Application of quantitative RT-PCR to the analysis of dopamine receptor mRNA levels in rat striatum

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MOLECULAR BRAIN RESEARCH

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ELSEVIER

Molecular Brain Research 34 (1995) 127-134

Research report

Application of quantitative RT-PCR to the analysis of dopamine receptor mRNA levels in rat striatum Sheila L. Vrana*, Bryan W. Kluttz, Kent E. Vrana Department of Physiology and Pharmacology and Centerfor the NeurobiologicalInvestigation of Drug Abuse, The Bowman Gray School of Medicine, Medical Center Blvd, Winston-Salem,NC 27157-1083, USA Accepted 13 June 1995

Abstract A quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) procedure has been developed which selectively amplifies and quantifies the two isoforms of the dopamine D2 receptor. Variability is corrected by the inclusion of a D2 dopamine receptor mRNA standard within each reaction. The internal standard was generated by introducing a point mutation within a D2 cDNA clone that created a unique restriction site within the amplified region. An in vitro transcribed RNA for the internal mutant control is added to the RNA isolated from brain tissue and the mixture is subjected to RT-PCR, digestion with the restriction enzyme, separation of the products by PAGE, and quantification by direct analysis of radioactivity incorporated during the PCR step. The standard is amplified, in the same reaction as the experimental RNA, using the same primers and RT-PCR conditions. In this manner, the effects of contaminants of the RNA preparation which could affect the amplification procedure are assessed. To insure that the amplification is linear, the number of PCR cycles is minimized. This adaptation avoids 'competitive PCR' and provides for a linear response. Moreover, to obviate non-specific co-amplification, primer annealing steps are performed at or above the melting temperature for the primers, thus increasing signal-to-noise ratios. Finally, primer pairs have been designed which permit amplification of specific fragments for each of the five rat dopamine receptor subtypes. These fragments have unique sizes and so can be differentiated when simultaneously amplified in the same RNA preparations. Keywords: RT-PCR; Dopamine receptors; mRNA; Quantification

1. Introduction CNS dopamine receptors (DAR) are encoded by five different genes (D1 through D5). In addition, the D2 subtype gene produces two major splice variant mRNAs (D21ong and D2short). The protein products of the five DAR genes can be divided into two classes based upon sequence homology, ligand binding, and second messenger coupling (reviewed in [8,21,22]). Unfortunately, subtype-selective ligands are not currently available for the definitive quantification and characterization of the five subtypes. To

Abbreviations: RT-PCR, reverse transcriptase-polymerase chain reaction; DAR, dopamine receptor; PAGE, polyacrylamide gel electrophoresis * Corresponding author. Fax: (1) (910) 716-8501; e-mail: [email protected]. 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

SSDI 0 1 6 9 - 3 2 8 X ( 9 5 ) 0 0 1 5 2 - 2

address the problem of multiple genetic isoforms, a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure has been developed which selectively amplifies each of the five DAR subtype mRNAs (including the two D2 isoforms) and quantifies the amount of mRNA. What is described in this report is a demonstration of the technique for the analysis of both splice variants of the D2 DAR, and the presence of all five DAR subtypes in rat striatum. Given the sensitivity of PCR, it would seem to be a perfect choice for measuring rare RNA species or RNA levels in small amounts of tissue. Procedures for quantitative RT-PCR were first described in 1989 and specific techniques have been developed in a number of laboratories since that time [1,2,4,9,19,23,27,29,30]. In addition, kits have been marketed which facilitate the synthesis of first-strand cDNAs from mRNA for use as templates in PCR amplifications (i.e., cDNA cycle kit, Invitrogen, AMV reverse transcriptase system, Gibco-BRL). In each of these

