This second page presents interesting papers, sampling technologies and links about single-cell or single-molecule qRT-PCR, using micro-manipulated or laser-capture microdissected tissue followed by real-time RT-PCR:

Limitations of mRNA amplification from small-size cell samples.

Nygaard V, Holden M, Loland A, Langaas M, Myklebost O, Hovig E.
BMC Genomics. 2005 Oct 27;6:147.
Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium
Hospital, Montebello, 0310 Oslo, Norway.

BACKGROUND: Global mRNA amplification has become a widely used approach to obtain gene expression profiles from limited material. An important concern is the reliable reflection of the starting material in the results obtained. This is especially important with extremely low quantities of input RNA where stochastic effects due to template dilution may be present. This aspect remains under-documented in the literature, as quantitative measures of data reliability are most often lacking. To address this issue, we examined the sensitivity levels of each transcript in 3 different cell sample sizes. ANOVA analysis was used to estimate the overall effects of reduced input RNA in our experimental design. In order to estimate the validity of decreasing sample sizes, we examined the sensitivity levels of each transcript by applying a novel model-based method, TransCount. RESULTS: From expression data, TransCount provided estimates of absolute transcript concentrations in each examined sample. The results from TransCount were used to calculate the Pearson correlation coefficient between transcript concentrations for different sample sizes. The correlations were clearly transcript copy number dependent. A critical level was observed where stochastic fluctuations became significant. The analysis allowed us to pinpoint the gene specific number of transcript templates that defined the limit of reliability with respect to number of cells from that particular source. In the sample amplifying from 1000 cells, transcripts expressed with at least 121 transcripts/cell were statistically reliable and for 250 cells, the limit was 1806 transcripts/cell. Above these thresholds, correlation between our data sets was at acceptable values for reliable interpretation. CONCLUSION: These results imply that the reliability of any amplification experiment must be validated empirically to justify that any gene exists in sufficient quantity in the input material. This finding has important implications for any experiment where only extremely small samples such as single cell analyses or laser captured microdissected cells are available.

Miniaturization applied to analysis of nucleic acids in heterogeneous tissues.

Day PJ.
Expert Rev Mol Diagn. 2006 Jan;6(1):23-8.
The University of Manchester, Centre for Integrated Genomic Medical Research
(CIGMR), Stopford Building, Oxford Road, Manchester, M13 9PT, UK.

Despite huge efforts in sample analysis, the measurement of marker nucleic acids within tissues remains largely nonquantitative. Gene analyses have benefited from sensitivity gains through in vitro gene amplification, including PCR. However, whilst these processes are intrinsically suited to highly reproducible, accurate and precise gene measurement, the term semiquantitative analysis is still commonly used, suggesting that other fundamental limitations preclude a generic quantitative basis to gene analysis. The most poorly defined aspect of gene analysis relates to the sample itself. The amount of cells and, particularly, cell subtype composition are rarely annotated before analysis; indeed, they are often extrapolated after analysis. To advance our understanding of pathogenesis, assay formats will benefit from resembling the dimensions of the cell, to assist in the analysis of cellular components of tissue complexes. This review is partly a perspective on how current miniaturization technologies, in association with molecular biology, microfluidics and surface chemistries, may evolve from the parts of a paradigm to enable the unambiguous quantitative analysis of complex biologic matter.

Gene-expression analysis at the single-cell level.

Dixon AK, Richardson PJ, Pinnock RD, Lee K.
Trends Pharmacol Sci. 2000 Feb;21(2):65-70.
Department of Pharmacology, University of Cambridge, Tennis Court Road,
Cambridge, UK CB2 1QJ.

The manner in which a cell responds to and influences its environment is ultimately determined by the genes that it expresses. To fully understand and manipulate cellular function, identification of these expressed genes is essential. Techniques such as RT-PCR enable examination of gene expression at the tissue level. However, the study of complex heterogeneous tissue, such as the CNS or immune system, requires gene analysis to be performed at much higher
resolution. In this article, the various methods that have been developed to enable RT-PCR to be performed at the level of the single cell are reviewed. In addition, how, when carried out in combination with techniques such as patch-clamp recording, single-cell gene-expression studies extend our understanding of biological systems is discussed.

MicroRNA expression profiling of single whole embryonic stem cells.

Tang F, Hajkova P, Barton SC, Lao K, Surani MA.
Nucleic Acids Res. 2006 Jan 24;34(2):e9.

Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental
Biology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.
MicroRNAs (miRNAs) are a class of 17-25 nt non-coding RNAs that have been shown to have critical functions in a wide variety of biological processes during development. Recently developed miRNA microarray techniques have helped to accelerate research on miRNAs. However, in some instances there is only a limited amount of material available for analysis, which requires more sensitive techniques that can preferably work on single cells. Here we demonstrate that it is possible to analyse miRNA in single cells by using a real-time PCR-based 220-plex miRNA expression profiling method. Development of this technique will greatly facilitate miRNA-related research on cells, such as the founder population of primordial germ cells where rapid and dynamic changes occur in a few cells, and for analysing heterogeneous population of cells. In these and similar cases, our method of single cell analysis is critical for elucidating the diverse roles of miRNAs.

Single-cell gene expression analysis: implications for neurodegenerative and neuropsychiatric disorders.

Ginsberg SD, Elarova I, Ruben M, Tan F, Counts SE, Eberwine JH, Trojanowski JQ,
Hemby SE, Mufson EJ, Che S.
Neurochem Res. 2004 Jun;29(6):1053-64.
Center for Dementia Research, Nathan Kline Institute, New York University School
of Medicine, Orangeburg, New York 10962, USA.

Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of
single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by single-cell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia, respectively.

Combined histochemical staining, RNA amplification, regional, and single cell
cDNA analysis within the hippocampus.

Ginsberg SD, Che S.
Lab Invest. 2004 Aug;84(8):952-62.
Center for Dementia Research, Nathan Kline Institute, New York University School
of Medicine, Orangeburg, NY, USA.

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

PCR amplification from single DNA molecules on magnetic beads in emulsion:
application for high-throughput screening of transcription factor targets.

Kojima T, Takei Y, Ohtsuka M, Kawarasaki Y, Yamane T, Nakano H.
Nucleic Acids Res. 2005 Oct 6;33(17):e150.
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural
Sciences, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.


Single neurons as experimental systems in molecular biology.

Hinkle D, Glanzer J, Sarabi A, Pajunen T, Zielinski J, Belt B, Miyashiro K,
McIntosh T, Eberwine J.
Prog Neurobiol. 2004 Feb;72(2):129-42.
Department of Pharmacology, University of Pennsylvania Medical School, 36th and
Hamilton Walk, Philadelphia, PA 19104, USA.

The cellular and the inter-connective complexity of the central nervous system (CNS) necessitate's analysis of functioning at both the system and single cell levels. Systems neuroscience has developed procedures that facilitate the analysis of multicellular systems including multielectrode arrays, dye tracings and lesioning assays, and at the single cell level there have been significant strides in assessing the physiology and morphology of individual cells. Until recently little progress had been made in understanding the molecular biology of single neuronal cells. This review will highlight the development of PCR and aRNA procedures for analysis of mRNA abundances in single cells. Also, other procedures for the analysis of protein abundances as well as the association of RNA with proteins will also be summarized. These procedures promise to provide experimental insights that will help unravel the functional mechanisms regulating the cellular components of the CNS.

Single-cell molecular biology:   Implications for diagnosis and treatment of neurologic disease.

Hinkle DA, Eberwine JH.
Biol Psychiatry. 2003 Aug 15;54(4):413-7.
Department of Pharmacology, University of Pennsylvania Medical Center,
Philadelphia, Pennsylvania, USA.

The continued discovery of basic pathologic mechanisms underlying neuropsychiatric illnesses will be critical to the development of improved diagnostic tests and more targeted therapeutic strategies. Molecular biological methods capable of evaluating gene expression at the single-cell level have great potential for advancing our knowledge of these processes. This review describes two techniques that are providing new insights into the intracellular regulation of ribonucleic acid trafficking and processing. These technologies promise to accelerate our understanding of both normal and abnormal molecular processes within neurons, and they have the potential for direct application to the study of human neurologic disease.

Methodological considerations regarding single-cell gene expression profiling for brain injury.

Davis JE, Eberwine JH, Hinkle DA, Marciano PG, Meaney DF, McIntosh TK.
Neurochem Res. 2004 Jun;29(6):1113-21.
The Head Injury Center, Department of Neurosurgery, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania, USA.

Genomic microarrays are rapidly becoming ubiquitous throughout a wide variety of biological disciplines. As their use has grown during the past few years, many important discoveries have been made in the fields of central nervous system (CNS) injury and disease using this emerging technology. In addition, single-cell mRNA amplification techniques are now being used along with microarrays to overcome many of the difficulties associated with the cellular heterogeneity of the brain. This development has extended the utility of gene expression profiling and has provided researchers with exciting new insights into the neuropathology of CNS injury and disease at a molecular and cellular level. New methodological, standardization, and statistical techniques are currently being developed to improve the reproducibility of microarrays and facilitate the analysis of large amounts of data. In this review, we will discuss the application of these techniques to experimental, clinically relevant models of traumatic brain injury.


