This page presents
interesting papers and about single-cell, single-molecule qRT-PCR, or
nano-liter volume range applieing
expression profiles measured by real-time PCR tomography in the Xenopus
Sindelka R, Jonák J, Hands R, Bustin SA,
Nucleic Acids Res. 2008 36(2):387-92.
Laboratory of Gene Expression, Institute of
Molecular Genetics, Academy of
Sciences of the Czech Republic, Videnska 1083, 14220
Prague 4, Czech Republic.
tomography is a novel, quantitative method for measuring localized RNA
expression profiles within single cells. We demonstrate its usefulness
by dissecting an oocyte from Xenopus laevis into slices along its
animal-vegetal axis, extracting its RNA and measuring the levels of 18
selected mRNAs by real-time RT-PCR. This identified two classes of
mRNA, one preferentially located towards the animal,
the other towards the vegetal pole. mRNAs within each group show
comparable intracellular gradients, suggesting they are produced by
similar mechanisms. The polarization is substantial, though not
extreme, with around 5% of vegetal gene mRNA molecules
detected at the animal pole, and around 50% of the molecules in the far
most vegetal section. Most animal pole mRNAs were found in the second
section from the animal pole and in the central section, which is where
the nucleus is located. mRNA expression profiles did not change
following in vitro fertilization and we conclude that the cortical
rotation that follows fertilization has no detectable
effect on intracellular mRNA gradients.
mRNA in single cells and modelling of RT-qPCR induced noise.
Bengtsson M, Hemberg M, Rorsman P, Stahlberg A.
BMC Mol Biol. 2008 9: 63.
Oxford Centre for Diabetes, Endocrinology and
Metabolism, University of Oxford,
The Churchill Hospital, Oxford, OX3 7LJ, UK.
expression has a strong stochastic element resulting in highly variable
mRNA levels between individual cells, even in a seemingly homogeneous
cell population. Access to fundamental information about cellular
mechanisms, such as correlated gene expression, motivates measurements
of multiple genes in individual cells. Quantitative reverse
transcription PCR (RT-qPCR) is the most accessible method which
provides sufficiently accurate measurements of mRNA in single cells.
concentration of guanidine thiocyanate
was used to fully lyse single pancreatic beta-cells followed by RT-qPCR
without the need for purification. The
accuracy of the measurements was determined by a quantitative
noise-model of the reverse transcription and PCR. The noise is
insignificant for initial copy numbers >100 while at lower copy
numbers the noise intrinsic of the PCR increases sharply, eventually
obscuring quantitative measurements. Importantly, the model allows us
to determine the RT efficiency without using artificial RNA as a
standard. The experimental setup was applied on single endocrine cells,
where the technical and biological noise levels were determined.
in single-cell RT-qPCR is insignificant compared to biological
cell-to-cell variation in mRNA levels for medium and high abundance
transcripts. To minimize the technical noise in single-cell RT-qPCR,
the mRNA should be analyzed with a single RT reaction, and a single
qPCR reaction per gene.
profiling in individual hematopoietic progenitors by digital RT-PCR.
Warren L, Bryder D, Weissman IL, Quake SR.
Proc Natl Acad Sci U S A. 2006 103(47): 17807-12.
Division of Biology, California
Institute of Technology, Pasadena, CA 91125, USA.
We report here a systematic, quantitative population analysis of
transcription factor expression within developmental progenitors, made
possible by a microfluidic chip-based "digital RT-PCR" assay that can
count template molecules in cDNA samples prepared from single cells. In
a survey encompassing five classes of early hematopoietic precursor, we
found markedly heterogeneous expression of the transcription factor
PU.1 in hematopoietic stem cells and divergent patterns of PU.1
expression within flk2- and flk2+ common myeloid progenitors. The
survey also revealed significant differences in the level of the
housekeeping transcript GAPDH across the surveyed
populations, which demonstrates caveats of normalizing expression data
to endogenous controls and underscores the need to put gene measurement
on an absolute, copy-per-cell basis.
expression profile of a single cell.
Tang F, Hajkova P, Barton SC, O'Carroll D, Lee C,
Lao K, Surani MA.
Nat Protoc. 2006 1(3): 1154-1159.
Wellcome Trust/Cancer Research UK Gurdon Institute
of Cancer and Developmental
Biology, University of Cambridge, Tennis Court Road,
Cambridge, CB2 1QN, UK.
