Towards a Comprehensive Single Cell Expression Profiling

Herbert Auer
IRB Barcelona, Spain

Abstract
Expression profiling, the measurement of all transcripts of a cell, is currently the most comprehensive method to describe its physiological state. Given that accurate profiling methods currently available require RNA amounts found in thousands to millions of cells, many fields of biology working with specialized cell types cannot use these techniques because available cell numbers are limited. Currently available alternative methods for expression profiling from picograms of RNA or from very small cell populations lack a broad validation of results to provide accurate information about the measured transcripts. Except for a few highly experimental methods like Single-Molecule-Sequencing, every attempted measurement of more than a few different transcripts needs a pre-amplification of cDNA. Here we present Pico Amplification, a novel workflow of RNA isolation and cDNA amplification for cell populations as small as a few or even a single cell. Micrograms of cDNA are generated, suitable for analysis by qPCR, microarrays or sequencing. Expression profiling of RNAs diluted to picograms showed that Pico Amplification virtually did not alter measurements of differential expression. MAQC samples A and B (Universal Reference RNA and Human Brain RNA) were utilized to provide transcriptome wide evaluation using microarrays and for comparison to qPCR measurements for over 800 transcripts. RNA isolated from 10 cells provided after Pico Amplification virtually identical information about the entire population as standard protocols do from thousands or millions of cells. This held true across the entire transcriptome. RNA isolated from individual cells provided insight into the heterogeneity of seemingly homogenous cell populations. Hundreds of transcripts were found to be differentially expressed between individual cells. Pico Amplification provides micrograms of cDNA from very small cell populations or even individual cells, highly representative of the original transcriptome. This allows the application of virtually all standard methods of expression profiling to interrogate the transcriptional status of an individual cell.

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Real time PCR on slide: a new tool for quantification of minute amounts of DNA

Jean-Christophe Avarre1, Angelique le Bras2, Régis Melizzi2, Maxime Rattier2, Gordana Cerovic2, Claude Weisbuch2, Marianna Alunni3, Martin Kantlehner3, Petra Hartmann3, Reinhard Bittner3, Wolfgang Mann3
1Institut de Recherche pour le Développement, Montpellier, France; 2Genewave SAS, Paris, France; 3Beckman Coulter Biomedical GmbH, Munich, Germany

Abstract
Detection, identification and quantification of micro-organisms represents one of the major challenges for our modern society. It concerns a wide variety of applications including bioprocess control, food technology, health care, environmental analysis and of course clinical diagnostics. In this regard, miniaturization of PCR protocols may offer many advantages including short assay time, high precision and sensitivity, high-throughput, low reagent consumption and portability. Though on-chip PCR is likely to become the ‘next-generation PCR’, translating microfluidics to biology labs is still limited by its cost and the expertise it requires. In this context, a new format system has been developed, which allows DNA extraction and real-time PCR amplification in 1-µl “reaction sites” on a slide. The system, called SicLive, enables to process 48 samples in parallel and offers the possibility to perform quantitative PCR amplifications within 2 hours, including DNA extraction. Results, obtained on different model systems, showed that reproducibility and dynamic range were comparable to those obtained in 20 µl with conventional real-time PCR. In terms of sensitivity, SicLive was able to amplify DNA from 1-5 copies of target as well as from single cells. By combining DNA extraction and amplification in a single reaction site, SicLive is particularly well adapted for quantifying precious samples containing very low amounts of genetic material, such as clinical or environmental samples. Finally, for single-cell applications, this slide format ensures an easy visual control, with standard microscopy, of both quality and quantity of the templates loaded.

