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Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Harold Gainer, Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 4D-20, Bethesda, Maryland 20892.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early ISHH studies using labeled oligonucleotide probes led to the view that expression of these peptide genes is mutually exclusive and occurs separately in the OT and VP magnocellular neurons (7). More recent studies using probes with higher specific activities reported that the OT and VP genes are coexpressed in 1–3% of the magnocellular neuronal population (8, 9) and that this can increase to 17% coexistence in female rats after 2 days of lactation (9). As all of these reports were based on qualitative data, we set out to quantitatively determine the OT and VP mRNA levels in single magnocellular neurons. Quantitative analyses of OT and VP mRNA levels in magnocellular neurons and total hypothalamus have been reported using ISHH (10, 11), solution hybridization-nuclease protection assay (RPA) (12, 13, 14), Northern blot (15), dot blot (16), as well as competitive RT-PCR methods (17). Only the quantitative ISHH method (10) was applicable to single cells; however, this method could only evaluate one mRNA species per cell and could not evaluate levels of OT and VP mRNA in the same cell.
Consequently, in the present study, we used competitive RT-PCR as the method to quantify OT and VP mRNA content in single dissociated magnocellular neurons from rat SON. Measurement of gene transcripts by RT-PCR has become a standard technique due to its simplicity and high sensitivity and because it also allows for the determination of multiple species of mRNA levels using small amounts of tissue. Many different quantitative RT-PCR methods have been developed (18, 19, 20). However, few studies have been performed at the single cell level. To better study OT and VP mRNA phenotypes in single magnocellular neurons using the quantitative RT-PCR method, we adapted a competitive RT-PCR method using a standard calibration curve (21) to quantify OT and VP mRNA levels in individual magnocellular neurons from the rat SON.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20 µm) has been
described previously (22). Briefly, 400-µm thick horizontal slices of
ventral hypothalamus were cut using a tissue chopper. The slices were
oxygenated in HBSS for 1 h at room temperature, and the SON were
then dissected. The tissues were incubated in trypsin, washed with low
calcium HBSS, and triturated. The cell suspension was maintained in
oxygenated artificial cerebrospinal fluid, and single putative
magnocellular neurons, recognized by their relatively large cell size,
were aspirated into a micropipette and transferred into 4 µl lysis
buffer (see below) in a 0.5-ml PCR tube on ice. The cells were stored
at -70 C until use. Animals were cared for according to the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication 80–23,1978) during the entire course of this research.
Primer design
Coding sequences in OT and VP genes were used to design the
primers for used in qualitative single cell RT-PCR analysis. These
primers were designed using Oligo 4.0 primer analysis software
(National Biosciences, Inc., Plymouth, MN). A review of GenBank using
the BLAST program showed that these primers are specific for OT and VP
mRNA. The sense primer for OT was 5'-GACGGTGGATCTCGGACTGAA-3', and the
antisense primer was 5'-CGCCCCTAAAGGTATCATCACAAA-3'. The sense primer
for VP was 5'-CCTCACCTCTGCCTGCTACTT-3', and the antisense primer was
5'-GGGGGGCGATG-GCTCAGTAGAC-3'.
For the quantitative RT-PCR studies, OT and VP PCR primers designed by LeMoullec et al. (17) were employed. These primers were synthesized by Genosys (Genosys Biotechnologies, Inc., The Woodlands, TX). The sense primer for VP was 5'-CGCAGTGCCCACCTATGCTCGCCA-3', and the antisense primer was 5'-TCGGCCACGCAGCTCTCATCGCTG-3'. The sense primer for OT was 5'-GAACACCAACGCCATGGCCTGCCC-3', and the antisense primer was 5'-TCGGTGCGGCAGCCATCCGGGCTA-3'. The latter PCR primers were used in the quantitative studies to amplify both the endogenous targets and the exogenous internal standards (see below).
Exogenous internal RNA standards and construction of full-length OT
and VP RNAs as calibration standards
The exogenous standard OT RNA used to evaluate sensitivity of
the RT-PCR procedure was constructed using the same OT PCR primers as
those used for the qualitative analysis (see above) and as described
previously (23). The exogenous internal standard RNA (pVOIS) used for
the quantitative RT-PCR analysis and the OT and VP full-length RNAs
used as standards for the calibration curves were synthesized by
in vitro transcription using a MEGAScript in
vitro transcription kit (Ambion, Inc., Austin, TX).
The RNA products were then run on formaldehyde-denatured agarose gels
to verify size and integrity. The RNAs were then extracted and
precipitated, and their concentrations were determined using a UV
spectrophotometer at 260 nm absorbance.
