Quantitative mRNA expression analysis in kidney glomeruli using microdissection techniques

The introduction of new tools for molecular analysis, such as RT-qPCR and microarrays, has provided researchers with powerful applications to study renal disease and development. However, the high cellular heterogeneity of the renal tissue complicates the molecular analysis of specific cells and cell groups such as glomerular or tubular cells. In the past, glomerular sieving and manual dissection were used for the isolation of glomeruli. However, these techniques cannot be used for the isolation of specific glomeruli or for the co-isolation of additional tissue fractions. In recent decades, new microdissection techniques such as laser-assisted microdissection have been developed. These applications allow the isolation of small cell groups from heterogeneous tissue for molecular analysis, including microarray and RT-qPCR. Although very promising, some drawbacks are associated with these techniques. The isolated sample material is generally small and requires sensitive assays. In addition, the long sample processing time may result in a considerable loss of RNA integrity. Careful optimization and rigorous quality analysis should overcome these drawbacks. In the present paper, the recent literature on the application of microdissection techniques in kidney research is reviewed, together with a discussion of the critical issues that are essential for the application of quantitative mRNA expression analysis with RT-qPCR on microdissected samples.     

改良手工显微切割法获取特定细胞提取高质量RNA

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100％无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.     

Expression microdissection adapted to commercial laser dissection instruments

Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection (xMD) is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators. This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis.     

鼻咽癌组织的显微切割及其 RNA 线性扩增

从微小体积鼻咽癌活检标本中获取纯净癌细胞一直是鼻咽癌分子生物学研究中的难题 . 为了寻找一种能从鼻咽癌活检组织中获得高纯度、高质量 RNA 来完成 cDNA 微阵列 (cDNA Microarray) 实验的简便实用方法,采用 RNAlater 技术保存鼻咽癌活检组织,显微切割技术来获得高纯度鼻咽癌细胞,利用 RNA 线性扩增技术得到 cDNA 微阵列实验所需 RNA. 结果表明：利用 RNAlater 技术可以很好地保持组织 RNA 的稳定,通过优化显微切割和 RNA 线性扩增的条件获得了 cDNA 微阵列实验所需的高纯度、高质量 RNA.     

小麦染色体的显微激光分离

探讨了应用氩离子激光进行植物染色体显微激光切割,分离的可行性,应用该技术对普通小麦的体细胞及特定染色体（１Ｂ染色体）实施切割,分离,并且以分离到的单细胞核或单条染色体为模板进行了ＰＣＲ ＤＮＡ扩增。该技术比玻璃针切割分离染色体技术,具有操作方便,容易掌握,且可对整个细胞核进行分离等优点,有利于促进染色体显微操作技术的普及应用。同时,探讨了染色体显微操作技术在细胞遗传学及分子生物学研究领域的应用前景     

Laser microdissection: exploring host-bacterial encounters at the front lines

The mucosal surfaces of tissues such as the stomach and intestines are in constant contact with indigenous bacterial populations and are major portals of entry for bacterial pathogens. Host responses to bacterial encounters at these surfaces frequently involve complex interactions between epithelial cells and immune cells, and are thus difficult to model in vitro. Laser microdissection is a technique in which pure populations of host cells are acquired from sections of complex tissue. When coupled with an expanding repertoire of techniques for molecular analysis of microdissected cells, laser microdissection allows host cellular responses to bacteria to be studied in their native tissue context. This approach has already yielded key insights into the nature of mucosal responses to commensal, as well as pathogenic bacteria, and promises to be an important addition to the cellular microbiologist's toolkit.     

Laser microdissection microscopy in parasitology: microscopes meet thermocyclers

The new methods of laser microdissection microscopy have received wide acceptance in biology and have been applied in a small number of parasitology investigations. Here, the techniques and applications of laser microdissection microscopy are reviewed with suggestions of how the systems might be used to explore applied questions in parasite molecular biology and host-parasite interactions.     

Molecular genetic analysis of the X-chromosomal nuclear envelope attachment region in nurse cells of the malaria mosquitoes Anopheles messeae Fall

DNA of the X-chromosomal nuclear envelope attachment region was isolated from malaria mosquito Anopheles messeae Fall. nurse cells by chromosome microdissection. A DNA library of the region was constructed using a plasmid vector. DNA sequencing revealed gene fragments, tandem repeats, and a great variety of transposable elements (TEs). The X-chromosomal nuclear envelope attachment region was concluded to correspond in molecular organization and cytogenetics to diffuse intercalary heterochromatin.     

Novel Cell and Tissue Acquisition System (CTAS): Microdissection of Live and Frozen Brain Tissues

We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS - Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.     

