Single-cell gene expression profiling using reverse transcription quantitative real-time PCR.
ABSTRACT Even in an apparently homogeneous population of cells there are considerable differences between individual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while the single-cell response might be binary and apparently irregular. The origin of this variability may be preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements of individual cells. Here, we describe a method to collect dispersed single cells either by glass capillaries or flow cytometry, followed by quantitative mRNA profiling using reverse transcription and real-time PCR. We present a single cell lysis protocol and optimized priming conditions for reverse transcription. The large cell-to-cell variability in single-cell gene expression measurements excludes it from standard data analysis. Correlation studies can be used to find common regulatory elements that are indistinguishable at the population level. Single-cell gene expression profiling has the potential to become common practice in many laboratories and a powerful research tool for deeper understanding of molecular mechanisms.
- SourceAvailable from: Jeremy ChienPLoS ONE 01/2012; 7(2). · 3.73 Impact Factor
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ABSTRACT: Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+) and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50). The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20) was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50). Within 14 days after ischemia (D3, D7, D14), additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3), transcriptionally active early reactive glia (mainly from D7) and permanent reactive glia (solely from D14). Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.PLoS ONE 01/2013; 8(8). · 3.73 Impact Factor
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ABSTRACT: We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs. The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)~0.76-0.80 between individual cells and R(2)~0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input. In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology.PLoS ONE 01/2012; 7(2):e30794. · 3.73 Impact Factor
Single-cell gene expression profiling using reverse transcription quantitative
Anders Ståhlberga,b,*, Martin Bengtssonb,c,1
aLundberg Laboratory for Cancer, Department of Pathology, Sahlgrenska Academy at University of Gothenburg, Gula Straket 8, 413 45 Gothenburg, Sweden
bTATAA Biocenter, Odinsgatan 28, 411 03 Gothenburg, Sweden
cOxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Churchill Hospital, Oxford OX3 7LJ, UK
a r t i c l ei n f o
Accepted 7 January 2010
Available online 11 January 2010
Single-cell gene expression
a b s t r a c t
Even in an apparently homogeneous population of cells there are considerable differences between indi-
vidual cells. A response to a stimulus of a cell population or tissue may be consistent and gradual while
the single-cell response might be binary and apparently irregular. The origin of this variability may be
preprogrammed or stochastic and a study of this phenomenon will require quantitative measurements
of individual cells. Here, we describe a method to collect dispersed single cells either by glass capillaries
or flow cytometry, followed by quantitative mRNA profiling using reverse transcription and real-time
PCR. We present a single cell lysis protocol and optimized priming conditions for reverse transcription.
The large cell-to-cell variability in single-cell gene expression measurements excludes it from standard
data analysis. Correlation studies can be used to find common regulatory elements that are indistinguish-
able at the population level. Single-cell gene expression profiling has the potential to become common
practice in many laboratories and a powerful research tool for deeper understanding of molecular
? 2010 Elsevier Inc. All rights reserved.
Cells have a remarkable ability to cooperate and jointly con-
struct complex structures such as tissues, organs and whole organ-
isms. These constructions are normally accurately tuned and
respond to stimuli with high precision. During development, cells
differentiate to specialized cell types, each with particular func-
tions in the environment they reside. In many aspects, individual
cells exhibit a high degree of variability and responses to identical
stimuli may be very different even in a seemingly homogeneous
Gene expression profiling is a pivotal research tool in molecular
biology. By default, measurements are made on large pools of cells
as this will increase reliability of the recordings. For tissues, differ-
ent cell types are mixed uncontrollably and the measured gene
expression profile has unknown contribution from different cell
types. In addition, cell population measurement will not reveal
how a particular transcript is distributed among the cells
(Fig. 1A–B). Bulk measurements easily miss potentially important
gene correlations (Fig. 1C–D) where single cell analysis would indi-
cate coupled transcriptional regulations, which might be con-
trolled by the same molecular mechanism (Fig. 1C–D) .
Observed heterogeneity may indicate the presence of specialized
cell types or originate in the random nature of the transcription
A typical single cell contains ?1 pg mRNA, which is equivalent
to a few hundred thousand molecules transcribed from about ten
thousand genes . The high sensitivity of reverse transcription
quantitative real-time PCR (RT-qPCR) makes it possible to detect
even a single molecule. RT-qPCR is also characterized by high
reproducibility and wide dynamic range [9–11]. These properties
make RT-qPCR suitable for single-cell gene expression profiling.
