A simple,chisel-assisted mechanical microdissection system for harvesting homogenous plant tissue suitable for the analysis of nucleic acids and proteins

Many physiological processes are limited to specific tissues or even specific cell types. Analysing entire plants or organs results in averaged data of all cell types contained in the sample; thus, specific metabolic functions cannot be assigned to individual cell types. A higher spatial resolution is required. By microdissecting plant organs, homogeneous material can be obtained. If a suitable amount of material is collected, standard analytical methods can be applied to elucidate cell type-specific processes. The collection of sufficient quantities of homogeneous material can be done by means of mechanical microdissection. This technique is a low-cost alternative to laser-coupled microdissection techniques. Here we describe a protocol for chisel-assisted mechanical microdissection of embedded plant material and demonstrate that the collected material is suitable to obtain nucleic acids and proteins.     

寡核苷酸芯片法、实时PCR 和测序法进行乙肝病毒 基因分型的比较

目的:比较寡核苷酸芯片法、实时荧光PCR和测序法在对慢性乙肝患者病毒基因分型的比较和方法学评价。方法:对126例不同基因型的慢性乙肝患者的血清样本分别用寡核苷酸芯片法、实时荧光PCR法和测序法进行基因分型,并评价各种方法的临床表现、所需时间和检测成本。结果:寡核苷酸芯片法、实时荧光PCR分别能检测到1%和0.1%比例的基因型。在126例慢性乙肝患者的临床样本中,寡核苷酸芯片法、实时荧光PCR和测序法分别检测出41(33%)、41(33%)和45(36%)例为B型,76(60%)、76(60%)、81(64%)例为C型。寡核苷酸芯片法、实时荧光PCR均检出9例B、C混合基因型。在三种检测方法中实时荧光PCR是最快速和廉价的。结论:寡核苷酸芯片法、实时荧光PCR能检出B、C混合基因型,而测序法只能检测出样本的主导基因型。     

A simple,cost-effective and flexible method for processing of snap-frozen tissue to prepare large amounts of intact RNA using laser microdissection

Understanding the molecular basis of disease requires gene expression profiling of normal and pathological tissue. Although the advent of laser microdissection (LMD) has greatly facilitated the procurement of specific cell populations, often only small amounts of low quality RNA is recovered. This precludes the use of global approaches of gene expression profiling which require sizable amounts of high quality RNA. Here we report a method for processing of snap-frozen tissue to prepare large amounts of intact RNA using LMD.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.     

不同方法检测鸡胴体中沙门菌结果的比较研究

对鸡胴体淋洗液样品进行沙门菌检测,样品经过前增菌和选择性增菌后,分别采用4种不同的方法进行检测,即普通PCR方法、实时荧光PCR方法、免疫学方法(VIDAS)和传统的微生物检验方法。共检测了56份样品,普通PCR检出阳性样品34份,实时荧光PCR阳性样品36份,VIDAS阳性样品28份;PCR和实时荧光定量PCR均无假阳性和假阴性结果。结果显示该3种检测方法均可以用于鸡胴体中沙门菌的快速检测。     

Single-tube multiplex PCR for rapid and sensitive diagnosis of subgenus B and other subgenera adenoviruses in clinical samples

We developed a new diagnostic method of subgenus (Sub) B adenovirus (Ad) in clinical samples using non-nested polymerase chain reaction (PCR). Sequences of the conserved hexon-coding region of representative strains of eight serotypes (3, 7, 11, 14, 16, 21, 34 and 35) of Sub B Ad were heterogeneous. In order to distinguish Ad serotype 3 (Ad 3) and Ad 7 from the other serotypes of Sub B Ad, and to differentiate Ad 3 and 7 from each other, 3 different downstream primers were designed based on the sequence heterogeneity. By a single-tube PCR method using a combination of 6 primers including the 3 new primers, Ads demonstrated to amplify 188, 206, 284, and 301 bp DNA fragments for Ad 3, Ad 7, other Sub B Ads, and non-Sub B Ads, respectively. A total of 114 clinical samples were selected to evaluate the direct applicability of our PCR. The results were compared with previous culture results. Sixty-seven out of 71 (94%) Sub B Ad culture-positive samples, and 15 out of 19 (79%) Sub C or E-positive samples amplified products of the expected size. Two of 20 (10%) culture-negative samples from pharyngoconjunctival fever patients were identified as Ad 3 by the PCR. Four samples, from which non-Ad viruses were isolated, were negative by the PCR. The present study might provide a rapid and sensitive diagnosis method for infections caused by Sub B Ads.     