Laser microdissection of fungal septa as visualised by scanning electron microscopy

Laser microdissection has been proven a successful technique to isolate single cells or groups of cells from animal and plant tissue. Here, we demonstrate that laser microdissection is suitable to isolate subcellular parts of fungal hyphae. Dolipore septa of Rhizoctonia solani containing septal pore caps were cut by laser microdissection from sections of mycelium and collected by laser pressure catapulting. Subsequently, microdissected septa were visualised using a wheat germ agglutinin labelling of cell walls, septa and septal pore caps and scanning electron microscopy. The use of laser microdissection on fungal cells opens new ways to study subcellular fungal structures and the biochemical composition of hyphal cells.     

Needle in a haystack: microdissecting the proteome of a tissue

Summary. Laser-assisted microdissection is a recent technology that enables cells to be harvested from tissue sections. Proteins can be extracted from the dissected cells for molecular analysis. This enables the analysis of proteins in specific cell types in an in vivo system. Although quantities of protein obtained from the dissected material can be small, it is possible to use established methods such as Western Blotting and 2D-PAGE, as well as newer technologies such as SELDI-MS, to analyse the proteins. This review describes the applications and technical considerations for using laser-assisted dissected cells in proteomics research.     

Molecular genetic analysis of the X-chromosome nuclear envelope attachment region in nurse cells of the malaria mosquitoes <Emphasis Type="Italic">Anopheles messeae</Emphasis> fall

DNA of the X-chromosomal nuclear envelope attachment region was isolated from malaria mosquito Anopheles messeae Fall. nurse cells by chromosome microdissection. A DNA library of the region was constructed using a plasmid vector. DNA sequencing revealed gene fragments, tandem repeats, and a great variety of transposable elements (TEs). The X-chromosomal nuclear envelope attachment region was concluded to correspond in molecular organization and cytogenetics to diffuse intercalary chromatin.     

Expression profiling using human tissues in combination with RNA amplification and microarray analysis: assessment of Langerhans cell histiocytosis

Summary. Advances in molecular genetics have led to sequencing of the human genome, and expression data is becoming available for many diverse tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as development, differentiation, homeostasis, and ultimately, disease pathogenesis. At present, an optimal methodology to assess gene expression is to evaluate single cells, either identified physiologically in living preparations, or by immunocytochemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quantity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction (PCR) based analyses, and linear RNA amplification including amplified antisense (aRNA) RNA amplification and a newly developed terminal continuation (TC) RNA amplification methodology have been used in combination with microdissection procedures such as laser capture microdissection (LCM) to enable the use of microarray platforms within individual populations of cells obtained from a variety of human tissue sources such as biopsy-derived samples {including Langerhans cell histiocytosis (LCH)} as well as postmortem brain samples for high throughput expression profiling and related downstream genetic analyses.     

Proteomic analysis of prolactinoma cells by immuno-laser capture microdissection combined with online two-dimensional nano-scale liquid chromatography/mass spectrometry

Background  

Pituitary adenomas, the third most common intracranial tumor, comprise nearly 16.7% of intracranial neoplasm and 25%-44% of pituitary adenomas are prolactinomas. Prolactinoma represents a complex heterogeneous mixture of cells including prolactin (PRL), endothelial cells, fibroblasts, and other stromal cells, making it difficult to dissect the molecular and cellular mechanisms of prolactin cells in pituitary tumorigenesis through high-throughout-omics analysis. Our newly developed immuno-laser capture microdissection (LCM) method would permit rapid and reliable procurement of prolactin cells from this heterogeneous tissue. Thus, prolactin cell specific molecular events involved in pituitary tumorigenesis and cell signaling can be approached by proteomic analysis.     

Molecular analysis of complex tissues is facilitated by laser capture microdissection

Every tissue contains heterogeneous cell populations. Laser capture microdissection (LCM) facilitates cell isolation from complex tissues followed by molecular analysis. LCM entails placing a transparent film over a tissue section or a cytological sample, visualizing the cells microscopically, and selectively adhering the cells of interest to the film with a focused pulse from an infrared laser. The film with the procured cells is then removed from the original sample and placed directly into DNA, RNA, or protein-extraction buffer for processing. LCM has revolutionized molecular analysis of complex tissues because it combines the topographic precision of microscopy with the power of molecular genetics, genomics, and proteomics. However, the success of molecular analysis still depends on the experimental design and requires the understanding of each technical step involved in specimen preparation. This review attempts to rationalize and demystify the choice of various technical options in upstream tissue processing supporting global analytical strategies.