Here we describe a
protocol for the detection of the
microRNA (miRNA) expression profile of a single cell by stem-looped
real-time PCR, which is specific to mature miRNAs. A single cell is
first lysed by heat treatment without further purification. Then, 220
known miRNAs are reverse transcribed into corresponding cDNAs by
stem-looped primers. This is followed by an initial PCR step to amplify
the cDNAs and generate enough material to
separate multiplex detection. The diluted initial PCR product is used
as a template to check individual miRNA expression by real-time PCR.
This sensitive technique permits miRNA expression profiling from a
single cell, and allows analysis of a few cells from early embryos as
well as individual cells (such as stem cells). It can also be used when
only nanogram amounts of rare samples are
available. The protocol can be completed in 7 d.
quantitation from a single cell by PCR using SYBR Green detection and
NATURE METHODS FEBRUARY 2008 sponsored
We describe a new,
highly sensitive and specific PCR
approach for quantitation of microRNAs (miRNAs): the miRCURY™ LNA
microRNA PCR system. The method, which allows detection of 10 copies of
miRNA, is enabled by the use of Locked Nucleic Acids (LNA™). The
LNA-conferred sensitivity facilitates accurate detection of miRNA
expression in a single cell.
single-cell RT-PCR analysis of Purkinje cells.
Ryosuke Kaneko, Yoshimi Kawamura & Takeshi Yagi
Nature Protocols 1, -
2143 - 2151 (2006)
protocol details a method for analyzing the expression of multiple
genes from a single Purkinje neuron, including the determination of
whether the gene expression is monoallelic or biallelic. The protocol
describes how to extract a single, living Purkinje cell for reverse
transcription, divide the cDNAs into three equal samples and subject
those to triplicate amplification of multiple targets by two rounds of
PCR (first a multiplex PCR then a gene-specific nested PCR) and finally
discriminate the allelic expression of the transcript by direct
sequencing of the PCR products. In optimal conditions, this method
permits the analysis of the expression of 18 genes in a single Purkinje
cell. This protocol can be completed in 5–6 d.
cDNA amplification to provide a template for representative
high-density oligonucleotide microarray analysis.
Kurimoto K, Yabuta Y, Ohinata Y, Saitou M.
Nat Protoc. 2007;2(3): 739-52.
Laboratory for Mammalian Germ Cell Biology, Center
for Developmental Biology,
RIKEN Kobe Institute, 2-2-3 Minatojima-Minamimachi,
Chuo-ku, Kobe 650-0047, Japan.
We describe here a
protocol for the representative amplification of global mRNAs from
typical single mammalian cells to provide a template for high-density
oligonucleotide microarray analysis. A single cell is lysed in a tube
without purification and first-strand cDNAs are synthesized using a
poly(dT)-tailed primer. Unreacted primer is specifically eliminated by
exonuclease treatment and second strands are generated
with a second poly(dT)-tailed primer after poly(dA) tailing of the
first-strand cDNAs. The cDNAs are split into four tubes, which are
independently directionally amplified by PCR, and then recombined. The
amplified products (approximately 100 ng) show superior representation
and reproducibility of original gene expression, especially for genes
expressed in more than 20 copies per cell, compared
with those obtained by a conventional PCR protocol, and can effectively
be used for quantitative PCR and EST analyses. The cDNAs are then
subjected to another PCR amplification with primers bearing the T7
promoter sequence. The resultant cDNA products are gel purified,
amplified by one final cycle and used for isothermal linear
amplification by T7 RNA polymerase to synthesize cRNAs for microarray
hybridization. This protocol yields cDNA templates sufficient for more
than 80 microarray hybridizations from a single cell, and can be
completed in 5-6 days.
single-cell cDNA amplification method for efficient high-density
Kurimoto K, Yabuta Y, Ohinata Y, Ono Y, Uno KD,
Yamada RG, Ueda HR, Saitou M.
Nucleic Acids Res. 2006 34(5): e42.