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applications, specific species of RNA are copied into DNA by the retroviral enzyme reverse transcriptase. This DNA product is then amplified using PCR technology. Since these steps are conducted with gene-specific primers, only the m R N A of interest is amplified and the amount of PCR product is proportional to the amount of starting RNA present in the original preparation. The RT-PCR approach has only recently received widespread acceptance because of the variability in the amplification procedure. The major concern is that the 'efficiency' of amplification can vary as a function of contaminants of the sample. In particular, the first strand cDNA synthesis by reverse transcriptase is notoriously sensitive to outside effectors. Therefore, two samples could exist containing identical amounts of a specific RNA, but the presence of inhibitory contaminants in only one of the samples would result in a lower amplification signal. One solution to this dilemma is the use of an internal RNA standard. That is, an RNA, which is constant between sample preparations, is amplified under the same conditions (often in the same reaction). Although this standard can be an endogenous housekeeping gene like fl-actin or glyceraldehyde-3-phosphate dehydrogenase, primer annealing efficiency may vary between the separate primers required for the mRNA-of-interest and the standard mRNA. In addition, secondary structures may preclude the same rate of RT-driven first strand synthesis for the two different RNAs. Therefore, it is best to use a standard which will utilize the same primers (generally this requires a variant of the same mRNA) and which amplify a fragment distinguishable from the endogenous gene. We describe here an approach to determine absolute levels of D21ong and D2short m R N A via RT-PCR procedures. Specifically, we have created an in vitro transcribed m R N A internal standard which allows us to quantify dopamine D2 m R N A levels from small amounts of total RNA. In addition, primer pairs have been designed which selectively and

differentially amplify each of the five DAR subtype mRNAs (D1 through D5).

2. Materials and methods 2.1. P r i m e r a n d i n t e r n a l c o n t r o l c o n s t r u c t i o n

The quantitative amplification of the D2 RNA will be utilized to describe the RT-PCR system. The D2 DAR gene has the peculiarity that alternative splicing of the region er~coding the third intracellular loop can remove sequences representing 29 amino acid residues from the final m R N A [7,10,15,17]. This gives rise to a long and a short form of the receptor (termed D21ong and D2short, respectively). In the present experiments, a PCR primer pair was designed (D2-5', 5'-GCAGTCGAGCTI-TCAGAGCC-3'; D2-3' , 5'-TCTGCGGCTCATCGTCT-I'AAG3'; amplifying sequences corresponding to amino acid residues 2 2 8 - 3 6 2 of the D21ong cDNA [5]; see Table 1) which will amplify both D21ong- and D2short-specific fragments. Specifically, the primers anneal on either side of the alternative splice site and therefore generate two different fragments following PCR (of 404 bp and 317 bp, respectively). As an internal control, a D21o"_ c D N A clone [15] was modified by divergent PCR [1]] to create a unique B a m H I restriction endonuclease site within the amplified region. A point mutation was introduced at a position (T924 to G924, creating the sequence GGATCC; numbering system of Monsma et al. [15]) so that digestion of the 404 bp amplification product with B a m H I would generate two fragments (160 bp and 244 bp), well-separated from the D21ong and D2short amplification products. This recombinant construct is designated D2Lm (for mutant of the D2~oog isoform of the DAR gene). The D2Lm clone is in the Bluescript S K - vector and is readily

Table 1 Dopamine receptor subtype-selective RT-PCR primers Subtype

Primer sequence a

Gene position b

RT-PCR product

Gene ref. c

DI

5'-CAGTCCATGCCAAGAATTGCC-3' 5'-AATCGATGCAGAATGGCTGGG-3' 5'-GCAGTCGAGCTITCAGAGCC-3' 5'-TCTGCGGCTCATCGTCTTAAG-3' 5'-TCCTGTCTGAGGTGCATCC-3' 5'-TCGAAGTGGTACTCCCCGAG-3' 5'-CTACTCAGGGTCCCCTCTI'C-3' 5'-TGATCTTGGCGCCTCTC'Iq~C-3' 5'-AGCATGCTCAGAGTTGCCGG-3' 5'-ACAAGGGAAGCCAGTCTIq'GG-3'

aa #235-310

225 bp

Zhou et al., 1990

aa #228-362 J aa #243-369

404 bp (long) c 317 bp (short) 381 bp

Grandy et al., 1989 Bunzow et al., 1988 Giros et al., 1991

aa #241-304

189 bp

O'Malley et al., 1992

aa #261-330

209 bp

Tiberi et al., 1991 [26]

D2 D3 D4 D5

The top primer of each amplification pair is the 5' primer, and the lower primer the 3' primer. The 3' primers are antisense. b Indicates the amino acid residues correspondingto the amplified sequences. In some cases, only a portion of a listed codon may be amplified. c Reference from which the primer sequences were derived. d Amino acids of the D2 (long) isoform of the mRNA. e Two fragments are amplified with these primers and correspond to the D2 (long) and D2 (short) isoforms.

