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Imaging
intracellular RNA distribution and dynamics in living cells.
Sanjay Tyagi
Nature Methods vol 6 no 5 2009: 331
Powerful methods
now allow the imaging of specific mRNAs in living cells. These methods
enlist fluorescent proteins to illuminate mRNAs, use labeled
oligonucleotide probes and exploit aptamers that render organic dyes
fluorescent. The intracellular dynamics of mRNA synthesis, transport
and localization can be analyzed at higher temporal resolution with
these methods than has been possible with traditional fixed-cell or
biochemical approaches. These methods have also been adopted to
visualize and track single mRNA molecules in real time. This review
explores the promises and limitations of these methods.
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mRNA-Seq
whole-transcriptome analysis of a single cell.
Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J,
Tuch BB, Siddiqui A, Lao K, Surani MA.
Wellcome Trust-Cancer Research UK Gurdon Institute of Cancer and
Developmental
Biology, University of Cambridge, Cambridge, UK.
Nat Methods. 2009 6(5): 377-82
Next-generation
sequencing technology is a powerful tool for transcriptome analysis.
However, under certain conditions, only a small amount of material is
available, which requires more sensitive techniques that can preferably
be used at the single-cell level. Here we describe a single-cell
digital gene expression profiling assay. Using our mRNA-Seq assay with
only a single mouse blastomere, we detected the expression of 75%
(5,270) more genes than microarray techniques and identified 1,753
previously unknown splice junctions called by at least 5 reads.
Moreover, 8-19% of the genes with multiple known transcript isoforms
expressed at least two isoforms in the same blastomere or oocyte, which
unambiguously demonstrated the complexity of the transcript variants at
whole-genome scale in individual cells. Finally, for Dicer1(-/-) and
Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes,
respectively, were abnormally upregulated compared to wild-type
controls, with 619 genes in common.
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Quantitative
analysis of gene expression in a single cell by qPCR.
Taniguchi K, Kajiyama T, Kambara H.
Hitachi Central Research Laboratory, Tokyo, Japan.
Nat Methods. 2009 6(7): 503-506
supl.
We developed a quantitative PCR method featuring a
reusable single-cell cDNA library immobilized on beads for measuring
the expression of multiple genes in a single cell. We used this method
to analyze multiple cDNA targets (from several copies to several
hundred thousand copies) with an experimental error of 15.9% or less.
This method is sufficiently accurate to investigate the heterogeneity
of single cells.
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Genomic
expression analysis by single-cell mRNA differential display of
quiescent
CD8
T cells from tumour-infiltrating lymphocytes obtained from in vivo
liver tumours.
Zhang W, Ding J, Qu Y, Hu H, Lin M, Datta A, Larson A, Liu GE, Li B.
Department of Biochemistry, Case Western Reserve University School of
Medicine,
Cleveland, OH 44106-4935, USA.
Immunology. 2009 May;127(1): 83-90.
We performed a genomic study combining single-cell
mRNA differential display and RNA subtractive hybridization to
elucidate CD8 T-cell quiescence/ignorance. By comparing actively
maintained quiescent CD8 T cells from liver tumour tumour-infiltrating
lymphocytes (TILs) with quiescent T cells at the single-cell level, we
identified differentially expressed candidate genes by high-throughput
screening and comparative analysis of expressed sequence tags (ESTs).
While genes for the T-cell receptor, tumour necrosis factor (TNF)
receptor, TNF-related apoptosis inducing ligand (TRAIL) and perforin
were down-regulated, key genes such as Tob, transforming growth factor
(TGF)-beta, lung Krüpple-like factor (LKLF), Sno-A, Ski, Myc,
Ets-2 repressor factor (ERF) and RE1-silencing transcription factor
(REST/NRSF) complex were highly expressed in the quiescent TIL CD8
cells. Real-time polymerase chain reaction (PCR) further confirmed
these results. A regulation model is proposed for actively maintained
quiescence in CD8 T cells, including three components: up-regulation of
the TGF-beta pathway, a shift in the MYC web and inhibition of the cell
cycle.
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Rac1
regulates pancreatic islet morphogenesis.
Greiner TU, Kesavan G, Stahlberg A, Semb H.
Stem Cell and Pancreas Developmental Biology, Stem
Cell Center, Lund University,
BMC B10, Klinikgatan 26, SE-221 84 Lund, Sweden.
BMC Dev Biol. 2009 Jan 6;9:2.
BACKGROUND:
Pancreatic islets of Langerhans originate from endocrine progenitors
within the pancreatic ductal epithelium. Concomitant with
differentiation of these progenitors into hormone-producing cells such
cells delaminate, aggregate and migrate away from the ductal
epithelium. The cellular and molecular mechanisms regulating islet cell
delamination and cell migration are poorly understood. Extensive
biochemical and cell biological studies using cultured cells
demonstrated that Rac1, a member of the Rho family of small GTPases,
acts as a key regulator of cell migration.