Laboratory for Mammalian Germ Cell Biology, Center
for Developmental Biology,
RIKEN Kobe Institute, 2-2-3 Minatojima-minamimachi,
Chuo-ku, Kobe, Hyogo 650-0047, Japan.
understanding of a small but essential population of cells in
development or adulthood (e.g. somatic stem cells) requires accurate
quantitative monitoring of genome-wide gene expression, ideally from
single cells. We report here a strategy to globally amplify mRNAs from
single cells for highly quantitative high-density oligonucleotide
microarray analysis that combines a small number of
directional PCR cycles with subsequent linear amplification. Using this
strategy, both the representation of gene expression profiles and
reproducibility between individual experiments are unambiguously
improved from the original method, along with high coverage and
accuracy. The immediate application of this method to single cells in
the undifferentiated inner cell masses of mouse
blastocysts at embryonic day (E) 3.5 revealed the presence of two
populations of cells, one with primitive endoderm (PE) expression and
the other with pluripotent epiblast-like gene expression. The genes
expressed differentially between these two populations were well
preserved in morphologically differentiated PE and epiblast in the
embryos one day later (E4.5), demonstrating that the
method successfully detects subtle but essential differences in gene
expression at the single-cell level among seemingly homogeneous cell
populations. This study provides a strategy to analyze biophysical
events in medicine as well as in neural, stem cell and developmental
biology, where small numbers of distinctive or diseased cells play
Levsky JM, Shenoy SM, Pezo RC, Singer RH.
Science. 2002 297(5582): 836-840.
Department of Anatomy and Structural Biology, Albert Einstein College
Medicine, 1300 Morris Park Avenue, Bronx, NY
A key goal of
is to relate the expression of specific genes to a particular cellular
phenotype. However, current assays for gene expression destroy the
structural context. By combining advances in computational fluorescence
microscopy with multiplex probe design, we devised technology in which
of many genes can be visualized simultaneously inside
cells with high spatial and temporal resolution. Analysis of 11 genes
cultured cells revealed unique patterns of gene expression within
cells. Using the nucleus as the substrate for parallel gene analysis,
a platform for the fusion of genomics and cell biology: "cellular
expression and the myth of the average cell.
JM, Singer RH.
Trends Cell Biol.
2003 13(1): 4-6.
of Anatomy and Structural Biology, Albert Einstein College of
1300 Morris Park Avenue, Bronx, NY 10461, USA.
all know that gene expression occurs within cells, yet we do not think
of expression in terms of its fundamental unit - a
single cell. Instead, we understand the expression of
genes in terms of a
cell population as all of our information comes from
samples containing millions
of cells. From a complex mixture of cells, we attempt
to infer the probable
state of an average cell in the population. In truth,
what we obtain is an
averaged cell, a contrivance for representing
biological knowledge beyond the limits
of detection. We never know the variation among the
members of the population
that our methods average into a mean. Recent
technological advances allow the
precise measurement of single-cell transcriptional
states to study this
variability more rigorously. How genes are expressed in
the population is
strikingly different to what we have assumed from
extrapolating to an average cell.
Does the average cell actually exist? As we discuss, it
increasingly clear that it doesn't.
capture microdissection with quantitative real-time PCR:
manipulation on RNA quality and gene expression.
Kerman IA, Buck BJ, Evans SJ, Akil H, Watson SJ.
J Neurosci Methods. 2006 153(1): 71-85.
Molecular and Behavioral Neuroscience Institute,
Department of Psychiatry,
University of Michigan, 205 Zina Pitcher Place,
Ann Arbor, MI 48109, USA.
capture microdissection (LCM) is increasingly being used in
quantitative gene expression studies of the nervous system. The current
study aimed at determining the impact of various tissue manipulations
on the integrity of extracted RNA in LCM studies. Our data indicate
that various tissue preparation strategies prior to microdissection may
decrease RNA quality by as much as 25%, thus affecting expression
profiles of some genes. To circumvent this problem, we developed a
strategy for reverse transcriptase real-time PCR that has considerable
sensitivity and can be used to calculate relative changes in gene
expression. This approach was validated in subregions of the rat
cerebellum. Accordingly, expression of glial gene markers -
myelin-associated glycoprotein and proteolipid protein 1 - was found
70-160-fold higher in the white matter layer of the cerebellar cortex
as compared to the neuron-enriched granular layer. In contrast,
expression of a specific neuronal maker, neuron-specific enolase, was
found seven-fold higher in the granular layer, as compared to the white
matter layer. Furthermore, this approach had high sensitivity and
specificity as we were able to detect a 38% decrease in the expression
of neuron-specific enolase without a change in the expression of glial
markers following administration of the neurotoxin, ibotenic acid.
These results demonstrate feasibility of performing accurate
semi-quantitative gene expression analyses in LCM samples.
profiling at the single molecule level.