The exogenous internal standard RNA, termed pVOIS, contained both the OT and VP PCR primer sequences, separated by 97 and 488 bp, respectively. Hence, the expected sizes of the OT and VP PCR products amplified from the internal standard RNA (pVOIS) are 97 and 488 bp, respectively. The details of construction of pVOIS has been previously described (17). The pVOIS plasmid and the plasmid containing full-length OT complementary DNA (cDNA; pOCY) were both obtained from Dr. F. Pinet. The plasmid containing full-length VP cDNA was provided by Drs. E. Mohr and D. Richter.
Single cell RT-PCR procedures
The general procedure used for single cell RT-PCR is similar to
that previously described (22), but with some modifications. Briefly,
the lysis buffer contained 1 x RT buffer (Life Technologies, Inc., Gaithersburg, MD); 5 U/ml Prime RNase
inhibitor (5'-3', Inc., Boulder, CO); 324 U/ml RNA guard
(Pharmacia Biotech, Piscataway, NJ); 0.5% Nonidet P-40;
0.5 mM each of deoxy (d)-ATP, dCTP, dGTP, and dTTP
(Life Technologies, Inc.); and 5 µM random
hexamers (Life Technologies, Inc.). The cells were lysed
at 72 C for 5 min, and 100 U superscript II reverse transcriptase
(Life Technologies, Inc.) was added. The RT reaction was
performed for 1 h at 42 C, and the reaction was terminated by
incubation for 15 min at 72 C and then chilled on the ice for 5 min.
Diethylpyrocarbonate-treated H2O was added to each tube and
brought to a final volume of 50 µl/tube. PCR was then carried out in
a 50-µl reaction volume using a hot start PCR as previously described
(23). Typically, 20% of the RT product (cDNA) was used in a PCR
reaction containing 1 x PCR buffer [60 mM Tris-HCl,
15 mM (NH4)SO4, and 2
mM MgCl2, pH 9.0]; 250 µM each
of dATP, dCTP, dGTP, and dTTP (pH 8.0; Invitrogen, San
Diego, CA); and 1.25 U Taq polymerase (Perkin-Elmer Corp., Branchburg, NJ). The Taq enzyme was in the top
layer of the tube separated from the lower buffer containing the cDNA
by wax purchased from Perkin-Elmer Corp. PCR was performed
in a Perkin-Elmer Corp. 9600 Thermal Cycler and consisted
of a 5-min preincubation at 95 C, followed by 40 or 44 cycles of
denaturing (94 C, 45 sec), annealing (62 C, 45 sec), and
extension (72 C, 90 sec), followed by a final extension of 7 min
at 72 C. PCR products were separated by 1.8% agarose gel
electrophoresis containing 0.5 mg/ml ethidium bromide, visualized using
an UV transilluminator, and then digitally photographed using a CCD
camera of Stratagene Eagle Eye System
(Stratagene, La Jolla, CA) and analyzed by the NIH IMAGE
software (NIH, Bethesda, MD) on a Power Macintosh 6100/60 computer
(Apple Computer, Inc., Cupertino, CA). The densitometry was
performed using the gel analysis macro of NIH IMAGE.
Quantitative and calibration of competitive RT-PCR
In this study, the standard calibration curves were generated
from full-length native RNAs by RT and competitive amplification
together with exogenous internal standard RNAs. This approach was used
to quantitatively measure mRNA levels of OT and VP in acute dissociated
single magnocellular neurons from the adult rat SON. The procedure was
similar to that described by LeMoullec et al. (17) and Tsai
and Whitbank (21) with some modifications for single cell analysis.
Traditional competitive RT-PCR methods use serial dilution of internal
standards, which require multiple reactions to determine the equal
molar point of the endogenous mRNA number (20, 24, 25). This approach
is highly time consuming and very costly if one wishes to examine
multiple genes in multiple samples. In addition, it requires higher
amounts of mRNA than are available in single cell samples. Therefore,
to simplify the competitive RT-PCR assay and to account for the
variations inherent in the assay, individual standard calibration
curves for the OT and VP mRNAs were generated. Different amounts of
full-length OT and VP cRNAs were reverse transcribed and amplified with
a constant amount of the exogenous internal standard RNA (pVOIS) (17).
