Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR
1 Division of Biological Sciences, Graduate School of Science, Hokkaido
University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan
2 Division of Innovative Research, Creative Research Initiative `Sousei'
(CRIS), Hokkaido University, North 21, West 10, Kita-ku, Sapporo 001-0021,
Japan
3 Laboratory of Functional Biology, Faculty of Pharmaceutical Sciences at
Kagawa Campus, Tokushima Bunri University, 1314-1 Shido, Sanuki 769-2193,
Japan
4 Field Science Center for Northern Biosphere, Hokkaido University, North 9,
West 8, Kita-ku, Sapporo 060-0809, Japan
5 Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N
4N1, Canada
* Author for correspondence (e-mail: eito{at}sci.hokudai.ac.jp)
Accepted 30 March 2005
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Summary |
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Key words: CREB, learning, Lymnaea stagnalis, manipulation, memory
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Introduction |
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One example where the determination of mRNA copy numbers would be
advantageous is given in single, identifiable neurons that play key and/or
necessary roles in transcription and translation mechanisms for new protein
synthesis in memory consolidation process, particularly in simpler model
central nervous systems (CNSs) of invertebrates
(Kandel and Pittenger, 1999;
Kandel, 2001
). It therefore
seems logical that we should try to determine the exact copy numbers of mRNAs
of transcription factors thought to play necessary roles in the molecular
cascade that leads to the formation of memory
(Yin and Tully, 1996
;
Abel and Lattal, 2001
;
Carew and Sutton, 2001
;
Pittenger and Kandel, 2003
;
Sangha et al.,
2003a
,c
).
A number of groups, including our own, have used the pond snail Lymnaea
stagnalis to analyze the behavioral, cellular and molecular mechanisms of
associative learning and subsequent memory formation
(Ito et al., 1999;
Benjamin et al., 2000
;
Lukowiak et al., 2003
;
Wagatsuma et al., 2004
;
Sakakibara et al., 2005
). The
results obtained from many experiments show that two identifiable neurons play
key and/or necessary roles in the memory consolidation process following
different forms of associative learning: (1) the cerebral giant cell for
aversive and appetitive feeding conditioning (Kojima et al.,
1997
,
2001
;
Sadamoto et al., 2000
;
Hatakeyama et al., 2004a
;
Korneev et al., 2005
) and (2)
the right pedal dorsal 1 cell for respiratory operant conditioning
(Sangha et al., 2003b
).
In the cerebral giant cell, a transcription factor, cyclic AMP (cAMP)
responsive-element binding protein (CREB), was shown to be present at both the
mRNA and protein levels (Ribeiro et al.,
2003; Sadamoto et al.,
2004a
,b
).
CREB has been proposed to act as a molecular switch for memory consolidation
(Bailey et al., 1996
;
Silva et al., 1998
;
Tully, 1998
;
Carew and Sutton, 2001
). We
hypothesized that in the paired cerebral giant cells the phosphorylation of
CREB following associative training of feeding behavior initiates a cascade of
altered gene activity and new protein synthesis that is necessary for memory
consolidation (Nakamura et al.,
1999
; Ribeiro et al.,
2003
; Sadamoto et al.,
2004a
,b
).
However, there are no accurate, rapid and appropriate methods to determine the
exact copy numbers of CREB mRNAs in a single neuron.
To develop a protocol for isolating a single cell and determining the mRNA copy numbers within it, we had to overcome two major technical difficulties. The first of these is the accurate and rapid preparation of mRNAs from a single cell, and particularly the preparation of those that turn over rapidly. The second obstacle was the determination of the exact copy number of each particular mRNA in this isolated cell.