Hesse J, Jacak J, Kasper M, Regl G, Eichberger T, Winklmayr M, Aberger
Sonnleitner M, Schlapak R, Howorka S, Muresan L, Frischauf AM, Schutz
Biophysics Institute, Johannes Kepler University Linz, A-4040 Linz,
Genome Res. 2006 Jun 29
developed a microarray platform for PCR amplification-independent
expression profiling of minute samples. A novel scanning system
combined with specialized biochips enables detection down to individual
fluorescent oligonucleotide molecules specifically hybridized to their
complementary sequence over the entire biochip surface of cm(2) size. A
detection limit of 1.3 fM target oligonucleotide
concentration-corresponding to only 39,000 molecules in the sample
solution-and a dynamic range of 4.7 orders of magnitude have been
achieved. The applicability of the system to PCR
amplification-independent gene-expression profiling of minute samples
was demonstrated by complex hybridization of cDNA derived from the
equivalent of only 10(4) cells, which matches results obtained in
ensemble studies on large samples. By counting each hybridized molecule
on the microarray, the method is insusceptible to gene-specific
variations of the labeling, thereby representing a principle advance to
conventional ensemble-based microarray analysis.
single-cell RT-PCR and Ca(2+) imaging in brain slices.
Durand GM, Marandi N, Herberger SD, Blum R,
Pflugers Arch. 2006 451(6): 716-726.
Institut fur Physiologie,
Ludwig-Maximilians-Universitat, Pettenkofer Strasse 12, 80336, Munchen,
have established a quantitative reverse transcriptase-PCR (RT-PCR)
approach for the analysis of RNA transcript levels in individual cells
of living brain slices. Quantification is achieved by using
rapid-cycle, real-time PCR protocols and high-resolution external cDNA
standard curves for the gene of interest. The method consists of
several procedures, including cell soma harvest, reverse transcription,
and an optimized cDNA purification step, which allowed us to quantify
transcripts in small types of neurons, like cerebellar granule cells.
Thus, we detected in single granule cells an average of 20 transcript
copies of the housekeeping gene
glyceraldehyde-3-phosphate-dehydrogenase. We combined two-photon
calcium imaging and quantitative RT-PCR in single Purkinje and granule
cells, respectively, and identified distinct glutamate
receptor-dependent Ca(2+) responses in these two cell types. The
approach was further tested by profiling the expression of the
ionotropic glutamate receptor subunits NR2B and NR2C in the cerebellum.
Our study revealed a developmental switch from an average of 15 NR2B
copies/cell at postnatal day 8 (P8) to about five NR2C copies/cell
after P26. Taken together, our results demonstrate that the new method
is rapid, highly sensitive, provides reliable results in neurons of
various sizes, and can be used in combination with Ca(2+) imaging.
strategies for small sample populations.
Methods. 2005 37(3): 229-237.
Center for Dementia Research, Nathan Kline Institute, Department of
and Physiology and Neuroscience, New York University School of Medicine,
Orangeburg, NY 10962, USA.
Advances in high
throughput cloning strategies have led to sequencing of the human
genome as well as progress in the sequencing of the genome of several
other species. Consequently, the field of molecular genetics is
blossoming into a multidisciplinary entity that is revolutionizing the
way researchers evaluate a myriad of critical concepts such as
development, homeostasis, and disease pathogenesis. There is tremendous
interest in the quantitative assessment of tissue-specific expression
of both newly identified and well characterized specific genes and
proteins. At present, an ideal approach is to assess gene expression in
single elements recorded physiologically in living preparations or by
immunocytochemical or histochemical methods in fixed cells in vitro or
in vivo. The quantity of RNA harvested from individual cells is not
sufficient for standard RNA extraction methods. Therefore, exponential
polymerase-chain reaction based analyses, and linear RNA amplification
including amplified antisense RNA amplification and a newly developed
terminal continuation RNA amplification methodology have been developed
for use in combination with microdissection procedures and
cDNA/oligonucleotide microarray platforms. RNA amplification is a
series of intricate procedures to amplify genetic signals from minute
quantities of starting materials for microarray analysis and other
downstream genetic methodologies. RNA amplification procedures
effectively generate quantities of RNA through in vitro transcription.
The present report illustrates practical usage of RNA amplification
technologies within the context of regional, population cell, and
single cell analyses in the brain.