The amount of mRNA in a single cell could then be directly determined
from the calibration curve. Briefly, a 1:2 serial dilution of OT or VP
full-length cRNA (0.840–0.026 attomoles, where 1 attomole equals
10-18 mol) along with 0.400 attomoles internal standard
RNA (pVOIS) were added in separate sets of tubes containing the RT
reaction mixture (same solution as lysis buffer for single cells
described above). The RT-PCR conditions and procedure were the same as
those used for the single cell RT-PCR protocol described above. After
densitometric analysis, the ratio of the full-length complementary RNA
(cRNA) OD to the internal standard RNA (pVOIS) OD was plotted against
the molar amount of OT or VP full-length cRNA (N). The calibration
curves for OT and VP mRNA were best fitted to a equation: ratio =
a x Nb + c x Nd, where a and c
(
0) are the coefficients of variable N (amount of cRNA), and b and d
are the exponents of N. Each calibration curve was generated from two
replicates. For all single cell samples, the same amount of internal
standard RNA (0.400 attomole) was routinely coamplified with single
cell RNA along with two replicates of the calibration curve at the same
time. All single cells were analyzed over 4 different days along with
calibration curves generated in the same experiment each day. The mRNA
content in the single cell sample was calculated from the
equation shown above. The number of molecules of mRNA can then be
computed from the attomole value using Avogadro’s number, or
6.023 x 1023 molecules/mol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Nearly all of the neurons tested
appeared to coexpress some OT and VP mRNA. These qualitative data are
similar to a previous preliminary report from our laboratory (22). The
PCR product intensities of OT and VP in the single neurons varied
greatly between the cells; hence, the qualitative RT-PCR could not
reliably determine the relative abundance between OT and VP in a given
neuron. Experimental variations in the RT and PCR reactions themselves
add to the experimental errors. Because of the exponential nature of
PCR, small variations in PCR efficiency will yield large changes in the
amounts of product. For example, because of these variations, we cannot
be certain whether VP or OT mRNA was or was not expressed in cells 4
and 5, respectively, in Fig. 1. Thus, it was necessary to analyze OT
and VP mRNA levels in a more quantitative manner.
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shows that all of these internal
standard RNAs could be detected using 40 cycles of PCR amplification
(our standard procedure, see Materials and Methods) and that
at least 30 molecules of OT mRNA could be detected by this method. The
intensities of the RT-PCR product bands increased with increasing RNA
molecules and appeared saturated between 3 x 103 and
3 x 104 copies of mRNA. To further examine the OT and
VP mRNA levels in the single magnocellular neurons in a quantitative
manner, we employed competitive RT-PCR with the internal standard,
pVOIS (see below), and calibration curves using the full-length OT and
VP RNA standards.
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illustrates our competitive
RT-PCR analysis of the target RNAs (full-length OT and VP cRNAs) and
the exogenous internal standard RNAs for OT and VP (pVOIS). Using 0.400
attomole (or 10-18 mol) internal standard RNA, both
full-length OT or VP cRNA in the 0.003–0.840 attomole range and
internal standard RNA can be detected (data not shown). Representative
calibration curves for the OT and VP mRNAs are shown in Fig. 3. In each
case, a photo of the ethidium bromide-stained gel with varying
full-length cRNA concentrations (upper photograph) is shown.
Calibration curves for the OT and VP RNAs in which the ratio of
full-length RNA (cRNA) OD to the internal standard (pVOIS) RNA is
plotted against the molar amount (in attomoles) of the OT or VP
full-length cRNA standards used are shown below each photo.
Given such calibration curves, the amounts of OT or VP mRNA in single
magnocellular neuron could be determined from the cRNA/pVOIS ratio in
that sample. Different amplification efficiencies between the OT and VP
PCR primers has been indicated by LeMoullec et al.
(17). Our calibration curves also show that the efficiencies of
amplification between endogenous targets and internal standard are
different. The variations in RT-PCR between the various reactions could
contribute to the variations in intensities between the PCR bands shown
in the figure. Hence, using an exogenous internal control procedure
such as pVOIS is essential for quantitative analysis.
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, we were able to determine the attomoles
of OT and VP mRNA in single magnocellular neurons. These data are
summarized in Table 1. Eleven individual
magnocellular neurons from the SON of a single normal female rat were
quantitatively analyzed for their OT and VP mRNA levels. The cells are
grouped according to their apparent phenotype in Table 1. We
operationally defined the phenotypes of single magnocellular neurons as
OT or VP based on their ratios of these two peptide mRNAs. The neurons
with ratios less than 3 are classified as OT- and VP
(OT/VP)-coexpressing cells. The neurons with ratios that are an order
of magnitude greater (i.e. with ratios >20) are considered
to be either OT or VP neurons, depending on the dominant mRNA species
in the cell. The average OT or VP mRNA molecule found for each
phenotype is shown in Table 1 and is about 0.674 or 0.777
attomole/neuron in the OT or VP neuronal phenotype, respectively.
