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single-cell handling
The purpose of this single-cell handling page is to provide researchers and clinicians with workflows and resources for single-cell molecular analysis such as RT-PCR, quantitative PCR, and sequencing and with a focus on the front-to-backhandling of single cells. Interest
in single
cell molecular analysis has risen dramatically over the last couple of
years, chiefly because single cell molecular analysis is the only way
to research genetic heterogeneity, i.e., differences
in copy number or gene expression levels
between individual cells, or genetically analyze very rare cells
such as circulating tumor or fetal cells. Single cell molecular
analysis has provided new insight in diverse research areas such as
immunology, oncology, stem cells or forensics. However, an often
overlooked aspect of single cell analysis is the difficulties of
handling single cells for molecular analysis associate with all three
major methods for single cell manipulation:
The
time
tested standard method for handling single cells is micromanipulation.
Typical micromanipulation systems consist of an inverted microscope
plus a joy-stick operated, motorized micromanipulation platform.
Applications include bacterial analysis, eproductive medicine, and
forensics. While micromanipulation is a powerful and versatile method
for capturing single cells, one of its drawbacks is the long distance
to transfer cells from a growth or storage medium into a tube or
microtiter plate (MTP) for molecular analysis. This increases the
amount of time needed for cell transfer thus limiting the throughput of
the method. In addition, once the micromanipulator leaves the optical
plane of the microscope it is no longer possible to visually control
correct transfer of the single cell or bacterium into the a tube or
MTP. As
a
result, some cells fail to transfer correctly into the bottom of a tube
or MTP well. Downstream analysis often includes amplification of
genetic material, including qPCR, but since there is no
straight-forward optical quality control (QC) option in tubes or MTPs,
investigators are left to guess whether a failed PCR reaction was due
to PCR failure, failed cell deposition, or whether there is a valid
biological reason. These problems can be overcome using a
two-dimensional layout which can be placed in nearest proximity to the
tweezers of the micromanipulator as provided by the Advalytix AmpliGrid
from Olympus allowing a convenient workflow as well as easy
verification of the correct cell placement.
Although
advanced in terms of speed, ease of use, and versatility to
micromanipulation, LCM also has to cope with the problem that proper
cell placement into in tubes or MTPs is hard to control. For example,
with the PALM Microlaser system cells can directly be deposited on
AmpliGrid slides taking the guess work out of cell placement. In
addition, using the RoboMover an automated time-saving selection,
relocation and harvesting of cells provides a time-saving downstream
analysis with best possible results.
Robert F. Bonner, Michael Emmert-Buck, Kristina Cole, Thomas Pohida, Rodrigo Chuaqui, Seth Goldstein, Lance A. Liotta Science 1997: Vol. 278. no. 5342, pp. 1481 - 1483  Cell
sampling applications with MicroLaser Systems
 Flow
cytometry
Flow cytometry is the high speed champion of single cell methods, with advanced systems capable of sorting over 50 million cells per hour based on more than 8 immuno parameters. For subsequent molecular analysis, cells are typically sorted into MTPs. Placement success into MTPs is typically in the 50-70% range, which is mainly due to the electric charge of plastic MTPS causing cells to be deflected before reaching the well bottoms. Substantial improvement of single cells placement success can be provided by direct placement of stained cells onto AmpliGrid slides. In addition, correct cell placement can easily be checked with a fluorescent microscope after a sort – that way, when interpreting PCR results, investigators know for sure that cells were present in the reaction.
CellSorter - facs in a petri
How to get the one you want? Single cells are in your hands. sort live cells right from the petri dish => http://www.facsinapetri.com Using a special micropipette you can easily sort single cells from your culture. CellSorter software scans in the region of interest with a motorized stage, and detects automatically the fluorescent cells. In a few seconds you can see a map of the culture indicating also the detected cells. Software calculates the optimal path to visit all detected cells by the micropipette and pick them up. Detected cells are collected from the Petri dish by picking them up with a glass micropipette. You can also detect cells manually, and sort them manually or automatically afterwards. User can evaluate and amend automatic cell detection.
Interviews, Talks and Posters:
New
techniques for isolation of
single prokaryotic cells. REVIEW
Frohlich J, Konig H. FEMS Microbiol Rev. 2000 Dec;24(5):567-72. Institut fur Mikrobiologie und Weinforschung, Johannes Gutenberg-Universitat, Becherweg 15, 55128, Mainz, Germany Since
the 1960s, several new
attempts have been made to improve the management of single prokaryotic
cells using
micromanipulator techniques. In order to facilitate the isolation of
pure
cultures we have recently developed an improved micromanipulation
method for
routine work. With the aid of this method single prokaryotic cells can
be picked
out
of a mixed community under direct visual control. The isolated aerobic
or
anaerobic cells can be grown in pure culture or can be subjected to
single cell
PCR. Other powerful and completely new approaches are the applications
of
laser micromanipulation systems, such as optical tweezers or laser
microdissection techniques. Of the latter two methods only optical
tweezers have been
successfully applied to cloning prokaryotic cells.