Optimization of real time RT-PCR methods for the analysis of gene expression in
mouse eggs and preimplantation embryos.
Jeong YJ, Choi HW, Shin HS, Cui XS, Kim NH, Gerton GL, Jun JH.
Mol Reprod Dev. 2005 Jul;71(3):284-9.
Laboratory of Reproductive Biology and Infertility, Samsung Cheil Hospital and
Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

This study was carried out to optimize conditions for using real time RT-PCR as an efficient and precise quantitative method for estimating the transcript levels of genes expressed in samples containing miniscule amounts of RNA, such as single mammalian oocytes and embryos. First, using mouse eggs and blastocysts, we tested three kinds of RNA isolation or collection methods: TRIZOL reagent, oligo-dT conjugated beads, or three freeze/thaw cycles with the reverse transcription buffer. There were no significant differences among three groups in mRNA quantity as assayed by real time RT-PCR analysis. Second, we compared the efficacy of real time analysis between TaqMan fluorescent probes and the SYBR-green dye system. The two systems presented similar real time RT-PCR profiles for the 16s ribosomal protein gene from oocytes to blastocysts. Third, RNA from mouse embryos at defined stages of preimplantation development were isolated and the levels of transcripts encoded by several housekeeping genes (GAPDH, beta-actin, ribosomal protein L7, 16s ribosomal protein, histone H2A.Z) were quantitatively analyzed by real time RT-PCR. The histone H2A.Z and 16s ribosomal protein slightly increased from the egg to blastocyst stages by approximately 10- and 30-fold, respectively. However, other transcripts increased more than 300-fold as a function of developmental stage from eggs to
blastocysts. Our results suggest that the simple freezing/thawing method for RNA collection, the economic SYBR-green dye system, and histone H2A.Z gene as an internal control should be useful for the real time RT-PCR analysis of single mouse eggs and preimplantation embryos.

Monitoring dynamics of single-cell gene expression over multiple cell cycles

Scott Cookson1, Natalie Ostroff1, Wyming Lee Pang1, Dmitri Volfson1,2 and Jeff Hasty1,*
1 Department of Bioengineering, University of California at San Diego, La Jolla, CA, USA and 2 Institute for Nonlinear Science, University of California at San Diego, La Jolla, CA, USA
* Corresponding author. Department of Bioengineering, University of California at San Diego, La Jolla, USA.
Molecular Systems Biology (2005) doi:10.1038/msb4100032

Recent progress in reconstructing gene regulatory networks has established a framework for a quantitative description of the dynamics of many important cellular processes. Such a description will require novel experimental techniques that enable the generation of time-series data for the governing regulatory proteins in a large number of individual living cells. Here, we utilize microfabrication to construct a Tesla microchemostat that permits single-cell fluorescence imaging of gene expression over many cellular generations. The device is used to capture and constrain asymmetrically dividing or motile cells within a trapping region and to deliver nutrients and regulate the cellular population within this region. We illustrate the operation of the microchemostat with Saccharomyces cerevisiae and explore the evolution of single-cell gene expression and cycle time as a function of generation. Our findings highlight the importance of novel assays for quantifying the dynamics of gene expression and cellular growth, and establish a methodology for exploring the effects of gene expression on long-term processes such as cellular aging.

Genome amplification of single sperm using multiple displacement amplification.

Jiang Z, Zhang X, Deka R, Jin L.
Nucleic Acids Res. 2005 Jun 7;33(10):e91.
Department of Environmental Health, Center for Genome Information, University of
Cincinnati College of Medicine 3223 Eden Ave, Cincinnati, OH 45267, USA.

Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 mul reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.

Single-molecule PCR:   an artifact-free PCR approach for the analysis of somatic mutations.

Kraytsberg Y, Khrapko K.
Expert Rev Mol Diagn. 2005 Sep;5(5):809-15.
Beth Israel Deaconess Medical Center & Harvard Medical School, 21-27 Burlington Avenue, Boston, MA 02215, USA.

A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future.

Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores.

Makino S, Cheun HI.
J Microbiol Methods. 2003 May;53(2):141-7.
Department of Applied Veterinary Science, Research Center for Animal Hygiene and
Food Safety, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR. One hundred liters of air were trapped on a filter of an air monitor device. After it was suspended in PBS, spores of B. anthracis were artificially added. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers. A single cell of B. anthracis was detected by real-time PCR within 1 h. Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics.