Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis.     

Gene expression analysis of in vivo fluorescent cells

Background

The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.

Methodology/Principal Findings

We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of ∼2∶1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).

Conclusions/Significance

Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.     

ISOLATION OF HIGH-QUALITY RNA FROM GRAINS OF DIFFERENT MAIZE VARIETIES

The study of gene expression in maize varieties represents a powerful tool aiming to increase vitamin A precursors. However, the isolation of RNA from different maize varieties is challenging because these varieties show different levels of polysaccharides, and most methods available for RNA isolation are inappropriate for grain samples. The polysaccharides co-purify and co-precipitate with RNA during isolation, resulting in low-quality RNA, compromising the use of RNA in subsequent applications. Thus, a cetyltrimethylammonium bromide (CTAB)-based method was adapted in this study and compared with six methods for RNA isolation, including commercial reagents and RNA and DNA isolation kits, in order to identify the most appropriate for maize grains from different varieties. Most of the methods evaluated were considered inadequate due to limitations in terms of purity and/or quantity of the isolated RNA, which affected the efficiency of subsequent RT-qPCR analysis, resulting in nonamplification of β-carotene hydroxylase gene (HYD3) or high deviation among replicates. However, the CTAB modified method allowed the study to obtain intact RNA, with high quality and quantity, from 25 maize varieties. Furthermore, this RNA was successfully used to evaluate the expression of HYD3 gene by real-time qualitative polymerase chain reaction (RT-qPCR), and thus represents a simple, efficient, and low-cost strategy.     

Current standards and pitfalls associated with the transfection of primary fibroblast cells

Cultured fibroblast cells, especially dermal cells, are used for various types of scientific research, particularly within the medical field. Desirable features of the cells include their ease of isolation, rapid cellular growth, and high degree of robustness. Currently, fibroblasts are mainly used to obtain pluripotent cells via a reprogramming process. Dermal fibroblasts, are particularly useful for gene therapies used for promoting wound healing or minimizing skin aging. In recent years, fibroblast transfection efficiencies have significantly improved. In order to introduce molecules (most often DNA or RNA) into cells, viral-based systems (transduction) or non-viral methods (transfection) that include physical/mechanical processes or lipid reagents may be used. In this article, we describe critical points that should be considered when selecting a method for transfecting fibroblasts. The most effective methods used for the transfection of fibroblasts include both viral-based and non-viral nucleofection systems. These methods result in a high level of transgene expression and are superior in terms of transfection efficacy and viability.     

Five-in-One: Simultaneous isolation of multiple major liver cell types from livers of normal and NASH mice

NASH is a chronic liver disease that affects 3%–6% of individuals and requires urgent therapeutic developments. Isolating the key cell types in the liver is a necessary step towards understanding their function and roles in disease pathogenesis. However, traditional isolation methods through gradient centrifugation can only collect one or a few cell types simultaneously and pose technical difficulties when applied to NASH livers. Taking advantage of identified cell surface markers from liver single-cell RNAseq, here we established the combination of gradient centrifugation and antibody-based cell sorting techniques to isolate five key liver cell types (hepatocytes, endothelial cells, stellate cells, macrophages and other immune cells) from a single mouse liver. This method yielded high purity of each cell type from healthy and NASH livers. Our five-in-one protocol simultaneously isolates key liver cell types with high purity under normal and NASH conditions, enabling for systematic and accurate exploratory experiments such as RNA sequencing.     

Comparison of RNA yield from small cell populations sorted by flow cytometry applying different isolation procedures.

BACKGROUND: RNA from sorted cell populations is crucial in many instances. We therefore compared four current protocols for RNA isolation, with regard to mRNA yield and purity. Moreover, we examined the effects on RNA recovery caused by different storage reagents. METHODS: Small populations of K562 cells or PMBC were sorted into the lysing reagent and subjected to RNA extraction, employing either phase separation extraction using an acidic guanidinium-isothiocyanate reagent (TriFast reagent), the silica-gel membrane-based spin-column technology (RNeasy Mini-/Micro-Kit), or the isolation via paramagnetic oligo(d)T-beads (microMACS). Cells designated for delayed RNA isolation were kept either in RNAlater, Qiagen Buffer RLT, TriFast or PrepProtect, or simply frozen after pelleting from PBS. The mRNA yield was determined by quantitative RT-PCR. RESULTS: Performing unpaired two-tailed t-tests revealed that RNA was extracted in significantly higher amounts using magnetic bead isolation. This method also allowed best discrimination of induced IL2 gene expression. In contrast, phase separation extraction showed the highest rate of failures. Intermediate storage reduced RNA yield. Contamination by genomic DNA was detected in several samples subjected to phase separation or silica-gel membrane-based spin-column extraction. CONCLUSIONS: Our results reveal advantages and disadvantages of RNA isolation procedures for small numbers of sorted cells and, therefore, facilitate the decision for the most appropriate protocol in a particular experimental context.     

Profiling gene expression in whole blood samples following an in-vitro challenge.