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Digital Competitive Allele-Specific TaqMan-Based Reverse Transcription-qPCR (castRT-qPCR) for Direct Detection and Enumeration of Circulating Tumor Cells

David Xingfei Deng, Yun Bao, Scott Sproul, Yu Wang, Fawn Wang, David Merrill, Pius Brzoska, Caifu Chen
Life Technologies, United States of America

Abstract
Molecular characterization and enumeration of circulating tumor cells (CTCs) promise to be valuable for cancer cell diagnosis, survival prognosis, and treatment guidance. However, current molecular assays require extensive blood sample enrichment process before analyzing extraordinarily rare CTCs. Here we reported a new approach for direct CTC molecular detection in whole blood samples without prior biophysical processing by combining sample partition and digital assay of a highly specific castRT-qPCR. CastPCR can detect rare copies of mutant alleles with a wide dynamic range of more than 6-log orders and < 5-copy sensitivity. Whole blood samples with spiked-in known mutation lung cancer cell lines and from lung cancer patients were partitioned in aliquots of 2.5 μL – 50 μL onto 96- or 384-well plate(s), such that each well contains either one cancer cell or none with per well 20,000 – 400,000 normal white blood cells and 10 – 200 million red blood cells. RNAs and/or DNAs were extracted by magnetic beads and directly or were pre-amplified prior to mutation detection. Genetic mutations and cell type specific markers (such as CK19 and/or CEA) for CTC identification and enumeration were determined by castRT-qPCR and/or TaqMan Gene expression assays. The sample partition process resulted in a relative CTC enrichment or digital enrichment of 20 – 400 folds (the relative ratio of CTC to normal cells) in a CTC-positive well. CastPCR clearly identified known mutation and CK19 in spiked-in samples of about 10 – 30 cells per mL whole blood, but detected no mutation signals in any sample well without cell spiked-in. Furthermore, cell type specific markers (CK19) and known EGFR mutation(s) were identified in the same sample wells, suggesting identified mutation is specific and from cancer cells not from normal cells. In five blood samples from lung cancer patients, EGFR mutation (p.L858R) was detected in all samples. Approximately 50% of circulating lung tumor cells in a patient with positive EGFR p.L858R mutation had also positive EGFR p.T790M mutation, an inducible drug-resistant CTC marker. For those samples with negative detection of EGFR mutation, corresponding wild type sequences were detected in all sample wells, suggesting the normal DNA amplification of those mutation-negative wells. Our data suggest that combination of digital sample enrichment and castRT-qPCR can be used to directly enumerate CTCs and detect cancer-related mutations in whole blood without prior biophysical sample enrichment. The new approach paves way for noninvasively CTC monitoring and individualized therapy.

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Relationship between cell attachment and bcr-abl cell expression heterogeneity within CML cell lines

Philip Day, Ehsan Karimiani
University of Manchester, United Kingdom

Abstract
Chronic myelogenous leukemia (CML) is believed to occur as a consequence of the clonal expansion of leukemic stem cells and to be maintained by an expanding population of hematopoietic stem cells that have acquired a BCR-ABL fusion gene. Recent studies indicate that primitive CML cells are less responsive to tyrosine kinase inhibitors and are a reservoir for the emergence of tyrosine kinase resistant subclones. Some studies have suggested that expression of BCR-ABL may also be required for altered cell adhesion and CML progression. The bcr-abl protein modulates cell adhesion and its effects in cell lines like K562 correlate with increased adhesion to fibronectin. It also has been reported that cell adhesion mediated resistance to apoptosis induced by BCR-ABL inhibitors, suggests that bcr-abl mediated cell adhesion may be involved in post-therapy residual disease of CML. However, some reports imply that a relatively small fraction (2%–20%) of blasts from patients with acute myeloid leukemia adhere to the plastic of the cell-culture dish. In this study we test if elevated BCR-ABL expression could be specifically identified in individual cells from attached cell populations and not unattached populations. The study developed a means to utilise flow assisted cell sorting of cell lines expressing BCR-ABL to derive individual attached and unattached cell sub-populations. Using a homogenous extraction procedure, cells individually flow sorted into microtitre plates were subjected to combinations of abl and bcr-abl qRT-PCR, and revealed the existence of low and high bcr-abl expressing cell line populations. The implications of this study will be discussed.