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shows that the ratio data in Table 1
falls into two clear groups: one OT/VP cell (ratio of 2) and the OT and
VP cells (whose ratios are >25). For the identified OT and VP neurons
in Fig. 4A there appears to be a tendency for the VP neurons to have
lower ratios than the OT neurons. However, many more cells will need to
be analyzed quantitatively before such a conclusion can be drawn. The
histogram in Fig. 4B plots the frequency distribution of total
nonapeptide (OT and VP) mRNA/cell, and although the differences among
various cells are less than 10, there appears to be no phenotypic trend
to the data.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Quantitative analysis of OT and VP gene expression in single
magnocellular neurons
To determine the levels of OT and VP mRNAs in single magnocellular
neurons, we adapted the quantitative competitive RT-PCR assay by also
using a calibration curve. This method has been found to be very
sensitive, reproducible, and relatively rapid (17, 21, 26). Our results
show that at least 2-fold differences in specific mRNAs can be
resolved, and that at least two genes can be quantitatively analyzed
from a single cell. By applying this method, we found that only 1 cell
of 11 tested was of the OT/VP phenotype, i.e. where the OT
and VP mRNA levels were comparable (in fact, the ratio in this cell was
2; see Table 1 and Fig. 4A). The low probability of finding this
coexisting phenotype was expected in cells from normal animals, as the
incidence of this phenotype in the magnocellular neuron population was
reported to be less than 5% in normal rats by the ISHH method (8, 9).
What is surprising from this study is that the OT and VP phenotypes
characterized by ISHH and immunocytochemistry are actually not
exclusively expressing only one peptide mRNA, but contain significant
amounts of both mRNAs. These neurons contain, on the average, 2 orders
of magnitude more mRNA for the major peptide (that defines the
phenotype) than the minor one (see Table 1), and hence, the phenotype
is a quantitative and not a qualitative property of peptide gene
expression.
The mRNA levels of the minor species estimated in OT and VP cells are
substantial. In the VP phenotype, which has an average of 0.777
attomoles VP mRNA, the level of OT mRNA is 0.012 attomoles/cell. The
latter OT mRNA is calculated (using the attomole values in Table 1 and
Avogadro’s number) to be about 7,230 OT mRNA molecules/cell, whereas
the major VP peptide mRNA is calculated to be 468,000 copies/cell.
Similar calculations for the OT phenotype gives 406,000 OT mRNA
molecules of OT mRNA/cell, and for the minor VP peptide mRNA gives a
value of 1,800 VP mRNA copies/cell. These values for the major peptide
species seem very high, especially in view of the estimate by Hastie
and Bishop (27), using mRNA-DNA hybridization techniques, that, on the
average, the total number of copies of a mRNA per brain cell is
563,505. However, it should be noted that hypothalamic magnocellular
neurons are much larger (i.e. about 20-µm average
diameter) than typical brain cells, and thus might contain more total
mRNA. Previous estimates of hypothalamic OT and VP mRNA levels and copy
numbers per neuron vary widely. Young et al. (10, 11)
estimated based on quantitative ISHH studies, that the magnocellular
neurons in normal rats contain about 30,000 OT or VP mRNA copies/cell.
A similar set of values was obtained by Sherman and Watson (28), who
used RPA of hypothalamic tissue punches. The latter study converted the
total mRNA measured per punch to per cell values by assuming, based on
previous studies, that there were 4,327 VP cells and 3,233 OT cells in
the punched tissues.
Other workers have reported on the quantitative levels of OT and VP mRNAs in total rat hypothalamus, using either RPA or quantitative RT-PCR assays. Kim et al. (29), using RPA, reports between 32 pg VP mRNA/µg total RNA in normal rat hypothalamus, and LeMoullec et al. (17) using quantitative RT-PCR reported 30 pg OT mRNA and 10 pg VP mRNA/µg total mRNA in normal rat hypothalamus. Assuming 50–100 µg total mRNA/rat hypothalamus (30) and an estimate of about 7,000 OT and VP cells each per total rat hypothalamus (31), we calculate values of 1.26–2.52 attomoles OT mRNA/neuron (759,000–1,510,000 molecules/neuron) and 0.36–2.29 attomoles VP mRNA/neuron (217,000–1,380,000 molecules/neuron) from these data. These calculations are close to the values we found. It should be noted, however, that in none of these studies, including our own, were the possibilities of RNA degradation in the initial sample accounted for; hence, the explanation for the differences in absolute copy numbers among these studies remains uncertain.
In summary, the RT-PCR procedure we have used is able to clearly distinguish among the OT, VP, and OT/VP phenotypes and is also able to quantitate the levels of expression of both of these two peptide mRNAs in the magnocellular neuronal phenotypes.
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Acknowledgments |
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Received February 19, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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