Francis L. Battye,
Amanda Light and David
M. Tarlinton
The Walter & Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia, Journal of Immunological Methods, Volume 243 (1-2) 2000, Pages 25-32  Cell sorters now allow the selection of cells and other bodies according to a range of quite diverse criteria. The additional refinement that allows the sorting of individual cells based on these criteria has seen application in many fields of research. Single cells may be sorted for microscopy, for culture and for genetic analysis by way of single cell PCR. In practical terms, in the setting up of an instrument for single cell sorting, there are additional requirements to ensure that each detected event is indeed a single cell or body, that this cell can be reliably sorted via saline droplet, separate from its fellow travelers, that the aiming of the droplet deflection is sufficiently precise to find the target vessel and that the cell will be undamaged on arrival. Among the diverse reported applications of the technique, two fields which have benefited greatly are lymphocyte development and haemopoiesis. In the former case, the analysis of gene rearrangements in lymphocytes, both in the pre- and post-antigenic phases of development, has been enabled by the combined technologies of single cell sorting and PCR. It is argued that such experiments could not have been done without that partnership. In a similar way, the single cell sorting technique has been found to be the perfect way to demonstrate precursor/progeny relationships between haemopoietic cells and, further, to demonstrate rigorously the effects of particular cytokines on the haemopoietic system. Sampling
efficiency of a single-cell capillary electrophoresis system.
Robert B. Brown 1 2, Julie Audet 1 2 * 1Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario, Canada 2Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), University of Toronto, Toronto, Ontario, Canada Cytometry part A; Volume 71A Issue 10, Pages 882 - 888 Capillary electrophoresis (CE) combined with a laser-induced fluorescence (LIF) detection scheme is a powerful approach for single-cell analysis. For measurements requiring a high temporal resolution, CE-LIF is often combined with cell lysis systems based on pulsed lasers. Although extremely rapid, laser lysis has raised some concerns about the efficiency at which the cell contents are sampled. We have assembled a single-cell CE-LIF mounted on the stage of a microscope. This system was coupled with a nanosecond pulsed laser for cell lysis. We have analyzed green fluorescent protein (GFP) expressed in single mammalian cells and developed a novel approach to estimate the cell sampling efficiency (SE) based on the use of fluorescent calibration microspheres and flow cytometry. A significant advantage of this method is that it does not require any knowledge or assumption regarding the cell volume. We have evaluated the SE for different laser pulse energies (from 2 to 9 J) and two different pulse focal positions in the xy plane (0-10 m from the center of the cell). We found the maximum SE at the lowest energy (2 J), with the pulse focused directly on the cell. We have demonstrated the utility of a novel method to measure the SE of a single-cell CE system. The measurements presented in this study indicate that rapid cell lysis with nanosecond lasers requires careful optimization of pulse parameters for maximum sampling of the cell contents. Dynamic
single cell culture array.
Dino Di Carlo, Liz Y. Wu and Luke P. Lee* Lab Chip, 2006, 6, 1445–1449, 1445 It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved highthroughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell–cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidicbased dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning y1 mm2 with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology. Quantitative
RT-PCR from one single cell using the AmpliGrid technology.
Dr. Diana Hops, Application Specialist and Petra Hartmann Pre-amplifying single cells with the AmpliGrid system followed by standard qPCR combines existing workflows with superior single cell sensitivity. This application note shows how AmpliGrid reverse transcription followed by an established Stratagene qPCR protocol allows to do gene expression analysis on single cells. Low
volume amplification and sequencing of mitochondrial DNA on a
chemically structured chip.
Sabine Lutz-Bonengel
& Timo Sänger & Marielle Heinrich & Ulrike Schön
& Ulrike Schmidt
Int J Legal Med (2007) 121:68–73 Abstract Low volume (LV) amplification (1 μL) of nuclear DNA (nucDNA) on a chemically structured chip is an appropriate and highly sensitive method to simultaneously amplify amelogenin and 15 forensically relevant short tandem repeats (STR). In this study, a combined method using on-chip LV amplification of mitochondrial DNA (mtDNA) and subsequent on-chip LV cycle sequencing was established to obtain a method, which is sensitive and robust enough to allow reliable analysis of DNA amounts representing the single cell level. All the necessary steps of the procedure except for the purification of the sequencing products - were accomplished within the same final 2-μL reaction volume. Low-volume
amplification on chemically structured chips using the PowerPlex16 DNA
amplification kit.