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Single Molecule RNA FISH: novel, simple, and accurate quantitative applications for gene expression analysis

Arjun Raj
University of Pennsylvania, United States of America

Abstract
We have developed a simple, direct method for specifically detecting individual RNA molecules in situ via fluorescence microscopy. Our RNA-FISH method relies on using large numbers of fluorescently labeled oligonucleotides designed to target particular RNA species. This approach is highly specific and highly sensitive and provides absolute, direct quantification of RNA abundance in single cells. Moreover, it is applicable to a broad range of biological sample types, ranging from microbes to human tissue section. We will discuss the method itself as well as address some biological applications in development and cancer.

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Identification of unknown cell populations and correlations using single-cell gene expression profiling

Anders Ståhlberg
University of Gothenburg, Sweden

Abstract
Single-cell gene expression levels show substantial variations among cells in seemingly homogenous populations. Access to fundamental information about cellular mechanism, such as correlated gene expression, motivates studies of multiple genes expressions in individual cells. Astrocytes perform many control and regulatory functions in the central nervous system. In contrast to neurons, we have limited knowledge about functional diversity of astrocytes and its molecular basis. We will show how subpopulations of cells can be identified at single-cell level using unsupervised algorithms and that gene correlations can be used to identify differences in activity of important transcriptional pathways. We identified two subpopulations of astrocytes with distinct gene expression profiles. One had an expression profile very similar to that of neurosphere cells, whereas
the other showed characteristics of activated astrocytes in vivo. Technical considerations related to reproducible and efficient sampling, lysis, reverse transcription and real-time PCR will also be presented. In addition to astrocytes, single-cell data from tumor cells, beta-cells and embryonic stem cells will be shown.


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Uncovering the Diversity of Individual Cells: Gene Expression Profiling with the BioMark System

Ken Livak
Fluidigm Corporation, United States of America

Abstract
Fluctuations in gene expression at the single cell level could be key for generating developmental signals and for understanding the progression of tumors. Data needs to be collected from a statistically significant number of single cells in order to determine the range of gene expression present in a population of cells. Furthermore, transcripts need to be quantified for a number of genes in order to obtain meaningful cell signatures. BioMark™ arrays provide a convenient and cost-effective system for performing multiple RNA expression assays on multiple single-cell samples. This system has been used to study single cell gene expression in embryonic stem cells, hematopoieticstem cells, cancer stem cells, and early stage embryos.

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25 years of PCR – from idea to subcellular expression profiling

Mikael Kubista
TATAA BIOCENTER, Sweden

Abstract
PCR was born from a great idea conceived by Kary Mullis and later refined into qPCR by Russ Higushi. Specificity was added by Ken Livak. The technique found immediate use in genetic engineering leading to several breakthrough innovations, and later PCR became the preferred platform for clinical diagnostics. The quantitative aspect of qPCR became particularly valuable in biological and medical research, and is today key technology in systems biology. Moreover, the extreme sensitivity of qPCR, allowing the detection of single molecular copies, and the relative ease of use, which allows qPCR to be integrated in a streamlined high throughput workflow, has led to the exciting area of single cell expression profiling. An astonishing heterogeneity in transcript levels among seemingly like cells has been found, while correlation between genes’ expression is a fingerprint of the type of cell. Most recently, qPCR tomography has been developed to measure intracellular mRNA profiles, revealing gradients of transcripts within the cell, preparing it for asymmetric cell division.
Anders Stahlberg, Daniel Andersson, Johan Aurelius, Maryam Faiz, Marcela Pekna, Mikael Kubista, Milos Pekny. Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations. Nucl. Acids Res. 1–12. doi:10.1093/nar/gkq1182 (2010).
Radek Sindelka, Monika Sidova, David Svec, Mikael Kubista. Spatial expression profiles in the Xenopus laevis oocytes measured with qPCR tomography. Methods 51:87-91(2010).
M. Bengtsson, A. Ståhlberg, P. Rorsman, and M. Kubista Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels. Genome Research 15, 1388-1392 (2005).
Research Highlights in Nature Review Genetics 6, 1758 (2005).


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