S.L. Vrana et al. /Molecular Brain Research 34 (1995) 127-134

QUANTITATIVE RT- PCR Tissue or Ceils (+/- treatment)

Transcription of mutant RNA

RNA Isolation

~

Generation of point mutation

A~tlonof J

mutant RNA

RT

PCN

Restrictiondigel~lon (of point mutation)

Gel slsctrophoresls

Quantification Fig. 1. Procedurefor performingquantitative RT-PCR using a homologous internal standard RNA. The flow diagram schematicallydescribes the proceduresfor quantitativeRT-PCR. The constructionof the mutant RNA and exact reactionconditions are described in Materials and methods.

transcribed using T7 RNA polymerase to generate a characterized and quantified RNA preparation. 2.2. RT-PCR protocol

The amplification protocol followed established procedures as described in Fig. 1. RNA was isolated from rat striatum by the single step procedure of Chomczynski [6] using commercially available TRI Reagent (Molecular Research Center, Inc; Cincinnati, OH). Efforts were taken to ensure removal of chromosomal DNA as detailed by the manufacturer. The purified total RNA was mixed with varying concentrations of the D2Lm in vitro transcription product and subjected to RT-PCR. Specifically, 1 /.tg of total striatal RNA was combined with 1-10 pg of D2Lm RNA and 1 /xM of the 3' primer. The mixture was incubated at 65°C for 10 min, then 22°C for 2 min to denature the secondary structure. The cDNA synthesis was then performed by adding the following reagents (final concentration): 500 U / m l RNAsin, 1.25 mM of each

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deoxynucleosidetriphosphate (dNTP), 4 mM Na4P207 (pyrophosphate), 250 U / m l reverse transcriptase, 100 mM Tris-HCl buffer (pH 8.3), 40 mM KCI, 10 mM MgCI 2, and 0.2 mM spermidine in a final reaction volume of 20 /zl. The reaction was carried out at 42°C for 1 h. In this process, the 3' primer anneals to all three of the target RNAs (endogenous D21ong and D2short and the exogenously added D2Lm) and is extended in the 5' direction via the RNA-dependent DNA polymerase activity of reverse transcriptase. The cDNA (2.5 /xl of the first strand synthesis reaction product) was mixed with (final concentrations): 0.2 m C i / m l [a-32p]dCTP, 0.5 mM of each dNTP, 1 /xM each of the 3' and 5' primers, 50 mM KCI, 2.5 mM MgC12, 10 mM Tris-Cl (pH 8.3), and 0.001% gelatin in a final volume of 25 /xl. The reaction was layered with mineral oil and Taq thermostable DNA polymerase (2.5 U; Promega; Madison, WI) added during a 5 min 'hot start' (94°C), performed to disrupt nonspecific hybridization of primer to template sequence. PCR amplification was conducted in a Perkin-Elmer thermocycler for 22 cycles under the following cycle conditions: 1.5 min at 94°C for denaturation; 2 min at 64°C for annealing and 2 min at 72°C for polymerase extension. The T,n for both primers is 64°C as calculated using the 2°C ( A / T ) and 4°C ( G / C ) method [12] or 60.4°C for the 5' primer and 58.3°C for the 3' primer calculated as described by Mueller et al. [16]. The reaction was radiolabelled with 0.2 m C i / m l [c~-32p]dCTP (with no reduction in unlabelled dCTP) to facilitate quantification of the resulting products. The use of radioactivity also increases the sensitivity of the system and so permits the use of fewer amplification cycles. Following amplification, samples (10 /xl) were removed from the RT-PCR reaction and digested with an excess of B a m H I (48 units of enzyme for 3 h) without purifying the DNA or modifying the buffer in any way. This approach is successful due to the fact that the restriction enzyme (although not under ideal buffer conditions) is present in large excess over the amplified fragment. The samples were subjected to 10% polyacrylamide gel electrophoresis (PAGE [20]), the gels dried, and the radioactivity visualized by autoradiography or direct analysis using a Betascope blot analyzer (Betagen-Intelligenetics; Mountainview, CA). 2.3. Single tube RT-PCR reactions