Schmidt U, Lutz-Bonengel S, Weisser HJ, Sänger T, Pollak S, Schön U, Zacher T, Mann W. Int J Legal Med. 2006 Jan;120(1):42-8. Epub 2005 Oct 18. Institute of Legal Medicine, Albert Ludwig University Freiburg, Albertstrasse 9, 79104, Freiburg, Germany. In forensic DNA analysis, improvement of DNA typing technologies has always been an issue. It has been shown that DNA amplification in low volumes is a suitable way to enhance the sensitivity and efficiency of amplification. In this study, DNA amplification was performed on a flat, chemically structured glass slide in 1-microl reaction volumes from cell line DNA contents between 1,000 and 4 pg. On-chip DNA amplification reproducibly yielded full allelic profiles from as little as 32 pg of template DNA. Applicability on the simultaneous amplification of 15 short tandem repeats and of a segment of the Amelogenin gene, which are routinely used in forensic DNA analysis, is shown. The results are compared to conventional in-tube amplification carried out in 25-microl reaction volumes. Real-time quantitative RT-PCR after laser-assisted cell picking. Fink L, Seeger W, Ermert L, Hanze J, Stahl U, Grimminger F, Kummer W, Bohle RM. Department of Pathology, Justus-Liebig-Universitat Giessen, Germany. Nat Med. 1998 4(11): 1329-1333. Effects of tissue
manipulation on RNA quality and gene expression.
Kerman IA, Buck BJ, Evans SJ, Akil H, Watson SJ. J Neurosci Methods. 2006 May 15;153(1):71-85. Epub 2005 Dec 6. Molecular and Behavioral Neuroscience Institute, Department of Psychiatry, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109, USA.  Laser
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.
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.
POSTER
qPCR
2005 Event: Distribution of mRNA
transcripts in single cells determined by quantitative RT-PCR.
Martin
Bengtsson1, Anders
Ståhlberg2, Patrik Rorsman(1, 3), Mikael Kubista2
A cell
contains approximately 20 pg of RNA, of which <5% is mRNA. That
corresponds to a
few hundred thousand transcripts, representing some 10,000 genes
expressed at one timepoint. The constitution of this expression
palette, or transcriptome, determines the fate of the cell and is a
record of its recent history. Gene expression is ultimately controlled
at the single cell level, but still, most
gene expression analysis studies of today are carried out using
thousands or millions of cells, for practical reasons. The measurements
become a representation of the average cell, and individual differences
in transcript levels remain undisclosed. Differences in a small
proportion of the cell population are not likely to be revealed when
looking at whole cultures or tissues.1: Department of Experimental Medical Science, Lund University, Lund, Sweden. 2: Department of Chemistry and Biosciences - Molecular Biotechnology, Chalmers University of Technology and TATAA Biocenter, Göteborg, Sweden. 3: The Oxford Centre for Diabetes, Endocrinology and Metabolism, The Churchill Hospital, Oxford, England. When a small number of molecules determine the fate of a chemical equilibrium, a certain randomness and stochasticity is observed. As the number of molecules increase as do the predictability of the reaction. The number of enhancer and transcription activator molecules in a cell is low, and a stochastic element is thus seen in gene expression analysis at the single cell level. It has been suggested that some genes are expressed in a binary, on or off, behavior, resulting in a binomial population distribution of the transcript levels. We have studied the gene expression of single cells in the pancreatic islets of Langerhans in mice using quantitative RT-PCR. The pancreatic islets are heterogeneous clusters of cells releasing major metabolic hormones, such as insulin. Precise quantification at this level has never before been carried out in tissues and data reveal intricate correlation between related genes while simultaneously showing a large spread between cells of the same type. Furthermore, we see a lognormal distribution of transcript levels in the single cell. Full
paper: Gene expression
profiling in
single cells from the pancreatic islets of Langerhans
reveals lognormal distribution of mRNA levels. Bengtsson M, Stahlberg A, Rorsman P, Kubista M. Genome Res. 2005 Oct;15(10):1388-92.
Improved
quantitative
real-time RT-PCR for expression
profiling of individual cells.