To reliably prepare mRNAs rapidly from a single cell, there appeared to be
two possibilities: (1) aspiration of the intracellular contents with a patch
pipette (Monyer and Lambolez,
1995; Surmeier et al.,
1996
; Baro et al.,
1997
; Liss et al.,
2001
; Tsuzuki et al.,
2001
; Liss, 2002
;
Song, 2002
); and (2) isolation
of the cell's soma by laser microdissection
(Schütze et al., 1997
;
Fink et al., 1998
;
Mawrin et al., 2003
). As with
any model system or experimental procedure, there are various `pluses and
minuses' that must be taken into account before proceeding. The aspiration
method has some serious limitations. For example, it is not certain whether
all the intracellular contents, e.g. mRNAs, can be collected completely into
the patch pipette by negative pressure. On the other hand, the laser
microdissection method cannot exclude the problem of contamination of other
small cells beneath the target one. Furthermore, in some instances, the
isolated cell required fixation, dehydration and embedding, and these steps
may have disadvantageous consequences for the preparation of mRNAs.
The other obstacle was the difficulty in determining the exact mRNA copy
number, which is expected to be small, in a single cell. Whereas traditional
methods, such as northern blot analysis and competitive polymerase chain
reaction (PCR), have yielded reproducible results
(Horikoshi and Sakakibara,
2000; Silbert et al.,
2003
), this methodology requires a large amount of mRNA and, thus,
cannot be easily used to determine the small amount of mRNA in a single cell.
By contrast, the recent development of the quantitative real-time reverse
transcription-PCR (qRT-PCR) method allows not only the quantification of a
small amount of mRNA in cells but also the rapid analysis of multiple gene
targets (Bustin, 2002
;
Bhandari et al., 2003
;
Hatakeyama et al., 2004c
;
Stram et al., 2004
). Using
qRT-PCR, research teams have begun to focus on single-cell quantitative
analyses of mRNAs in identifiable cells
(Spijker et al., 1999
;
Schacher et al., 1999
;
Schacher et al., 2000
;
Steuerwald et al., 2000
;
Tkatch et al., 2000
;
Eberwine, 2001
;
Becker et al., 2002
;
Storm et al., 2002
;
Parhar et al., 2003
).
In the present study, we therefore used the qRT-PCR method on a single cerebral giant cell isolated with newly developed micromanipulators to determine the exact copy numbers of mRNAs of the activator and repressor types of CREB. We show here in a single cerebral giant cell isolated from a naïve snail that we are able to determine reliably the exact copy number of mRNA of the repressor type of CREB (CREB2) but not the activator one (CREB1). Because the protocol is not only relatively simple, but highly specific and reliable, we will be able in future experiments to determine if the amounts of other mRNAs change in a single cell as a result of development, immunity, learning and memory.
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Materials and methods |
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Isolation of a single, identifiable neuron
The CNS was removed from the anesthetized snail, and carefully desheathed
in cold Lymnaea saline. Then the CNS was exposed to 2 mg
ml1 trypsin (Sigma-Aldrich, St Louis, MO, USA) in
Lymnaea saline at room temperature for 10 min. This CNS dissection
method did not interfere with mRNA stability, as will be described later.
After washing off the enzyme solution (at least three times) with cold
Lymnaea saline, the cerebral giant cells were isolated in a cold cell
culture medium [50% modified Leibovit's L-15 medium (special order;
Invitrogen, Carlsbad, CA, USA) and 50% Lymnaea saline] containing 30
mmol l1 D-glucose.
Single cerebral giant cells were bored with glass microelectrodes constructed with a tip diameter of about 10 µm that was rounded by fire-melting and capped. The glass microelectrode was advanced into the cerebral giant cell with state-of-the-art micromanipulators (Narishige Scientific Instrument Laboratory, Tokyo, Japan) under a stereoscopic microscope, without damaging the soma because of the capped dull tip. In addition, a small length of primary neurite was also isolated with the somata (Fig. 1).
|
Lysis of isolated neurons
The soma with its primary neurite was first gently removed from the
ganglion, and then it was placed into a lysis solution with an RNase inhibitor
and rapidly frozen, as described below. The following procedures were
performed to certify that the loss of mRNA was minimal.
The isolated cerebral giant cell in 0.5 µl of cell culture medium was
transferred to a tube containing 4.5 µl of lysis solution [0.75 µl of 50
µmol l1 oligo d(T)16 primer (PE Applied
Biosystems, Foster City, CA, USA), 0.2 µl of 1 mg ml1
yeast tRNA (Roche, Basel, Switzerland), 0.05 µl of 30 U
µl1 Prime RNase inhibitor (Eppendorf, Hamburg, Germany),
and 6x104 copies of salmon gonadotropin hormone (GTH)
2 RNA with 0.2 µl of 10% NP40 (Calbiochem, Darmstadt, Germany), all
of which were stocked in DEPC water]. The salmon GTH
2 RNA was used as
an internal control to check the technical variation between determinations.
Although housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase,
are generally used as internal controls for qRT-PCR assays
(Aerts et al., 2004
), no such
suitable gene has been found in Lymnaea. Therefore, we prepared
salmon GTH
2 RNA by in vitro synthesis. The samples were
rapidly frozen in liquid nitrogen and then stored at 80°C.
Preparation of primers, probes and standard curves for Lymnaea CREB1 and CREB2 mRNAs and salmon GTH 2 RNA
The nucleotide sequences of Lymnaea CREB1 and CREB2 mRNAs
(accession numbers: AB041522 and AB083656, respectively; for details, see
Sadamoto et al., 2004b) were
used to design the primers and probes for qRT-PCR. We used the Primer Express
software (version 1.0; PE Applied Biosystems) to design appropriate primers
and fluorogenic probes for qRT-PCR assays.
For Lymnaea CREB1 mRNA, the primers were 5'-GTT GGT GAC GAA AAG TAC GTA ATT G-3' and 5'-CTC ACA TGG ACC ACT GAA ATG C-3', and the probe was 5'-FAM-TTT TCA ATG TCA GCT GTT CCA GGA CCA T-TAMRA-3'. For Lymnaea CREB2 mRNA, the primers were 5'-CCT AGC TAC GGC TGC TAT ATC TAC AAA-3' and 5'-GTC AAC AAG TCC AGG TCC CAT T-3', and the probe was 5'-FAM-CTG CCA AGC AGC AAA TCT TCG TTC CA-TAMRA-3'. The melting temperatures are described later.
For the control experiments, as mentioned above, we also determined the
copy number of salmon GTH 2 RNA. The specific primers were 5'-AAT
CTT CCC CAA CAT CAT ACA GTG-3' and 5'-TCA CCG GGA AGC CAT
CCT-3', and the probe was 5'-FAM-TTG CAA CGC AGC ATG TGG CTT
CAG-TAMRA-3'.
The standard RNAs were synthesized from the target cDNA templates by an
in vitro RNA synthesis kit (MAXIscript; Ambion, Austin, TX, USA), and
the copy numbers of RNA products were calculated on the basis of their
absorbance values. The RNA products were serially diluted to prepare standard
RNA solutions, and were subjected to reverse transcription. The volume of
reaction mixture (15 µl) and the components were the same as those for test
samples that consisted of the lysis and reverse transcription solutions.
Standard RNA solutions that were reverse-transcribed were referred to as
standard cDNA solutions. In the qRT-PCR assay, several doses of standard cDNAs
(1x10 to 1x105 copies for CREB1 cDNA, 1x10,
2x10, 5x10 and 1x102 to 1x105
copies for CREB2 cDNA, and 2.4x10 to 2.4x104 copies for
GTH 2 cDNA) were applied in duplicate in each run. In cases in which
there were fewer than 10 copies of standard RNA, reproducible amplification
could not be shown (see Results).
Quantitative RT-PCR assay for Lymnaea CREB1 and CREB2 mRNAs in a single neuron
The samples that were frozen with the lysis solution and stored at
80°C were warmed to 65°C for 1 min to break the cell membrane
completely, and then quenched on ice. Then 10 µl of a solution for reverse
transcription was added. The reverse transcription solution contained 0.375
µl of 50 U µl1 MultiScribe reverse transcriptase (PE
Applied Biosystems), 1.5 µl of 10x TaqMan Buffer A (PE Applied
Biosystems), 3.24 µl of 25 mmol l1 MgCl2 (PE
Applied Biosystems), 3.0 µl of 2.5 mmol l1 dNTP (Toyobo,
Tokyo, Japan), and 0.1 µl of 30 U µl1 Prime RNase
inhibitor (Eppendorf), all of which were stored in DEPC water. The final
concentrations of components in 15 µl of the reverse transcription solution
were 1.25 U µl1 MultiScribe reverse transcriptase, 1/10
volume of 10x TaqMan Buffer A, 5.5 mmol l1
MgCl2, 0.5 mmol l1 dNTP, 2.5 µmol
l1 oligo d(T)16 primer, 13.3 µg
ml1 yeast tRNA, 0.3 U µl1 Prime RNase
inhibitor, and 0.13% NP40 in DEPC water.
On the basis of the protocol provided by the manufacturer (PE Applied Biosystems), reverse transcription was performed at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. The cDNA samples (6 µl) were applied for qRT-PCR in duplicate to avoid technical variation. For real-time PCR, a PCR reaction solution (19 µl; PE Applied Biosystems) was added to the above reverse-transcribed solution (6 µl). The final components of the reaction mixture (25 µl) for real-time PCR were 0.025 U µl1 AmpliTaq Gold DNA polymerase (PE Applied Biosystems), 1/10 volume of 10x TaqMan Buffer A, 3.5 mmol l1 MgCl2, 0.2 mmol l1 dNTP, 0.1 µmol l1 each of the forward and reverse primers, and 50 nmol l1 fluorogenic probe (PE Applied Biosystems) in distilled deionized water. The conditions were as follows. For CREB1 mRNA, PCR consisted of 1 cycle at 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 62°C for 30 s. For CREB2 mRNA, the PCR conditions were 1 cycle at 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 58°C for 30 s.
The intensity of reporter dye fluorescence was captured at each cycle of PCR with an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems), and the amplification plots were traced. A threshold for the increase in fluorescence was arbitrarily determined in the exponential phase of the amplification plots, and a standard curve was drawn to show the starting copy number of the standard RNA vs the threshold cycle.
Assessment of qRT-PCR assay for Lymnaea CREB2 mRNA in a single neuron in the reverse transcription solution with cell debris
RT-PCR was carried out immediately after lysis of a single cerebral giant
cell without centrifugation. Therefore, to examine the RT-PCR efficiency in
samples containing cell debris, the efficiencies were compared between
standard RNA solutions and sample mRNA solutions, which were prepared from 19
cerebral giant cells. Both standard CREB2 RNA solutions and serially diluted
sample mRNA solutions were subjected to qRT-PCR in triplicate. We thus
obtained curves between the starting quantity of RNA and the threshold cycle
for both solutions, and checked the linear range of the curves and the
parallelism between them. The amount of total mRNA in the sample was
arbitrarily assigned a value of one.
In addition, we examined whether the target mRNA was degraded during the
aforementioned procedures with endogenous RNase in isolated neurons, and also
the effects of freeze-thaw. For this purpose, a known amount of salmon GTH
2 RNA (6x104 copies) was added to the lysis solution
(4.5 µl) with an isolated, single cerebral giant cell, and its copy number
was determined by qRT-PCR of 1 cycle at 95°C for 10 min, followed by 50
cycles of 95°C for 15 s and 58°C for 30 s.