A modification of the RT-PCR procedure was developed which permitted single tube amplifications. RT reactions (20 g l final volume) were assembled in PCR thermocycle tubes as follows (final concentrations): 1 /.Lg striatal RNA, (1 × ) RT-PCR buffer (1 × = 10 mM Tris-HC1 (pH 8.3), 50 mM KCI, 2.5 mM MgCI 2, 0.005% NP-40, 0.005% Tween-20, 10 /~g/ml gelatin), 500 U / m l RNAsin, 1.25 /.~M 3' primer, 1.25 mM each dNTP, 250 U / m l AMV reverse transcriptase (Seikagaku America; Rockville, MD). The reactions were overlaid with mineral oil and placed in

S.L. Vrana et aL / Molecular Brain Research 34 (1995) 127-134

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a Perkin-Elmer thermocycler at 42°C for 1 h. At the end of the reverse transcription reaction, the thermocycler was programmed to increase temperature to 94°C for 5 min (hot start). During this period, 5 / z l of a mixture containing 5 /zM 5' primer, 1 × RT-PCR buffer, 1 /~Ci/ml [ a 32p]dCTP, and 1.25 U / ~ l Amplitaq DNA polymerase (Perkin-Elmer; Norwalk, CT) were added to the reaction. The thermocycler was then programmed to proceed with the amplification protocol described in the preceding section.

MW 1

2

3

4

(bp)

5

39@

2.4. Differential RT-PCR amplification o f the five dopamine receptor subtype mRNAs

Primer pairs were designed which selectively hybridize and amplify the five DAR subtypes (Table 1). Several important criteria were employed in the design of the primers: (1) the primer pairs were selected from areas of minimal homology so that there would be no cross-hybridization under high stringency conditions, (2) the amplification product for each subtype possesses a unique size (187-404 bp) so that the subtype-specific fragments are readily differentiated, (3) the relative TmS for the annealing of every primer are the same, and (4) where feasible, the primer pairs spanned splice sites (D2 and D3; D1 and D5 are intronless genes). In this way, all of the individual subtype m R N A molecules can be amplified under the same conditions (even in the same reaction) and then differentiated following resolution on polyacrylamide gels. The five subtype-specific products were amplified from striatal total R N A exactly as described for single tube

22O

600

[] D2 long Z~ D2 short • D2Lm (short ond long fragments c o m b i n e ~ / ~

500 400

13_. 300

100

0

1

2

3

4

MWM

0

I 2

t 4

t

a

i

6

8

10

D2Lm RNA

12

(pg)

Fig. 3. Quantificationof the D2 mRNA content of striatum using a D2Lm standard curve. 1 /.~gof striatal RNA was amplified in the presence of increasing amounts of D2Lm internal standard RNA. Following the RT-PCR reactions, the products were digested with BamH| and resolved by PAGE. The polyacrylamidegel was dried and subjected to autoradiography (top panel) and direct quantification on a Betagen betascope analyzer (bottom panel). Top: Lane 1, RT-PCR amplification of 1 Ng of striatal RNA; lanes 2-5, amplification of striatal RNA in the presence of 1.0, 2.5, 5.0 and 10.0 pg of in vitro transcribed D2Lm RNA, respectively. Bottom: Graphic representation of the quantification of the RT-PCR signals as determined by direct measurement of the radioactivity within each band (represented in the top panel).

Fig. 2. RT-PCR analysis of D21ong and D2short in the presence and absence of the internal standard D2Lm mRNA. Samples were subjected to RT-PCR followed by PAGE and autoradiography. Lane 1, amplification of 1 /zg of striatal RNA; lane 2, amplification of 1 /.tg of striatal RNA followed by BamHI digestion; lane 3, amplification of 5 pg D2Lm RNA; lane 4, amplification of D2Lm RNA followed by BamHI digestion.

reactions, and analyzed via PAGE and autoradiography. Owing to the widely varying amounts of each m R N A within the striatum, the number of amplification cycles was empirically balanced to make the signals readily visible on the same exposure (see Fig. 4 legend for details). In separate experiments, PCR products from the five different

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subtypes were digested with restriction enzymes (known to be present within the individual fragments) to ensure the identity of the amplified fragments (data not shown).

