB use, and discusses current considerations with transfusing WB.     

DNA microarray analysis reveals novel gene expression profiles in collagen-induced arthritis

Global gene expression was analyzed in early and late collagen-induced arthritis (CIA). Of 8734 cDNAs analyzed, 330 were induced and 55 downregulated greater than twofold in early or late disease. Hierarchical clustering of these 385 cDNAs demonstrated five distinct expression patterns differentiating early from late disease and correlating with histopathologic changes in the paw. Of the 385 cDNAs, 185 are known, characterized genes, the majority of which are not described as playing a role in arthritis. However, several of these genes are involved in pathological processes relating to arthritis, including apoptosis, inflammation, and cellular proliferation. One interesting gene, follistatin-like gene, is highly expressed along the margin of contact between inflammatory synovial pannus and eroding bone, suggesting a role in joint destruction. These results demonstrate that global gene expression profiles distinguish early and late CIA and reveal several genes novel to arthritis the further characterization of which will advance our understanding of arthritis.     

Use of whole blood as the routine transfusion product in Africa

In many countries in sub‐Saharan Africa (sSA) whole blood is more commonly available from blood transfusion services than red cell concentrates. Although in recent years, many countries have made significant progress in the implementing component preparation, this has largely been facilitated by external funding support. The large majority of rather than none of the sSA countries are leucocyte‐reducing or irradiating blood for transfusion. Systems for the routine detection of adverse consequences of blood transfusions (haemovigilance) only exist where transfusion safety has been identified as a health priority by the government. As a resource, the availability of blood transfusion in these countries is limited since less than 5 units of blood were donated per 1000 population far below the recommended requirement of 20 units/1000 per year. Young children are the main users of blood for transfusion in these sSA regions, largely due severe anaemia secondary to infection and sickle cell anaemia. Outcomes for children with severe anaemia are poor, even in those receiving a transfusion. Although it has been speculated that this may be due to transfusion‐related cardiac or pulmonary events, available data from observational studies and clinical trials indicate that these are rare complications of transfusion. Evidence from clinical physiology studies including those examining myocardial functions before and after the receipt of whole blood provide reassuring evidence that volume overload is rare and clinical trials reporting outcomes in children receiving whole blood transfusion, including a Phase II trial examining higher volumes, indicate that there is no evidence of cardiac or pulmonary overload events.