Effects of enzyme treatment during dissection of the central nervous system on the copy numbers of mRNAs
We examined the effects of enzyme treatment for dissection of the CNS on
the copy numbers of mRNAs. Total RNA was extracted from the CNS samples
treated with 2 mg ml1 trypsin for 10 min at room temperature
or with 2 mg ml1 protease type IX (Sigma) for 10 min at room
temperature (Hatakeyama et al.,
2004b) in Lymnaea saline by the acid guanidium
thiocyanate-phenol-chloroform method
(Chirgwin et al., 1979
). The
amount of total RNA extracted from a single CNS ranged from 1.4 to 3.0 µg.
The copy number of CREB2 mRNA in total RNAs (120 ng each) was determined in
duplicate by qRT-PCR.
Statistical analysis
The data are expressed as the means ± S.E.M. One-way
analysis of variance (ANOVA) was applied to determine the statistical
significance (P<0.05).
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Results |
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Accurate and rapid isolation of a single, identifiable neuron
The newly developed micromanipulators with glass microelectrodes, which
were fabricated with a fire-melted round and capped tip (ca. 10 µm
diameter), were used for isolation of a single cerebral giant cell under a
stereoscopic microscope. As mentioned previously, a small length of primary
neurite of the cerebral giant cell was also isolated
(Fig. 1). Our procedure enabled
us to isolate a single cerebral giant from the CNS within 20 min. This period
is much shorter than that by use of a patch pipette or laser microdissection
(Monyer and Lambolez, 1995;
Surmeier et al., 1996
;
Baro et al., 1997
;
Schütze et al., 1997
;
Fink et al., 1998
;
Tkatch et al., 2000
;
Liss et al., 2001
;
Tsuzuki et al., 2001
;
Liss, 2002
;
Song, 2002
;
Mawrin et al., 2003
).
The morphology of isolated cerebral giant cells was observed under a stereoscopic microscope to confirm that the somata were intact and that no other cells were attached to the target neurons. To further confirm that we isolated only a single cerebral giant cell without any other cells, the isolated neuron was fixed and stained with Hoechst33258, followed by observation under a fluorescence microscope. Only the nucleus of the cerebral giant cell was observed, confirming that no other cells were removed with the single cerebral giant cell (Fig. 1). We therefore considered that RNAs in the somata of isolated neurons were completely maintained and that no RNAs were contaminated by other neurons or glial cells.
Validity of qRT-PCR assay for determination of the mRNA copy number in a single neuron even in the presence of cell debris
When the qRT-PCR method is used for a whole cell, sample solutions include
cell debris, such as genome DNA, proteins, lipids, and so on. By comparison,
standard RNA solutions are pure, as they are synthesized from the cDNA
templates. Accordingly, we need to confirm the efficiency of RT-PCR for a
sample mRNA solution that contains cell debris.
We first obtained the standard curve of CREB2 RNA solutions, and then obtained the curve for sample solutions of CREB2 mRNA that were prepared from 19 cerebral giant cells. The standard curve was linear within the range of 2x10 to 1x105 copies, and the curve of sample mRNA was parallel to the standard curve at 10 to 3x102 copies (Fig. 2). The slopes of the lines were 3.98 for the standard solutions, and 3.94 for the sample solutions. These results showed that RNA can be amplified with the same efficiency both in standard RNA solutions and in sample mRNA solutions by qRT-PCR.
|
|
Furthermore, we diluted the salmon GTH 2 RNA from 100,000 copies (1,
1/10, 1/100, 1/1000, 1/10000, 1/100000), and theoretically obtained 1 copy
(Fig. 3D). The error at 10
copies, that is 1/10000, shows that the S.E.M. is low enough (2.93,
N=6 samples). These results indicated that we can disregard
degradation of target mRNA by either endogenous RNase in the isolated cerebral
giant cells or by the freeze-thaw procedure.
Determination of the copy number of Lymnaea CREB2 mRNA in a single cerebral giant cell
We determined the copy number of Lymnaea CREB2 mRNA in a single
cerebral giant cell (Fig.