EXPRESSION OF SINGLE CHONDROCYTES
SV; Shieh, AC; Athanasiou, KA
University, Houston, TX
normalization method for mRNA measurement by quantitative PCR and
Ken-ichi Aisaki, Katsuhide Igarashi, Noriyuki Nakatsu, Atsushi Ono,
Kodama and Taku Nagao
BMC Genomics2006, 7:64
Division of Cellular
and Molecular Toxicology, National Institute of Health Sciences,
1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan and President,
National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku,
Tokyo 158-8501, Japan
Transcriptome data from
quantitative PCR (qPCR) and DNA microarrays are typically obtained
from a fixed amount of RNA collected per sample. Therefore, variations
in tissue cellularity
and RNA yield across samples in an experimental series compromise
of the absolute level of each mRNA species per cell in any sample.
Since mRNAs are copied from genomic
DNA, the simplest way to express mRNA level would be as copy number per
template DNA, or
more practically, as copy number per cell.
Results: Here we report a method
(designated the "Percellome" method) for normalizing the expression of
values in biological samples. It provides a "per cell" readout in mRNA
and is applicable to both quantitative PCR (qPCR) and DNA microarray
studies. The genomic DNA content
of each sample homogenate was measured from a small aliquot to derive
the number of cells
in the sample. A cocktail of five external spike RNAs admixed in a
dose-graded manner (dose-graded
spike cocktail; GSC) was prepared and added to each homogenate in
proportion to its DNA
content. In this way, the spike mRNAs represented absolute copy numbers
per cell in the
sample. The signals from the five spike mRNAs were used as a
dose-response standard curve for
each sample, enabling us to convert all the signals measured to copy
cell in an expression profile-independent manner. A series of samples
was measured by qPCR and Affymetrix
GeneChip microarrays using this Percellome method, and the results
showed up to 90% concordance.
Conclusion: Percellome data can be
compared directly among samples and among different studies, and
different platforms, without further normalization. Therefore,
"percellome" normalization can
serve as a standard method for exchanging and comparing data across
different platforms and among
for the detection of multiple miRNA species in small samples.
Lao K, Xu NL, Yeung V, Chen C, Livak KJ, Straus NA.
Biochem Biophys Res Commun. 2006 343(1): 85-89.
Applied Biosystems, 850 Lincoln Centre Dr.,
Foster City, CA 94404, USA.
(approximately 22 nucleotides), non-coding RNAs that play critical
gene regulation and may be used as rapid precise diagnostic indicators
stages of cancer. The small size of these RNAs makes detection of
microRNA species in very small samples problematic. Here we investigate
parameters associated with multiplexing RT-PCR to obtain relative
profiles of multiple microRNAs in small sample sizes down to the amount
found in a single cell.
individual bovine nuclear transfer blastocysts.
somatic cell nuclear transfer the gene expression profile of the donor
cell has to be changed or reprogrammed extensively to reflect that of a
normal embryo. In this study we focused on the switching on of
embryonic genes by screening with a microarray consisting of 5,000
independent cDNA isolates derived from a bovine blastocyst library
which we constructed for this purpose. Expression profiling was
performed using linearly amplified RNA from individual day 7 nuclear
transfer (NT) and genetically half-identical in vitro produced (IVP)
blastocysts. We identified 92 genes expressed at lower levels in NT
embryos whereas transcripts of 43 genes were more abundant in NT
embryos (P 0.05, 1.5-fold change). A range of functional
was represented among the identified genes, with a preponderance of
constitutively expressed genes required for the maintenance of basal
cellular function. Using a stringent quantitative SYBR-green real time
RT-PCR based approach we found, when comparing the means of the
expression levels of a larger set of individual embryos, that
differences were small (2-fold) and only significant for two of the
seven analysed genes (KRT18, SLC16A1). Notably, examination of
transcript levels of a single gene in individual embryos could not
distinguish an NT from a control embryo. This unpredictability of
individual gene expression on a global background of multiple gene
expression changes argues for a predominantly stochastic nature of
Joanna Somers, Craig Smith,
Martyn Donnison, David N Wells, Harold Henderson, Lance
McLeay and P L Pfeffer
(2006) 131 1073–1084
quantitative measurement of gene expression directly from a small
amount of whole blood.
Zheng Z, Luo Y, McMaster GK.
Clin Chem. 2006 52(7): 1294-1302.
Panomics, Inc., 6519 Dumbarton Circle,
Fremont, CA 94555, USA.
