使用RNeasy Mini Kit纯化的总RNA具有高质量，适用于多种下游操作。实验方案还包括部分纯化的RNA、体外转录本和酶反应RNA的回收。不提供溶壁酶、酵母裂解酶或玻璃珠（酵母样本需要）。因样本来源的发育阶段、种属和生长条件的不同，从样本中分离的RNA量也各异。由于RNA酶步骤富集的RNA种属>200 nt，因此RNA产量不包括5S rRNA、tRNA或其他低分子量RNA。
使用RNeasy Mini Kit从图示数目的HeLa细胞中分离的总RNA的RT-PCR。使用不含RNA酶的DNA酶酶切10 µl（1/5）洗脱液，并使用oligo-dT引物进行逆转录。使用2.5 µl（1/20）的cDNA混合物进行50 µl PCR。扩增452 bp的GAPDH片段。C-：阴性对照；C+：阳性对照；M：100 bp分子量标准。
Performance
RNeasy Mini Kit可从至多30 mg动物或人类组织样本，或从100–1 x 107个动物或人类细胞样本或酵母中纯化总RNA（参见”RT-PCR of RNA from as few as 100 cells”和”High-quality RNA from a variety of samples”）。
· Evaluation of single cells to detect heterogenous populations
评价检测单个细胞异质种群
· Whole blood assay: No isolation procedures and optimal culture medium
全血检测：没有隔离程序和培养基
· Physiological stimulans for phagocytes: Bacteria and fMLP
吞噬细胞生理：细菌和fMLP
· Dose response: Low and high stimulant
剂量反应:低和高的
· No electrostatic artifacts in polystyrol tubes compared to latex beads
没有静电工件比较乳胶珠子聚苯乙烯管内
· Standardized test procedure
标准化测试程序
· Exclusion of aggregation artifacts by DNA staining
通过DNA染色排除聚合工件
· Compatible with whole blood of mice and rats
兼容的小鼠和大鼠全血
· Fast assay: Whole assay time is 1.5 hours
快速测定:整个试验时间是1.5小时
100 analyses. Clinical Diagnostic for the Quantitative Analysis of Leukocyte Oxidative Burst in Human Whole Blood. Evaluation by flow cytometry.
100次分析。临床诊断人体全血白细胞氧化破裂的定量检测。通过流式细胞术进行评估。
Clinical Diagnostic for the Quantitative Analysis of Leukocyte Oxidative Burst in Human Whole Bloods
GLYCOTOPE公司PHAGOBURST白细胞氧化突发定量分析10-0200
SUMMARY and EXPLANATION
BURSTTEST (PHAGOBURST) allows the quantitative determination of leukocyte oxidative burst in heparinized whole blood. It contains unlabeled opsonized bacteria (E.coli), phorbol 12-myristate 13-acetate (PMA) and the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) as stimulants, Dihydrorhodamine (DHR) 123 as a fluorogenic substrate and necessary reagents. It determines the percentage of phagocytic cells which produce reactive oxidants (conversion of DHR 123 to R 123) and their enzymatic activity (amount of R 123 per cell).
The evaluation of these bioactivities should be performed by flow cytometry.
BURSTTEST(PHAGOBURST)允许定量测定白细胞氧化闯入肝素化全血。 它包含未标记的促进调理作用的细菌(大肠杆菌),佛波醇12十四烷酸乙酸13(PMA)和趋化作用的多肽n甲酰遇见亮氨酸板式换热器(fMLP)作为xing奋剂,二氢若丹明(DHR)123作为一个荧光衬底和必要的试剂。它决定了吞噬细胞产生活性氧化剂的比例(转换DHR 123 到 R 1233)及其酶活性(每个细胞R 123的数量)。
这些生物活性的测定应该有流式细胞术完成。
APPLICATIONS
The diagnostic kit is intended to investigate the altered oxidative burst activity found in various disorders and to evaluate the effects of drugs.
Reduced or missing burst activity is observed in inborne defects like the chronic granulomatous disease (CGD). CGD is a heterogenous group of inherited disorders that usually manifests itself during the first two years of life (3, 4). The disease is characterized clinically by repeated and life-threatening infections caused by bacterial and fungal organisms. These infections typically consist of pneumonia, lymphadenitis, or abscesses that involve lymph nodes, lungs, and liver. The NADPH oxidase is the enzyme system responsible for producing superoxide anion, which is quickly converted to hydrogen peroxide and hydroxyl radicals. Abnormalities in the constituent peptides of the NADPH oxidase enzyme system lead to the dysfunctions characteristic of CGD. Neutrophils from CGD patients fail to produce a significant oxidative burst following stimulation. Different forms of CGD are described (classical X-linked CGD and autosomal recessive patterns). BURSTTEST (PHAGOBURST?) is a rapid and sensitive method for the diagnosis of CGD and for the detection of X-linked carriers.
The oxidative burst of granulocytes is impaired in transplant patients and patients with AIDS (6). The spontaneous and fMLP-induced neutrophil respiratory burst was shown to be increased in neonates with laboratory signs of infection (7). Various immunomodulators (e.g., cytokines (GM-CSF, G-CSF, TNF) or drugs) seem to have effects on the oxidative burst. By using fMLP as a low stimulant one can investigate additive or priming effects (8) of test substances.
The diagnostic kit is also applicable on blood of mice, rats, rabbits, dogs, cattle and other species.
TEST PRINCIPLES Phagocytosis by polymorphonuclear neutrophils and monocytes constitutes an essential arm of host defense against bacterial or fungal infections. The phagocytic process can be separated into several major stages: chemotaxis (migration of phagocytes to inflammatory sites), attachment of particles to the cell surface of phagocytes, ingestion (phagocytosis) and intracellular killing by oxygen-dependent (oxidative burst) and oxygen-independent mechanisms (1, 2).
BURSTTEST (PHAGOBURST?) allows the quantitative determination of leukocyte oxidative burst. The BURSTEST kit contains unlabelled opsonized E.coli bacteria as particulate stimulus, the protein kinase C ligand phorbol 12-myristate 13-acetate (PMA) as high stimulus and the chemotactic peptide N-formyl-MetLeuPhe (fMLP) as low physiological stimulus, Dihydrorhodamine (DHR) 123 as a fluorogenic substrate (5) and necessary reagents. Heparinized whole blood is incubated with the various stimuli at 37°C, a sample without stimulus serves as negative background control. Upon stimulation, granulocytes and monocytes produce reactive oxygen metabolites (superoxide anion, hydrogen peroxide, hypochlorous acid) which destroy bacteria inside the phagosome. Formation of the reactive oxidants during the oxidative burst can be monitored by the addition and oxidation of DHR 123. The reaction is stopped by addition of LYSING SOLUTION, which removes erythrocytes and results in a partial fixation of leukocytes. After one washing step with WASHING SOLUTION, DNA STAINING SOLUTION is added to exclude aggregation artifacts of bacteria or cells. The percentage of cells having produced reactive oxygen radicals are then analyzed as well as their mean fluorescence intensity (enzymatic activity)
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