3. Results

Experiments were designed to show that both long and short forms of the D2 receptor could be amplified from striatal RNA, and that the D2Lm internal control could be amplified as well. 1 /xg of total striatal RNA or 5 pg of in vitro transcribed D2Lm was amplified by RT-PCR (Fig. 2). Concentrations had previously been established such that amplification of the two preparations would generate comparable endpoint signals. Note that the amplification primers yield signals for both D21ong and D2shor t. Moreover, the relative ratio of the two species is 3:1 as quantified by direct measurement of the amplified D N A on a Betagen Betascope. This is in agreement with previous reports of the relative abundance of these two m R N A species [10,15]. As expected, amplification of the D2Lm in vitro transcript produces the same molecular weight band as D21ong (Fig. 2, lane 3; they differ in the identity of only one nucleotide). Digestion of the D2Lm at its unique B a m H I site converts it to two smaller fragments which are well resolved from the D21ong and D2shor t (lane 4). Note that these conditions yield complete fragment digestion. When the endogenous D2 m R N A species are amplified in the presence of the D2Lm standard and then digested with B a m H I , not only can the four species be resolved, but they can be readily quantified (data not shown; see Fig. 3). Amplification of extremely small amounts of D2Lm RNA standard, in the absence of striatal RNA (or carrier RNA) produces slightly higher signals than amplification in the

C y c l e s - ~ 18 D1

(-) RT 18 22 30 D2

D3

I)4

presence of total RNA (data not shown). On the other hand, the presence of the standard D2Lm RNA does not alter the amplification of the endogenous D2 species (see Fig. 3). This latter point is important for several reasons. If the number of amplification cycles becomes large enough, the products can begin to compete for limiting amounts of primer a n d / o r deoxynucleotides. This is the basis of 'competitive PCR' [9,23,27]. Moreover, although very unlikely based on Cot analysis, there is a possibility that the PCR products could reach high enough levels to create mixed hybrids (D21ong/D2Lm) which would not digest with B a m H I (increasing the apparent D2tnng signal). Neither of these possibilities has proven to be a problem as long as the number of cycles is minimized and the internal standard never greatly exceeds the amount of endogenous signal ( < 10-fold excess). Studies were conducted to prove that increasing concentrations of the internal standard did not affect the amplification efficiency of the m R N A of interest. Fig. 3 displays the amplification of an endogenous striatal D2 signal and subsequent quantification using a range of D2Lm internal standard m R N A concentrations. Specifically, 1 /xg of striatal total RNA was amplified in the presence of 1 pg, 2.5 pg, 5.0 pg, and 10 pg of D2Lm m R N A (top panel, lanes 2-5). Following RT-PCR amplification, the products were digested with B a r n H ! to resolve the internal standard from the endogenous signal after PAGE. Clearly, increasing the amount of D2Lm input increases the output signal following quantification (bottom panel). In fact, there is a linear response between input RNA and output signal. The titration of internal standard had no effect on the amplification signal from the unknown striatal m R N A (cf., constant signals for D2~ong and D2shor t in the top panel and the horizontal line in the bottom panel). Finally, the point

22

t8

(+) RT 18 22 30

22

D1

D2

I)6

I:)3

I)4

60S 398 - 344 --

MWM s99-120 ~ . 201 164 •

Fig. 4. Selective amplification of the D1 through 1)5 dopamine receptor subtypes from the same striatal RNA preparation using a single tube RT-PCR procedure. The five DAR subtype-specific products were amplified from 1 /.tg total RNA with the specific primers described in Table 1 using the single tube protocol as detailed in Methods. The amplification products were analyzed via PAGE and autoradiography; the specific subtype amplified is indicated directly above each lane. The first five lanes illustrate the lack of amplification of D1 through D5 signals in the absence of reverse transcriptase ( - RT), indicating that the amplification conditions are RNA-dependent. The last five lanes show the amplification of all five DAR subtypes from rat striatum in the presence of reverse transcriptase ( + RT). The number of amplification cycles performed for each subtype was varied to optimize visualization of the PCR products on the same gel, and is indicated above each lane. Note that the amplification products migrate at the sizes predicted in Table 1. The non-specific labeling at the top of the + RT, D4 lane is a result of the high number of thermocycles necessary to visualize the rare D4 message. These results will have to await quantitative determinations (using internal standard RNAs) before definitive levels and relationships can be established.

