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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:
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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