Even if single-cell gene expression profiling using RT-qPCR has
been successfully applied to several different applications [6,12–
14], it has still not become common practice for laboratories. In
this paper, we describe the workflow of single cell RT-qPCR includ-
ing: cell collection, cell lysis, RT, qPCR and data analysis.
2. Description of method
Single cell RT-qPCR constitutes several sequential steps, out-
lined in Fig. 2. Our intention with this paper is to present the most
common experimental approaches with suitable references and in
1046-2023/$ - see front matter ? 2010 Elsevier Inc. All rights reserved.
* Corresponding author. Address: Lundberg Laboratory for Cancer, Department of
Pathology, Sahlgrenska Academy at University of Gothenburg, Gula Straket 8, 413
45 Gothenburg, Sweden. Fax: +46 31828733.
E-mail address: email@example.com (A. Ståhlberg).
1Present address: Nuevolution A/S, Ronnegade 8, 2100 Copenhagen, Denmark.
Methods 50 (2010) 282–288
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ymeth
detail describe the most appropriate experimental setup for analy-
sis of cells in suspension. For successful single-cell gene expression
profiling good laboratory practice is essential. All RNA work re-
quires completely RNase free conditions. Furthermore, PCR con-
tamination must be avoided, since even negative (zero values)
are used in data analysis. Physical separation, i.e. different rooms,
of pre-PCR, PCR and post-PCR is recommended.
3. Single cell collection and lysis
A majority of single-cell studies published deploy one of three
methods to collect cells: Flow cytometry, glass capillaries, and la-
ser capture. This review will describe the two former while laser
capture and laser microdissection are described in detail elsewhere
[15–17]. For some applications, the origin and surroundings of a
cell is vital information and this excludes flow cytometry as collec-
tion method, leaving laser capture as best option. Where high
numbers of cells are needed, and where collections of intact cells
are of importance, flow cytometry is recommended. Glass capillar-
ies, as we use them in this article, will also collect intact cells.
Single cell suspensions are prepared from tissue using mechan-
ical separation, enzymatic treatment, and/or non-physiological
buffers. The yield of functionally viable, dissociated cells will be
dependent on several parameters such as concentrations, incuba-
tion times and temperatures. An informative website for different
cell dissociation approaches is: www.tissuedissociation.com. As
the expression of some genes may be altered by the cell treatment
it is recommended that the expression of the genes of interest is
quantified also in an untreated sample. For example, one part of
the biological sample is saved for total RNA isolation and the
remaining is used for cell dissociation and collection.
3.1. Single cell sorting using flow cytometry
Several flow cytometry instruments, such as FACSDiva and
FACSVantage (both BD Biosciences, San Jose, CA, USA) can be used
to sort out individual cells . PCR plates (96-well) with lysis buf-
fer should be prepared in advance. In addition to standard flow
cytometry calibration, the instrument needs to be carefully cali-
brated to deposit single cells in the center of each collection tube.
This can easily be tested by sorting ?50 beads/cells on the plastic
Fig. 1. Cell heterogeneity and correlated transcript levels. Single cell measurements can distinguish the cases in (A) and (C) from (B) and (D), respectively, while cell
population measurements cannot.
Fig. 2. Overview of single-cell gene expression profiling using RT-qPCR.
A. Ståhlberg, M. Bengtsson/Methods 50 (2010) 282–288
film covering the 96-well PCR plate. It can also be worthwhile to
check the calibration every second plate, because the sorting arm
may be displaced over time. A small volume of lysis buffer will in-
crease the risk of a cell sticking to the wall of the tube, while too
large volume will likely interfere with downstream reactions. We
have found that ?5 ll lysis buffer is a suitable volume to work with
using flow cytometry.
For practical reason, a significant number of cells are needed for
calibration, and thousands of cells are wasted by the flow cytome-
try instrument. For most cell types it is recommended to keep the
cells on ice before sorting. If specific antibodies are used to sort out
subpopulations, cells need to be kept in medium that do not inter-
fere with the fluorophores used (PBS with ?2.5% serum is usually
compatible with both the fluorescence-activated cell sorting proce-
dure and keeping the cells viable). If no fluorophores are used, cells
should be kept on cell type-specific medium to avoid altered gene
expression. Viable cells can be enriched by flow cytometry by add-
ing 7-aminoactinomycin D (Sigma–Aldrich) before sorting. As a po-
sitive control 1–2 wells may be used to collect ?30 cells. This
number of cells will level out most single cell variability, and if
these controls result in negative expression data, reliable single cell
data for that particular gene will most likely be hard to obtain.
