A Powerful Method for Transcriptional Profiling of Specific Cell Types in Eukaryotes: Laser-Assisted Microdissection and RNA Sequencing

The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms

Robust computational reconstitution – a new method for the comparative analysis of gene expression in tissues and isolated cell fractions

BACKGROUND: Biological tissues consist of various cell types that differentially contribute to physiological and pathophysiological processes. Determining and analyzing cell type-specific gene expression under diverse conditions is therefore a central aim of biomedical research. The present study compares gene expression profiles in whole tissues and isolated cell fractions purified from these tissues in patients with rheumatoid arthritis and osteoarthritis. RESULTS: The expression profiles of the whole tissues were compared to computationally reconstituted expression profiles that combine the expression profiles of the isolated cell fractions (macrophages, fibroblasts, and non-adherent cells) according to their relative mRNA proportions in the tissue. The mRNA proportions were determined by trimmed robust regression using only the most robustly-expressed genes (1/3 to 1/2 of all measured genes), i.e. those showing the most similar expression in tissue and isolated cell fractions. The relative mRNA proportions were determined using several different chip evaluation methods, among which the MAS 5.0 signal algorithm appeared to be most robust. The computed mRNA proportions agreed well with the cell proportions determined by immunohistochemistry except for a minor number of outliers. Genes that were either regulated (i.e. differentially-expressed in tissue and isolated cell fractions) or robustly-expressed in all patients were identified using different test statistics. CONCLUSION: Robust Computational Reconstitution uses an intermediate number of robustly-expressed genes to estimate the relative mRNA proportions. This avoids both the exclusive dependence on the robust expression of individual, highly cell type-specific marker genes and the bias towards an equal distribution upon inclusion of all genes for computation

Highly Parallel Genome-Wide Expression Analysis of Single Mammalian Cells

We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)~0.76-0.80 between individual cells and R(2)~0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology

Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes

Identification of mecA-related oxacillin resistance in staphylococci by the E test and the broth microdilution method

A set of 165 strains of different staphylococcal species, 67 Staphylococcus aureus, 71 novobiocin-sensitive coagulase-negative staphylococci (CNS) and 27 novobiocin-resistant CNS was used. The oxacillin and methicillin MICs were recorded after 24 and 42 h of incubation at 35 degrees C and at 30 degrees C. Significantly higher MICs were recorded at 30 degrees C compared with 35 degrees C. While a poor discrimination between mecA-positive and mecA-negative strains was obtained with methicillin, the oxacillin MICs enabled identification of resistant strains under certain conditions. The distribution of MICs differed between the three groups of species. Separation of uninduced mecA-positive (> or = 4.0 mg oxacillin/L) and mecA-negative ( or = 1.0 mg/L). The mecA-positive and mecA-negative strains of novobiocin-resistant CNS were clearly separated after 24 h of incubation by either method

Evaluation of four qualitative methods for detection of beta-lactamase production in Staphylococcus and Micrococcus species

Four qualitative methods for the detection of beta-lactamase production in Staphylococcus and Micrococcus species were evaluated and compared with a quantitative macroiodometric reference method. The disc diffusion test with penicillin G and the cloverleaf method could not separate beta-lactamase-positive from beta-lactamase-negative strains. Two applications of the chromogenic cephalosporin test, using uninduced strains and strains grown on blood agar plates, gave a large number of false negative and false positive results. False negative reactions were most common among uninduced strains, while the false positive reactions were most often recorded for Staphylococcus saprophyticus. A high degree of efficiency was recorded for the nitrocefin spot test, using induced strains grown on antibiotic susceptibility agar, and for the starch-iodine plate method. The starch-iodine plate with methicillin as inducer gave the most reliable results