The antitumor effect of tumor-draining lymph node cells activated by both anti-CD3 monoclonal antibody and activated B cells as costimulatory-signal-providing cells

To establish an efficient cell-culture system for adoptive immunotherapy, we attempted to use lipopolysacharide (LPS)-activated B cells (LPS blasts) as costimulatory-signal-providing cells in the in vitro induction of antitumor effector cells. Both normal and tumor-draining lymph node cells were efficiently activated by both anti-CD3 monoclonal antibody (mAb) and LPS blasts, and subsequently expanded by a low dose of interleukin-2 (IL-2; anti-CD3 mAb and LPS blasts/IL-2). The expanded cells were predominantly CD8⁺ T cells and showed a low level of tumor-specific cytotoxic T lymphocyte (CTL) activity. The adoptive transfer of B16-melanoma-draining lymph node cells expanded by anti-CD3 mAb and LPS blasts/IL-2 showed significant antitumor effect against the established metastases of B16 in combination with intraperitoneal injections of IL-2. This treatment cured all B16-bearing mice. In addition, these mice also showed tumorspecific protective immunity against B16 at the rechallenge. Considering that activated B cells express several kinds of costimulatory molecules, these findings thus indicate an efficacy of costimulation that is derived from activated B cells for the in vitro induction of tumor-specific CTL, in co-operation with anti-CD3 mAb. The culture system presented here may thus be therapeutically useful, providing potent effectors for adoptive immunotherapy against various types of cancer.     

Use of the PCR and fluorescent probes to recover SSU rRNA gene sequences from single cells of the ciliate protozoon Spathidiutn

A two-stage heminested PCR approach was developed to amplify small subunit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucary-otes. The method was evaluated using the ciliate protozoon Spathidiutn when PCR products were obtained from nine of 10 cells tested. Southern blotting demonstrated that all fragments contained the same sequence in a region of SSU rDNA which is normally highly variable between species. A fluorescent oligonucleotide probe was used to demonstrate that this sequence also occurred in fixed cells of Spathidiutn. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background auto-fluorescence. The Spathidiutn sequence is closely related to that from the haptorid Homalozoon vermiculare.     

Short communication: Detection of non-culturable Vibrio cholerae O139, by PCR and fluorescent antibody methods,in laboratory microcosms

Non-culturable Vibrio cholerae O139 was detected in microcosms by PCR and fluorescent-antibody (FA) techniques. When survival of V. cholerae O139 in microcosms was assessed by viable counting on culture media, the vibrio became non-culturable after 44 days and remained non-culturable for an additional 7 weeks.     

Genomic profiles for human peripheral blood T cells, B cells, natural killer cells, monocytes, and polymorphonuclear cells: comparisons to ischemic stroke, migraine, and Tourette syndrome

Blood genomic profiling has been applied to disorders of the blood and various organ systems including brain to elucidate disease mechanisms and identify surrogate disease markers. Since most studies have not examined specific cell types, we performed a preliminary genomic survey of major blood cell types from normal individuals using microarrays. CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ natural killer cells, and CD14+ monocytes were negatively selected using the RosetteSep antibody cocktail, while polymorphonuclear leukocytes were separated with density gradient media. Genes differentially expressed by each cell type were identified. To demonstrate the potential use of such cell subtype-specific genomic expression data, a number of the major genes previously reported to be regulated in ischemic stroke, migraine, and Tourette syndrome are shown to be associated with distinct cell populations in blood. These specific gene expression, cell-type-related profiles will need to be confirmed in larger data sets and could be used to study these and many other neurological diseases.     

数据库搜索及ISSR-抑制PCR法开发香菇微卫星标记

采用数据库搜索及ISSR-抑制PCR法开发香菇微卫星标记。由数据库搜索法开发出21对引物,11对有多态性,各位点平均产生3.3个等位基因;通过ISSR-抑制PCR法开发出8对引物,5对具多态性,各位点平均产生3个等位基因。结果表明,在香菇SSR开发中,两种方法均是行之有效的。     

丁坝对鱼类栖地的影响范围评估

利用磁珠富集法和5’锚定PCR法开发背瘤丽蚌的微卫星标记,将获得的多态性引物用于群体的遗传多态性分析,以期在比较两种开发微卫星标记方法的基础上同时获得一批有用的微卫星引物。从磁珠富集法获得的微卫星序列阳性克隆率为69.2%,重复次数超过10的占总数的70.2%,从设计的28对引物中筛选得到多态性引物11对,开发效率为39.3%。这11对引物用于养殖群体的遗传多样性分析,结果显示,等位基因数范围为4～13,观测杂合度、期望杂合度范围分别为0.205～0.738、0.566～0.839。而5’锚定PCR法获得的微卫星序列阳性克隆率为97.8%,重复次数超过10的占总数的24.7%,从设计的56对引物中筛选得到多态性引物19对,开发效率为30.4%。这19对引物用于养殖群体的遗传多样性分析,结果显示,等位基因数范围为3～10,观测杂合度、期望杂合度范围分别为0.208～0.894、0.431～0.896。实验结果表明,磁珠富集法所获微卫星序列质量高,开发微卫星标记效率较高;而5’锚定PCR法实验操作更简便,所获得的引物遗传多样性指数更高。两种方法开发的引物均可用于背瘤丽蚌和近缘种的野生种质资源遗传多样性研究。