Quantification of gene expression in single cells

Martin Bengtsson

    Research output: ThesisDoctoral Thesis (compilation)

    66 Downloads (Pure)

    Abstract

    Studies of apparently homogeneous cell populations and single cells often give highly divergent results. Cells exhibit varying responsiveness to stimuli and gene expression and they are in many aspects stochastic and unpredictable. We have developed a method to measure gene expression quantitatively in individual cells with real-time RT-PCR.

    mRNA for hormones, ion channels and enzymes in pancreatic alpha- and beta-cells were quantified. The distribution of transcript levels were highly skewed and was best described by a lognormal distribution. Thus, the geometric--and not the commonly used arithmetic--mean value is the appropriate measure of average expression level. In beta-cells, insulin mRNA levels were increased in response to glucose stimulation; an effect due to an increased fraction of cells with high expression. The insulin genes Ins1 and Ins2 have similar promoter regions and were indeed co regulated within single beta-cells.

    Na-channels in alpha- and beta-cells display very different inactivation properties (being separated by 40 mV). We measured hormone mRNA and all Na-channel isoforms in single cells and correlated gene expression with patch-clamp recordings. Cell type-specific expression of Na-channel isoforms can partly explain the divergent inactivation.

    Early differentiation of human embryonic stem cells involves the transcription factors Pou5f1, Nanog and Sox2. We quantified their expression in single stem cells and observed that they are not correlated with each other. Instead Pou5f1 correlates with the transcription factors Id1 and Id3.

    We conclude that quantitative gene expression measurements on single cells allow: 1) studies of cell population heterogeneity and noise in gene expression; 2) exploration of genes that are co-regulated; and 3) correlation of gene expression with functional properties such as electrophysiological properties.
    Original languageEnglish
    QualificationDoctor
    Awarding Institution
    • Department of Clinical Sciences, Malmö
    Supervisors/Advisors
    • Rorsman, Patrik, Supervisor
    Award date2007 May 4
    Publisher
    ISBN (Print)978-91-85559-41-1
    Publication statusPublished - 2007

    Bibliographical note

    Defence details

    Date: 2007-05-04
    Time: 13:15
    Place: Lilla Aulan, MFC Ing 59 UMAS Malmö

    External reviewer(s)

    Name: Schuit, Frans
    Title: Prof
    Affiliation: Katholieke Universiteit Leuven, Belgium

    ---


    <div class="article_info">Martin Bengtsson, Anders Ståhlberg, Patrik Rorsman and Mikael Kubista. <span class="article_issue_date">2005</span>. <span class="article_title">Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels</span> <span class="journal_series_title">Genome Research</span>, <span class="journal_volume">vol 15</span> <span class="journal_pages">pp 1388-1392</span>.</div>
    <div class="article_info">Martin Bengtsson, Patrik Rorsman and Anders Ståhlberg. <span class="article_issue_date"></span>. <span class="article_title">Single-cell mRNA quantification with real-time RT-PCR</span> (submitted)</div>
    <div class="article_info">Anders Ståhlberg, Martin Bengtsson and Henrik Semb. <span class="article_issue_date"></span>. <span class="article_title">Quantitative transcription factor analysis of undifferentiated single human embryonic stem cells</span> (submitted)</div>
    <div class="article_info">Martin Bengtsson, Chris Partridge, Reshma Remrachaya, Albert Salehi, Anna Wendt, Yang Zhang de Marinis, Matthias Brain, Lena Eliasson and Patrik Rorsman. <span class="article_issue_date"></span>. <span class="article_title">Widely different Na+-current inactivation properties in a- and b-cells may result from cell-specific expression of distinct Na+-channel subunits</span> (manuscript)</div>

    Subject classification (UKÄ)

    • Endocrinology and Diabetes

    Keywords

    • Medicin (människa och djur)
    • Medicine (human and vertebrates)
    • Biomedical sciences
    • Biomedicinska vetenskaper
    • quantitative gene expression analysis
    • single cell PCR
    • islets of Langerhans

    Fingerprint

    Dive into the research topics of 'Quantification of gene expression in single cells'. Together they form a unique fingerprint.

    Cite this