Liss B. Nucleic Acids Res 2002 Sep 1;30(17):e89 University
Laboratory of Physiology and MRC Anatomical Neuropharmacology Unit,
New
quick
method for isolating RNA from laser captured cells stained
byimmunofluorescent immunohistochemistry;
RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR. Jack M. Gallup, Kenji Kawashima, Ginger Lucero and Mark R. Ackermann Biol. Proced. Online 2005; 7(1): 70-92. We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IFIHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, timeconsuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead. Small-Sample
Total RNA Purification: Laser Capture Microdissection and Cultured Cell
Applications.
Karen E. Dolter and Jeffrey C. Braman Stratagene, La Jolla, CA, USA BioTechniques 30:1358-1361 (June 2001) Gene expression studies require analysis of RNA, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. The Absolutely RNA‘ microprep kit provides a convenient method for isolating total RNA from small numbers of cells such as those harvested by laser capture microdissection (LCM). The protocol includes binding of RNA to a solid support, thus eliminating the need for organic extraction and alcohol precipitation. DNase digestion on the solid support reduces or eliminates DNA contamination and minimizes RNA handling. Efficient washing removes contaminants, and elution in a small volume of buffer results in high-purity RNA at a concentration appropriate for demanding applications such as RT-PCR. RNA isolated from as few as 200 laser capture microdissected brain tumor cells resulted in detection of low, medium, and highly expressed genes by conventional and real-time RT-PCR. 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.
Analysis
of connective tissues by laser capture
microdissection
and reverse transcriptase-polymerase chain reaction. Robin Jacquet, Jennifer Hillyer, William J. Landis* Department of Biochemistry and Molecular Pathology Northeastern Ohio Universities College of Medicine, Rootstown, OH, USA Studies of gene
expression from bone, cartilage, and other tissues are complicated by
the fact that their RNA, collected and pooled for analysis, often
represents a wide variety of composite cells distinct in individual
phenotype, age, and state of maturation. Laser capture microdissection
(LCM) is a technique that allows specific cells to be isolated according
to their phenotype, condition, or other marker from within such
heterogeneity. As a result, this approach can yield RNA that is
particular to a subset of cells comprising the total cell population of
the tissue. This study reports the application of LCM to the gene
expression analysis of the cartilaginous epiphyseal growth plate of
normal newborn mice. The methodology utilized for this purpose has been
coupled with real-time quantitative reverse transcriptase-polymerase
chain reaction (QRT-PCR) to quantitate the expression of certain genes
involved in growth plate development and calcification. In this paper,
the approaches used for isolating and purifying RNA from phenotypically
specific chondrocyte populations of the murine growth plate are detailed
and illustrate and compare both qualitative and quantitative RT-PCR
results. The technique will hopefully serve as a guide for the further
analysis of this and other connective tissues by LCM and RT-PCR.
Laser-Capture
Microdissection: Refining Estimates
of the Quantity and Distribution
of Latent Herpes Simplex Virus 1
and Varicella-Zoster Virus DNA in Human Trigeminal Ganglia at the Single-Cell Level. Kening Wang,* Tsz Y. Lau, Melissa Morales, Erik K. Mont,† and Stephen E. Straus Medical Virology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland There
remains
uncertainty and some controversy about the percentages and types of
cells in human sensory nerve ganglia that harbor latent herpes simplex
virus 1 (HSV-1) and varicella-zoster virus (VZV) DNA. We developed and
validated laser-capture microdissection and real-time PCR (LCM/PCR)
assays for the presence and copy numbers of HSV-1 gG and VZV gene 62
sequences in single cells recovered from sections of human trigeminal
ganglia (TG) obtained at autopsy. Among 970 individual sensory neurons
from five subjects, 2.0 to 10.5% were positive for HSV-1 DNA, with a
median of 11.3 copies/positive cell, compared with 0.2 to 1.5% of
neurons found to be positive by in situ hybridization (ISH) for HSV-1
latency-associated transcripts (LAT), the classical surrogate marker
for HSV latency. This indicates a more pervasive latent HSV-1 infection
of human TG neurons than originally thought. Combined ISH/LCM/PCR
assays revealed that the majority of the latently infected neurons do
not accumulate LAT to detectable levels. We detected VZV DNA in 1.0 to
6.9% of individual neurons from 10 subjects. Of the total 1,722 neurons
tested, 4.1% were VZV DNA positive, with a median of 6.9 viral
genomes/positive cell. After removal by LCM of all visible neurons on a
slide, all surrounding nonneuronal cells were harvested and assayed: 21
copies of HSV-1 DNA were detected in 5,200 nonneuronal cells, while
nine VZV genomes were detected in 14,200 nonneuronal cells. These data
indicate that both HSV-1 and VZV DNAs persist in human TG primarily, if
not exclusively, in a moderate percentage of neuronal cells.
Further
single-call qPCR papers on => http://singelcell.gene-quantification.info
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