After finished sorting, each 96-well PCR plate should be sealed
and put on dry ice and stored at ?80 ?C until RT. As a control for
contamination, some liquid without cells can be collected from
the flow cytometry instrument to eliminate the chance that
mRNAs from lysed cells have contaminated the collected cells.
3.2. Single cell collection using glass capillaries
This collection method requires a phase contrast microscope,
preferably inverted, mounted with micromanipulators [6,19].
Shortly prior cell collection, the dissociated cells should be re-pla-
ted on Petri dishes, allowing cell adhesion. Usually, several dishes
can be prepared and maintained in optimal conditions until single
cell collection. Depending on the use of fluorophores, dissociated
cells should be kept on cell specific medium or medium which
do not interfere with the fluorophores used. Cell collection is sim-
plified if the cells have attached slightly to the dish and are phys-
ically separated from each other. Cells may be washed before
collection to remove dead cells and loose debris that could contam-
inate the pipette.
Borosilicate glass capillaries (Hilgenberg, GmbH) with outer
diameter of 1.5–1.6 mm and wall thickness of 0.16 mm can be
pulled to pipettes using a patch-clamp pipette puller (Heka PIP5).
The diameter of the tip should be ?10 lm, but the width is ad-
justed to cell size. This is substantially wider than standard
patch-clamp pipettes and large enough to allow passage of an in-
tact cell ensuring collection of all mRNA. An alternative method
is to penetrate the cell membrane with a patch-clamp pipette
(diam. ?0.5 lm) and only collect the cytoplasm [20,21]. This has
the advantage of allowing mRNA harvest from intact tissue, and
in conjunction with patch-clamp recording. However, it is techni-
cally very challenging which limits the number of samples that
can be collected within reasonable time limits. In addition, normal-
ization of expression levels poses a particular difficulty when an
unknown fraction of the cell’s mRNA is harvested.
The glass pipette is mounted on a hydraulic micromanipulator
bya flexibleplastictubing to the mouthof the researcher.If the pip-
pipette and collect individual cells with minimum volume of extra-
cellular solution (?0.1 ll). Pipettes should be emptied in standard
PCR tubes containing 2 ll lysis solution. This small volume allows
high concentration of detergents needed for cell lysis and RNase
inhibition. To simplify the process of emptying the glass capillary,
tubefacilitates completeemptying of the capillary.We havenot de-
tected any inhibition of RT or difference in transcript number for
cells with or without small traces of borosilicate glass (data not
shown). PCR tubes containing single cells should be stored on ice
during collection and kept in ?80 ?C until RT. As a control for con-
tamination and inhibition of downstream reactions, ?1 ll of the
lyzed together with the single-cell samples.
3.3. Cell lysis
The amount of protein, salts and debris in the single-cell sam-
ples is very small, allowing us to omit mRNA purification. We have
Fig. 3. Glass capillary emptying. The tip of the glass capillary contains a single cell that is emptied into the PCR tube containing 2 ll lysis buffer. To speed up and simplify the
procedure, our local workshop constructed a stand for the tube and a moveable capillary holder allowing precise emptying.
A. Ståhlberg, M. Bengtsson/Methods 50 (2010) 282–288
pooled up to ten cells, each collected individually, and not ob-
served any inhibitor effects on reverse transcription . However,
for efficient and complete RT we need to ascertain that the cell is
lysed and that all available mRNA is accessible to the enzyme. Cell
lysis can be performed with heat, mechanic forces, osmotic pres-
sure, enzymatic treatment and detergents. In addition to efficient
cell lysis, RNase activity needs to be blocked. RNase activity can
be inhibited by RNase inhibitors, such as RNaseOUT (20 U, Invitro-
gen), and chaotropic salts. For mammalian cells, especially mouse
b-cells, we have found that the use of guanidine thiocyanate is effi-
cient to lyse cells. In addition, low concentrations (?50 mM) of
guanidine thiocyanate is compatible with RT, for some genes it
may even enhance the RT efficiency and act as an RNase inhibitor
. The maximum concentration of guanidine thiocyanate that
may be used in the lysis buffer depends on the dilution factor be-
tween lysis and reverse transcription. For mouse b-cells, 2 ll lysis
buffer containing 250 mM guanidine thiocyanate is sufficient for
efficient lysis and also compatible with 10 ll RT reactions. RNA/
DNA carrier may be added to the lysis buffer, but we have not
found any positive effects from the use of carrier in our experimen-
tal setup. We have also evaluated heat incubation during lysis
(80 ?C, 5 min) but it does not significantly affect mRNA recovery
(data not shown). Commercial single cell lysis buffers, such as
CellsDirect (Invitrogen) and CelluLyser (TATAA Biocenter), may
also be used without additional extraction or purification.