Genomics tools (gene- and protein-expression studies) can be used to find possible target genes involved in a quantifiable trait or disease state. However in many instances, cells and tissues directly involved in the trait's expression, for example, brain tissue, are not amenable for gene expression analysis. Whole blood cells share a molecular make-up for cellular communication and gene regulation systems with many other cell types, for example, neuronal cells, and have the advantage of being very accessible for gene profiling. We investigated the feasibility of nationwide blood sample collection for lymphocyte RNA isolation and real-time PCR analysis to quantify genomic responses. We tested several designs for blood collection and storage: blood sampling in PAXgene blood collection tubes and storage at -20 degrees C, blood sampling in heparin tubes and decanting the samples (with or without in-vitro stimulus) into either PAXgene blood collection tubes and storage at -20 degrees C, or polypropylene tubes followed by snap-freezing and storage at -80 degrees C. The latter procedure is the best cost-wise when only small amounts of total RNA are needed for downstream applications. Lymphocyte gene expression studies are most likely hampered by the quality of isolated RNA rather than the sampling method. We show that large-scale nationwide sample collections did not alter RNA quality or gene expression levels when compared to sampling and processing in a more controlled way. To this end, we present an optimized protocol for easy and standardized isolation of high quality RNA using the PAXgene isolation kit. Based on these results, we suggest that whole blood genomic data can be used as a genomic probe in experimental and clinical research.     

FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

Use of a simple method to isolate intact RNA from partially hydratedSelaginella lepidophylla plants

Isolation of high-quality RNA is a necessary step in gene expression analysis. Although many methods can be used to isolate RNA from plants where contamination of preparations with complex carbohydrates or phenolic compounds is a problem, the application of these methods toSelaginella lepidophylla tissues has failed to obtain good-quality RNA. Here we introduce 2 modifications to the method developed by Chomczynski and Sacchi (1987), generating a simple and rapid method that allows the isolation of intact RNA fromS. lepidophylla-dehydrated tissues. Although the introduced modifications are not new, their addition proved to be decisive for success in RNA isolation. Quality of the RNA obtained was evaluated by electrophoresis in agarose and by 3 different PCR-based techniques—RT-PCR, RNA differential display, and synthesis of a cDNA library.     

PCR技术在猴免疫缺陷病毒(SIV)感染模型中的应用

目的(1)建立RT PCR方法,定性测定SIV感染猴血浆中病毒RNA,比较其与传统血浆病毒分离方法的敏感性;(2)建立DNA PCR方法,检测SIV感染猴外周血淋巴细胞(PBMCs)中的前病毒DNA。(3)检验DNA PCR和RNA PCR方法在猴SAIDS模型应用中的实用性和可操作性。方法用SIVmac251静脉感染恒河猴,定期采血,从血浆中提取病毒RNA,以RNA为模板通过RT PCR法扩增,凝胶电泳定性;从感染猴PBMC中提取带有整合的SIV前病毒DNA的细胞基因组DNA,巢式PCR扩增,凝胶电泳定性。结果DNA PCR和RNA PCR经两轮扩增后均得到一长度为477bp的特异条带,测序鉴定确为目的片段。9只实验猴感染SIV后7d,RNA PCR结果为79阳性,DNA PCR结果为100%阳性,而血浆病毒分离只有59阳性;此后一直到感染后的42d,RNA PCR和DNA PCR的结果一直为100%阳性,而血浆病毒分离阳性率在感染后35d下降到49,到42d时下降为零。结论PCR方法比病毒分离方法的敏感性高。尤其是DNA PCR,既可检测具有活跃病毒复制的受感染细胞,又可检测那些携带病毒处于转录休眠期的细胞,所以在感染的早期和中后期———血浆病毒水平较低的情况下或病毒处于潜伏感染的阶段,它作为猴艾滋病(SAIDS)模型病毒学指标之一有其必要性和重要性。这个指标的检测方法应该是较血浆病毒RNA检测更为敏感。     

A simple and effective method for the isolation of inner cell mass samples from human blastocysts for gene expression analysis

The isolation of pure inner cell mass (ICM) and trophectoderm (TE) cells from a single human blastocyst is necessary to obtain accurate gene expression patterns of these cells, which will aid in the understanding of the primary steps of embryo differentiation. However, previously developed pure ICM isolation methods are either time-consuming or alter the normal gene expression patterns of these cells. Here, we demonstrate a simple and effective method of ICM samples isolation from human blastocysts. In total, 35 human blastocysts of all stages with expanded and good morphology were incubated in calcium/magnesium-free HEPES medium for 5 min before micromanipulation. With the aid of a laser, a biopsy pipette was inserted directly into the blastocoel for the suction-based removal of ICM samples. The ICM samples were obtained through simple mechanical pulling force or laser assistance, and each isolation process required 3–4 min. The isolated ICM and TE fractions were subjected to single-cell real-time quantitative RT-PCR to evaluate keratin 18 (KRT18) expression. Finally, 33 paired ICM and TE samples were verified using gene expression analysis. KRT18 was readily detectable in all TE cells but absent in 30 ICM counterparts, indicating a pure ICM isolation rate of 90.9% (30/33). The relative KRT18 expression of three TE samples compared with their three contaminated ICM counterparts was 19-fold (P?<?0.001), indicating that the contamination was very weak. These results demonstrate that our ICM isolation method is simple and effective.