4A,B). In 6 µl of the sample solution (in duplicate from 15
µl solution for a single neuron), the copy number was determined to range
from 10 to 180 by the following procedures. The threshold was set under the
condition that the PCR amplifications for all the single neuron samples showed
exponential patterns (Fig. 4A).
The threshold cycles were then exported to the standard curve prepared with
the standard RNA solutions (Fig.
4B). The copy number of 10180 obtained from the
determination of CREB2 mRNA (N=11 single cells) was included in the
region parallel to the standard curve, as shown in
Fig. 2. Thus, the copy number
obtained here was considered reliable. By calculating the average value of
duplicated samples for each neuron, it was estimated that the copy number of
CREB2 mRNA in a single cerebral giant cell was between 30 and 240.
|
Effects of enzyme treatment on the copy number of Lymnaea CREB2 mRNA
Enzyme treatment is required for desheathing the CNS to isolate a single
neuron rapidly. We therefore examined the effects of enzyme treatment on the
copy number of target mRNA. Total RNAs of control CNSs and those of CNSs
treated with 2 mg ml1 trypsin for 10 min or 2 mg
ml1 protease type IX for 10 min in the Lymnaea
saline were purified, and 120 ng of total RNA of each group was
reverse-transcribed. Then, the copy number of Lymnaea CREB2 cDNA was
determined by qRT-PCR. No significant differences were observed between the
control and the sample treated with trypsin or between the control and the
sample with protease (control, N=8, 102498±7065; trypsin,
N=5, 110202±9312; protease type IX, N=10,
123005±10296). These results indicate that the use of enzymes is
feasible for the dissection of CNS samples.
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Discussion |
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Reliability of our assay system
One of the first questions that must be raised concerns the reliability of
our assay. On the basis of the data presented here, we concluded that our
assay is reliable. We based this conclusion on the following points: (1) the
standard curves were linear within the range of 10105
copies; (2) the curve obtained from the serially diluted sample mRNA solutions
was parallel to the standard curve; and (3) the intra-assay variation was
within 15% in our assay system. We were also confident in regard to our limit
of detection, which is 25 mRNA copies in a single cell. Our assay system can,
therefore, reliably determine the numbers of mRNAs in a single cell as long as
there are at least 25 copies of the target mRNA being assayed.
Advantages of our assay system compared with the others
As described in the Introduction section, some studies at single-cell level
have been previously reported (Baro et al.,
1997; Tkatch et al.,
2000
; Liss et al.,
2001
; Tsuzuki et al.,
2001
). First, the studies by Baro et al.
(1997
) and Tsuzuki et al.
(2001
) measured the density of
PCR bands using conventional methods. Thus, the quantification was less
sensitive than reported by us here. Second, Tkatch et al.
(2000
) and Liss et al.
(2001
) used qRT-PCR. In the
Tkatch et al. (2000
) study,
brain slices from mice were made after decapitation and then incubated with
artificial saline. The slices were then digested by protease and dissociated
mechanically; and only then were single neurons picked up for analysis.
However, these neurons were not uniquely identified. Furthermore, because the
experiment took a relatively long time, it could not be used to measure mRNAs
that turned over rapidly. Liss et al.
(2001
) used a patch pipette
for aspiration of intracellular contents. However, it is not certain whether
all the contents of the cell could be collected into the patch pipette.
Recently, a few other new methods have been proposed to accomplish the
single-cell task (e.g. Kamme et al.,
2003; Ginsberg and Che,
2004
; Korneev et al.,
2005
). However, our assay surpasses all of them from the
viewpoints of accuracy, rapidity and relevancy. In particular, we should add
some comments on the method by Korneev et al.
(2005
). They measured the
amounts of nitric oxide synthase (NOS) and other mRNAs along with the isolated
total RNA from single cerebral giant cells with an RNA purification kit.