3.4. RNA spike
As a control for the entire workflow from single cell collection
to data analysis, an RNA spike may be added in the lysis buffer.
Fig. 4. RT priming. Identical amounts of purified total RNA was used as starting material and four genes were measured (Ins1, Ins2, Rps29 and Hprt). (A) Determination of
optimal RT primer concentration using oligo(dT), random hexamers and gene specific primer. The gene specific primer was identical to reverse qPCR primer. Agarose gel
electrophoresis analysis for Ins2 revealed that increasing concentration of gene specific primer resulted in formation of erroneous PCR products. Melting curve analysis could
not distinguish between correct and erroneous PCR products. Unspecific PCR products were observed for Ins2, Hprt and Rps29 using gene-specific primers. (B) Relative RT
reaction yields are shown for various primer combinations. 2.0 lM oligo(dT), 2.0 lM random hexamers and 0.25 lM GSP was used for all RT primer combinations. Agarose gel
inspection showed formation of erroneous PCR products for Hprt and Ins2 using gene specific primer. The Ins1 assay was specific for all conditions while Rps29 occasionally
formed erroneous PCR products using gene specific primer. Any combination of priming methods was invariably superior or equal to the single best priming method used.
Relative cDNA yield was arbitrarily set to a value of one for the gene with lowest expression in the group of genes with the lowest RT primer concentration tested in (A) and in
left group (Olig) in (B) . Values are means ± SEM for 3 separate experiments. Abbreviations used: Olig, oligo(dT); Rand, random hexamer; GSP, gene specific primer.
A. Ståhlberg, M. Bengtsson/Methods 50 (2010) 282–288
However, the use of RNA spike will not reveal any information
about cell quality, lysis efficiency, mRNA accessibility and mRNA
quality. Setting up single-cell gene expression profiling for the first
time or changing the experimental setup, RNA spikes may be use-
ful to include as a control for RNA stability, RT and qPCR. From our
experience, data from the RNA spike always results in almost iden-
tical Cq-values if all single cells are run in parallel. RNA spikes can
either be commercial or generated by in vitro transcription .
The sequence of the RNA spike should be unique compared to
the transcriptome of the single cells analyzed. Alternatively, the
RNA spike should be used in high excess compared to the endoge-
3.5. Reverse transcription
High RT efficiency is needed to ensure that low transcript num-
bers can be detected and quantified by qPCR. There are several dif-
ferent reverse transcriptases and priming methods available. We
have previously shown that the variability in RT efficiency is highly
gene dependent [22,23]. Therefore, we prefer to use a blend of oli-
go(dT) and random hexamers to prime the reverse transcription
reaction (Fig. 4). Gene-specific primers can bind unspecifically to
the mRNA  leading to cDNA similar to that of random hexamer
priming, but with the gene specific primer in the 30end. If the gene
specific primer is identical to the reverse PCR primer, the specific-
ity will be reduced in the PCR and the number of products unre-
lated to the intended target will increase. Use of gene specific
primer identical to the reverse primer should therefore be carefully
optimized before use or avoided completely. It may also be worth-
while to test different reverse transcriptases, as the efficiency var-
ies greatly and gene-dependently .
For many genes we have seen that the use of SuperScript III
(Invitrogen) gives sufficient cDNA yield. We frequently use the fol-
lowing reverse transcription protocol: A mix of a single cell in 2 ll
lysis buffer (containing 50 mM guanidine thiocyanate, Sigma–Al-
drich), 0.5 mM dNTP (Sigma–Aldrich), 2.0 lM oligo (dT15, Eurofins
MWG Operon), 2.0 lM random hexamers (Eurofins MWG Operon)
is incubated at 65 ?C for 5 min in a total volume 6.5 ll. dNTP, oli-
go(dT15) and random hexamers can be added directly to the frozen
cells without thawing the samples. Beware of batch differences of
oligo(dT) and random hexamers; check new batches before using
them. Then cool the samples on ice or cooling block to <25 ?C.
Add 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 5 mM
dithiothreitol, 20 U RNaseOUT and 50 U SuperScript III (all Invitro-
gen) to total volume of 10 ll. The final concentration of guanidine
thiocyanate in RT should be 650 mM. The following temperature
profiles may be used for RT: 25 ?C for 5 min, 50 ?C for 60 min,
55 ?C for 15 min and 70 ?C for 15 min. Only minor differences have
been observed using other incubation times and temperatures as
long the reverse transcriptases have not been inactivated by heat.