RESULTS: To address the functional role of Rac1 in
islet morphogenesis, we generated transgenic mice expressing dominant
negative Rac1 under regulation of the Rat Insulin Promoter. Blocking
Rac1 function in beta cells inhibited their migration away from the
ductal epithelium in vivo. Consistently, transgenic islet cell
spreading was compromised in vitro. We also show that the EGF-receptor
ligand betacellulin induced actin remodelling and cell spreading in
wild-type islets, but not in transgenic islets. Finally, we demonstrate
that cell-cell contact E-cadherin increased as a consequence of
blocking Rac1 activity.
CONCLUSION: Our data support a model where Rac1 signalling controls
islet cell migration by modulating E-cadherin-mediated cell-cell
adhesion. Furthermore, in vitro experiments show that betacellulin
stimulated islet cell spreading and actin remodelling is compromised in
transgenic islets, suggesting that betacellulin may act as a regulator
of Rac1 activity and islet migration in vivo. Our results further
emphasize Rac1 as a key regulator of cell migration and cell adhesion
during tissue and organ morphogenesis.
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A
novel single-cell quantitative real-time RT-PCR method for quantifying
foot-and-mouth disease viral RNA.
Huang X, Li Y, Zheng CY.
State Key Laboratory of Virology, College of Life
Sciences, Wuhan University, Wuhan 430072, China.
J Virol Methods. 2009 155(2): 150-156
Foot-and-mouth disease virus is a positive-sense,
single-stranded RNA virus with a negative strand as its replication
intermediate, which can cause severe acute infection in sensitive cell
lines. To investigate better the actual state of virus infection, there
is a need to measure the amount of FMDV RNA in a single acutely
infected cell rather than in a large number of cells. Therefore, in the
present study, a strand-specific single-cell quantitative real-time
RT-PCR was developed to analyze the RNA or FMDV. This new method uses
two techniques in concert with each other: a technique for isolating
single cells with micromanipulators, which is coupled to an assay for
detecting viral RNA by real-time RT-PCR. In the assay of acute
infection, 185 of 224 (82.6%) single-cell samples were positive and
contained viral genome copies ranging from several to thousands, and up
to 1,000,000 copies. However, not all cells were infected and there
were differences in the number of viral RNA copies between cells. A
single-cell quantitative RT-PCR was validated to be feasible and
effective.
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Quantification
of circulating endothelial and progenitor cells: comparison of
quantitative PCR and four-channel flow cytometry.
Steurer M, Kern J, Zitt M, Amberger A, Bauer M, Gastl G, Untergasser G,
Gunsilius E.
Tumor Biology and Angiogenesis Laboratory, Division of Hematology and
Oncology,
Innrain 66, Innsbruck Medical University, 6020 Innsbruck, Austria.
BMC Res Notes. 2008 28;1: 71
BACKGROUND:
Circulating endothelial cells
(CEC) and endothelial precursor cells (CEP) have been suggested as
markers for angiogenesis
in cancer. However, CEC/CEP represent a tiny and heterogeneous cell
population,
rendering a standardized monitoring in peripheral blood difficult.
Thus, we
investigated whether a PCR-based detection method of CEC/CEP might
overcome the
limitations of
rare-event flow cytometry.
FINDINGS: To test the sensitivity of both
assays endothelial colony forming cell clones (ECFC) and cord blood
derived
CD45- CD34+ progenitor cells were spiked into peripheral blood
mononuclear cells
(PBMNC) of healthy volunteers. Samples were analyzed for the expression
of CD45,
CD31, CD34, KDR or CD133 by 4-color flow cytometry and for the
expression of CD34,
CD133, KDR
and CD144 by qPCR. Applying flow cytometry, spiked
ECFC and progenitor
cells were detectable at frequencies >/= 0.01%, whereas by qPCR a
detection
limit of 0.001% was achievable. Furthermore, PBMNC from healthy
controls (n = 30),
patients with locally advanced rectal cancer (n = 20) and metastatic
non-small cell
lung cancer (NSCLC, n = 25) were analyzed. No increase of CEC/CEP was
detectable by
flow cytometry. Furthermore, only CD34 and KDR gene expression was
significantly elevated in patients with metastatic NSCLC. However, both
markers are
not specific for endothelial cells.
CONCLUSION: QPCR is more sensitive, but
less specific than 4-channel flow cytometry for the detection of
CEC/CEP
cell types. However, both methods failed to reliably detect an increase
of CEC/CEP
in tumor patients. Thus, more specific CEC/CEP markers are needed to
validate
and improve the detection of these rare cell types by PCR-based assays.