However, and this is the important difference between our work and theirs,
they did not determine the exact copy numbers of target mRNAs for NOS or any
other proteins in a single cerebral giant cell, whereas we have. They have
only reported the relative levels of target mRNAs.
The methodology in the Korneev et al.
(2005) paper is different in a
number of important ways than ours. (1) To obtain the relative levels of
target mRNAs, they used mRNA for ß-tubulin as a reference point. However,
it must be remembered that the shape of neurons changes (e.g. the dendritic
tree) even with learning and memory (in fact morphological change at either
the pre- or post-synaptic specialization is probably one of the most important
physical manifestations of memory formation; e.g. Alkon et al.
(1990
) and Bailey and Kandel
(1993
). Because such changes
involve alterations in ß-tubulin activity, it is probably not the most
accurate reference point to use as its mRNA levels are most likely changing.
(2) There may also be differences in PCR efficiency. These differences may
arise from a number of factors the most relevant being: (a) differences in the
efficiency of how primers anneal to templates; (b) differences in the
efficiency of polymerization of PCR products; and (c) a different affinity of
SYBR Green to PCR products. (3) A loss of mRNA in their isolation procedure
cannot be ruled out. Taken together, the data presented in the Korneev et al.
(2005
) paper are weaker than
the data presented in our present paper. Because only relative amounts of
mRNAs were measured in the Korneev et al.
(2005
) paper against a
probably changing reference point, the conclusions that could and should be
drawn are relatively weak regarding their biological meaning.
By contrast, the protocol developed by us and presented in the present
paper has perfectly settled all these problems. (1) Our present protocol
clearly gives the exact copy numbers of target mRNAs in a single cell, and
this is important to elucidate the biological meaning of transcription. (2)
The copy numbers of target mRNAs are determined by comparison with the
standard curves prepared from the cDNA solutions corresponding to the serially
diluted solutions of the target mRNAs themselves in our present protocol. This
indicates that the PCR efficiency is not questionable. (3) The RNA loss is
highly improbable in our RNA isolation protocol. We, therefore, claim that our
present protocol completely surpasses all other published methods including
that described by Korneev et al.
(2005).
Why is the copy number of CREB2 mRNA variable between single neurons?
Whereas our intra-assay variation was on the order of 15%
(Fig. 3), we found a much
larger variation in the copy number of CREB2 mRNA between single cerebral
giant cells (Fig. 4B). In
addition, the estimated copy number of CREB1 mRNA in single cerebral giant
cells also showed a slight variance (Fig.
4D). For CREB2, the range was from 30 to 240 mRNA copies obtained
from single cerebral giant cells from different snails. The difference in the
mRNA copy numbers should be regarded as meaningful and important. There are a
number of possible reasons for this range in the mRNA copy numbers,
particularly as regards CREB2. First, preliminary experiments showed that
various environmental and experimenter-applied stimuli are able to up-regulate
the expression of the CREB2 gene in the cerebral giant cells (H. Sadamoto and
E. Ito, unpublished data). Second, the mammalian homolog of Lymnaea
CREB2, ATF4, is also upregulated by many different extracellular signals
(Hai and Hartman, 2001). In
addition, several intracellular stress pathways are known to activate ATF4
(Rutkowski and Kaufman, 2003
).
Third, ATF proteins are also involved in such important homeostatic functions
as glucose metabolism and trophic factor-dependent survival. Thus, various
physiological and environmental stimuli can easily alter the expression of
CREB2 in single cerebral giant cells from different snails, even when the
snails are maintained under similar conditions.
In conclusion, the results from our study showed that it is possible to isolate a single, identifiable cell accurately and rapidly, and to determine the exact copy numbers of particular mRNAs within it. We chose to examine the CREB transcripts and found that in the cerebral giant cells there were more mRNA copies of the repressor type, CREB2, than there were mRNA copies of the activator type, CREB1.
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Acknowledgments |
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