To increase the amount of mRNA, pre-amplification methods may
be applied [24,25].
The quantitative contribution from genomic DNA to the expres-
sion signal is generally insignificant, as it would only increase the
copy number by two for most genes. Nevertheless, PCR primers
should whenever possible be designed to cross introns. DNase
treatment is another option, but difficult to evaluate in single-cell
samples. For +/? measurements however, both these precautions
3.6. Quantitative real-time PCR
The number of genes that can be analyzed from a single cell is
limited by the number of transcripts of the studied genes. We have
shown that >?20 target molecules per PCR are needed for accurate
quantification . Furthermore, we have shown that highest
reproducibility for measurements of limited samples is achieved
when the dilution between RT and qPCR is kept at a minimum,
even at the expense of qPCR-replicates . For example, the accu-
racy in qPCR will be higher if all cDNA is quantified in a single qPCR
reaction instead of splitting the cDNA sample in two and analyze
the duplicate. However, loading too much of the RT reaction may
inhibit the qPCR, in part due to inhibitory effects of reverse trans-
criptase enzyme on PCR [19,21,26–28]. The inhibition depends on
the amount of reverse transcriptase and Taq polymerase used in
the two reactions. Using SuperScript III (Invitrogen) and JumpStart
(Sigma–Aldrich) the ratio should be <2 (U/U). This ratio may be dif-
ferent using other enzymes. Practically, about 5–10 genes can suc-
cessfully be analyzed from the same cell, a higher number would
require pre-amplification of cDNA.
Pre-amplification of single-cell cDNA allows virtually limitless
number of measured genes, and has been used on single cells in
concert with microarrays . In a pioneering work by Brady
and Iscove , it was shown that polyA-cDNA can be globally
amplified, followed by gene-specific PCR on the resulting pool.
Alternatively, a multiplex PCR on the single-cell cDNA, using low
concentrations of all gene-specific primers, results in a population
of PCR products that are quantified by qPCR. This way, up to 40
 and 100s (TaqMan?PreAmp Master Mix, p/n 4391128, Ap-
plied Biosystems) of transcripts have been analyzed in a single cell.
However, pre-amplification introduces one extra source of poten-
tial bias which needs to be verified. For more information on
pre-amplification methods, the reader is referred elsewhere
Normal qPCR guidelines are used to design working assays for
single-cell gene expression profiling [9,32]. Assays should be gene
specific, have high sensitivity, dynamic range and reproducibility
[9,32]. For most real-time PCR instruments, typical quantification
cycle (Cq) values are between 30 and 40 analyzing single cells.
Many single cells will and should not result in any detectable
PCR product (no Cq-value). Using melting curve analysis, non-spe-
cific PCR products may be identified. To eliminate detection and
quantification bias, assays should not give rise to any primer-di-
mers, at least not before Cq = 40. From our experience is the assay
specificity, i.e. lack of erroneous PCR products such as primer-di-
mers, one of the most important parameter when optimizing sin-
gle cell assays. If non-specific PCR fragments are formed in
addition to the correct PCR product, data should only be used qual-
itatively. Probe-based qPCR assays can be used but they will not re-
veal important information about interfering PCR products that are
formed. Another alternative is to use one-step RT-qPCR assays.
Running RT and qPCR in the same reaction tube eliminates one
handling step and consequently reduces the risk of contamination.
The main drawback with one-step RT-qPCR is that RT and qPCR
most often have different optimal reaction conditions, reducing
the sensitivity, efficiency and reproducibility of respective reac-
tions. All assays should be validated by gel electrophoresis, even
if probes are used.
The following qPCR protocol has been proven to work well:
10 ll reactions containing 10 mM Tris (pH 8.3), 50 mM KCl,
3 mM MgCl2, 0.3 mM dNTP, 1 U JumpStart Taq polymerase (all Sig-
ma–Aldrich), 0.5 ? SYBR Green I (Invitrogen), 200–400 nM of each
primer (Eurofins MWG Operon) and 1–4 ll cDNA (cDNA should be
diluted to avoid qPCR inhibition from the RT reaction). Premade
qPCR mixes can also be used. However, assay performance usually
differs somewhat between qPCR mixes and should not be changed
between runs for samples to be compared. Primers should be de-
signed according to standard procedure [9,11]. Primer3 (http://
one freely available primer design software that may be used.
For samples with low RNA quality short amplicon lengths are rec-
ommended [33,34]. The following temperature profile may be ap-
A. Ståhlberg, M. Bengtsson/Methods 50 (2010) 282–288