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Prognosis
of non-small cell lung cancer patients by detecting circulating cancer
cells
in the peripheral blood with multiple marker genes.
Sher YP, Shih JY, Yang PC, Roffler SR, Chu YW, Wu
CW, Yu CL, Peck K.
Institute of Biomedical Sciences, Academia Sinica,
Taipei, Taiwan, Republic of China.
Clin Cancer Res. 2005 Jan 1;11(1): 173-179
PURPOSE: Current
lung cancer staging and prognosis methods are based on imaging methods,
which may not be sensitive enough for early and accurate detection of
metastasis. This study aims to validate the use of a panel of markers
for circulating cancercell detection to
improve the accuracy of cancer staging, prognosis, and
as a rapid assessment of therapeutic response.
EXPERIMENTAL DESIGN: We analyzed the National Cancer
Institute-Cancer Genome Anatomy Project database to identify potential
marker genes for the detection of circulating cancer cells in
peripheral blood. Nested real-time quantitative PCR and a scoring
method using cancer cell load Lc were employed to correlate the amount
of circulating cancer cells with clinical outcomes in 54 non-small cell
lung cancer (NSCLC) patients. The Kaplan-Meier method was employed for
analysis of prognostic variables.
RESULTS: A panel of four marker genes was identified and experimentally
validated. With these marker genes, we achieved an overall positive
detection rate of 72% for circulating cancer cells in the peripheral
blood of NSCLC patients. Patients who had higher Lc values had worse
outcomes and shorter survival times. Patients with poor therapeutic
response were revealed by positive detection of circulating cancer
cells after therapy. The results correlated well with the patients'
survival time.
CONCLUSION: Circulating cancer cell detection by a panel of markers and
the Lc scoring method can supplement the current tumor, node,
metastasis staging method for improved prognosis and for rapid
assessment of therapeutic response. Together, they may facilitate the
design of better therapeutic strategies for the treatment of NSCLC
patients.
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Nanoliter
reactors improve multiple displacement amplification of genomes from
single cells.
Marcy Y, Ishoey T, Lasken RS, Stockwell TB, Walenz
BP, Halpern AL, Beeson KY, Goldberg SM, Quake SR.
Department of Bioengineering, Stanford University,
Stanford, California, USA.
PLoS Genet. 2007 Sep;3(9): 1702-1708
Since only a small
fraction of environmental bacteria are amenable to laboratory culture,
there is great interest in genomic sequencing directly from singlecells.
Sufficient DNA for sequencing can be obtained from one cell by theMultiple
Displacement Amplification (MDA) method, thereby eliminating the need todevelop
culture methods. Here we used a microfluidic device to isolate
individual Escherichia coli and amplify genomic DNA by
MDA in 60-nl reactions. Our resultsconfirm a report that
reduced MDA reaction volume lowers nonspecific synthesis that
can result from contaminant DNA templates and unfavourable interaction between
primers. The quality of the genome amplification was assessed by qPCR
and compared favourably to single-cell amplifications
performed in standard 50-microl volumes. Amplification
bias was greatly reduced in nanoliter volumes, thereby providing
a more even representation of all sequences. Single-cell amplicons from
both microliter and nanoliter volumes
provided high-quality sequence data by high-throughput
pyrosequencing, thereby demonstrating a straightforward route to sequencing
genomes from single cells.
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Fluidigm
Dynamic Arrays provide a platform for single-cell gene expression
analysis.
Fluidigm application note 2009 (1)
Historically,
single-cell gene expression experiments have been difficult and
expensive to perform. Now, however, single-cell gene expression results
from single-cell samples can be inexpensive and easily reproducible
using Fluidigm’s Dynamic Array™ integrated fluidic circuits and
BioMark™ system for genetic analysis. This method is ideally suited for
high-throughput cell-line studies to determine individual cell behavior
in a homozygous population. To demonstrate this capability, we chose
single human cells from eight-cell-stage embryos, collected and
analyzed for expression of 46 developmental genes.
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Quantification
of
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.
BACKGROUND:
Gene
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.
RESULTS: Low
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.
CONCLUSION: Noise
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.
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Intracellular
expression profiles measured by real-time PCR tomography in the Xenopus
laevis oocyte.
Sindelka R, Jonák J, Hands R, Bustin SA,
Kubista M.
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.
Real-time PCR
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.
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Intracellular
Gene Expression Profi les Revealed with Real-time PCR Tomography
The BioMark system enabled measuring diffenntiation on the single-cell
level with high accuracy and throughput.
Fluidigm application note 2009 (2)
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