Accurate and precise
quantification of mRNA in whole blood is made difficult by gene
expression changes during blood processing, and by variationsand biases
introduced by sample preparations. We sought to develop a quantitative
whole-blood mRNA assay that eliminates blood purification, RNA
isolation, reverse transcription, and target amplification while
providing high-quality data in an easy assay format.
performed single- and
multiplex gene expression analysis with multiple hybridization probes
to capture mRNA directly from blood lysate and used branched DNA to
amplify the signal. The 96-well plate singleplex assay uses
chemiluminescence detection, and the multiplex assay combines
Luminex-encoded beads with fluorescent detection.
single- and multiplex assays could quantitatively measure as few as
6,000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles),
respectively, in up to 25 microL of whole blood. Both formats had CVs
< 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed
quantitative measurement of gene expression in the minority of cells in
whole blood. The signals from whole-blood lysate correlated well with
signals from purified RNA of the same sample, and absolute mRNA
quantification results from the assay were similar to those obtained by
quantitative reverse transcription-PCR. Both single- and multiplex
assay formats were compatible with common anticoagulants and
PAXgene-treated samples; however, PAXgene preparations induced
expression of known antiapoptotic genes in whole blood.
the singleplex and the multiplex branched DNA assays can quantitatively
measure mRNA expression directly from small volumes of whole blood. The
assay offers an alternative to current technologies that depend on RNA
isolation and is amenable to high-throughput gene expression analysis
of whole blood.
of small cellular samples in cancer: less is more.
Glanzer JG, Eberwine JH.
Br J Cancer. 2004 90(6): 1111-1114.
Department of Pharmacology, University of
Pennsylvania Medical Center, Philadelphia, PA 19104-6058, USA.
profiling of tumours from cancer patients has uncovered several genes
that are critically important in the progression of a normal cell to an
oncogenic phenotype. Leading the way in these discoveries is the use of
microarrays, a technology that is currently in transition from basic
science applications to use in the clinic. Microarrays can determine
the global gene regulation of an individual cancer, which may be useful
in formulating an individualised therapy for the patient. Currently,
cells used in breast cancer microarray studies often come from either
homogenous cultures or heterogeneous biopsy samples. Both cell sources
are at a disadvantage in determining the most accurate gene profile of
cancer, which often consists of multiple subspecies of cancerous cells
within a background of normal cells. Therefore, acquisition of small,
but highly specific biopsies for analysis may be required for an
accurate expression analysis of the disease. Amplification methods,
such as polymerase chain reaction (PCR) and amplified antisense RNA
(aRNA) amplification, have been used to amplify the mRNA signal from
very small samples, which can then be used for microarray analysis. In
this study, we describe the acquisition, amplification, and analysis of
very small samples (<10000 cells) for expression analysis and
demonstrate that the ultimate resolution of cancer expression analysis,
one cell, is both feasible and practical.
of single disseminated tumour cells in minimal residual cancer.
Blankenstein TJ, Schmidt-Kittler O, Petronio M, Polzer B, Stoecklein,
NH, Riethmuller G.
2002 360(9334): 683-689.
Immunologie, Ludwig-Maximilians-Universitat Munchen, D-80336 Muenchen,
Because cancer patients
with small tumours often relapse despite local and
systemic treatment, we investigated the genetic variation of the
of distant metastasis at the stage of minimal residual disease.
tumour cells can be detected by epithelial markers in mesenchymal
and represent targets for adjuvant therapies.
METHODS: We screened 525 bone-marrow, blood,
and lymph-node samples from 474 patients with breast, prostate,
and gastrointestinal cancers for single disseminated cancer cells by
with epithelial-specific markers. 71 (14%) of the samples contained
two or more tumour cells whose genomic organisation we studied by
single cell genomic hybridisation. In addition, we
tested whether TP53 was mutated. Hierarchical
clustering algorithms were used to determine the degree of clonal
relatedness of sister cells that were isolated from individual patients.
Irrespective of cancer type, we saw an unexpectedly high genetic
in minimal residual cancer, particularly at the level of chromosomal
Although few disseminated cells harboured TP53 mutations at this stage
of disease, we also saw microheterogeneity
of the TP53 genotype. The genetic heterogeneity was
strikingly reduced with the emergence of clinically evident
INTERPRETATION: Although the heterogeneity of primary tumours
has long been known, we show here that early disseminated cancer cells
genomically very unstable as well. Selection of clonally expanding
cells leading to metastasis seems to occur after
dissemination has taken place. Therefore, adjuvant
therapies are confronted with an extremely large reservoir of
variant cells from which resistant tumour cells can be selected.