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where the line for the endogenous signal intersects the titration line for the D2Lm represents the absolute value for the amount of D2 mRNA which was present in the striatal sample. Based on this experiment, there are approximately 5 pg of D21ong and 1.5 pg of D2short mRNA in 1 /.~g of total striatal RNA. Note that this calculation is only made possible since the standard RNA is full-length in nature. Therefore, the signal intensities can be compared directly without resorting to correction factors for relative lengths and specific activities. If poly(A ÷) mRNA is 2% of total, that means that the D2 receptor mRNA (both long and short variants) represents approximately 0.033% of the striatal messenger RNA. This procedure therefore permits the determination of the absolute amount of D2 receptor mRNA present in a sample. Using the primers shown in Table 1, all five genetic subtypes of the DAR were amplified from striatal RNA. These primers were designed to have the same Tm so that the subtypes could be amplified at the same time under the same conditions (in this case, in different reactions). Fig. 4 shows the selective amplification of the five DAR subtypes from 1 /zg of striatal RNA. Initial experiments (not shown) revealed that there were widely varying amounts of the DAR when equal numbers of thermocycles were employed. Fig. 4 is the result of the empirical selection of varying numbers of cycles. In addition, the presence of contaminating genomic DNA was discounted by control reactions performed in the absence of reverse transcriptase. Preliminary experiments indicated that traditional guanidinium thiocyanate isolation of RNA can produce amplification artifacts generated from genomic DNA (data not shown). Moreover, the DNA contamination does not produce uniform artifactual signals for all of the subtypes.

4. Discussion Quantitative RT-PCR has several significant advantages over traditional in situ hybridization, solution hybridization, Northern blot and RNA dot blot procedures for the analysis of differential gene expression. The advantages include the following. (i) When necessary, this procedure can be absolutely quantitative to yield values of pg mRNA per /.~g total RNA. For instance, it is presently impossible to establish the precise relationships between various DAR subtype mRNAs using Northern and in situ hybridization techniques with different probes. In this regard, the present procedure is similar to solution hybridization; however, it requires much less starting material. (ii) The high sensitivity of the system will permit the analysis of relative gene expression using small amounts ( < 1 /xg) of RNA. As a result, it will be possible to study the DAR subtype mRNAs in preparations from very small tissue samples. Recently, this approach has been applied to the analysis of RNA from individual cells (D.J. Surmeier, S.L. Vrana and K.E. Vrana, unpublished results). (iii) Finally, the proce-

dure is much faster (and more convenient) than in situ hybridization for analyzing and quantifying a large number of samples, and it is more sensitive than Northern blot analysis. Like Northern blots and solution hybridization, this RT-PCR procedure lacks the anatomical resolution of in situ hybridization. However, because of its sensitivity, this approach can be applied to very small amounts of tissue permitting fine anatomical dissections. As discussed previously, RT-PCR suffers from a well-characterized variability. This issue has been addressed through the design and use of a homologous internal standard for a given gene. A number of modifications have been incorporated into traditional quantitative RT-PCR in order to refine the procedure and enhance the output. First, the use of the point mutant internal standard RNA has several major advantages. The use of RNA (as opposed to a DNA standard) corrects for the variable RT step. The fact that the standard is a full-length variant of the endogenous gene product means that the same primers and conditions (i.e., the same reaction) will be employed. The ability to digest the standard following the reaction allows for the discrimination of the standard and endogenous signals so that the amplifications can be conducted in the same tube. Therefore, the standard can be utilized to correct for contaminants present within the experimental sample. Similar approaches have been previously reported in which the standard RNA is a deletion variant of the wild-type gene. Whereas this is an acceptable technique, we prefer the point mutant because it will maintain nearly perfect secondary structure and therefore RT reaction efficiency. However, the point mutant does introduce the need for post hoc restriction digestion of the PCR products. An interesting variant of this technique has recently been reported involving the use of interspecies variants of the same gene (with post hoc restriction analysis) as internal standards [14]. Secondary modifications of standard RTPCR protocols involved the use of low cycle numbers (typically 18 to 22), high annealing temperatures (at or slightly above the primer T m values), and radioactivity incorporation for sensitivity. When combined, these adaptations generate a linear standard curve for the D2Lm RNA. Most competitive PCR procedures require determination of cross-over points for two logarithmic curves or the use of a logarithmic standard curve [9,18,23,27] and so have increased intrinsic variability. In the present procedure, such small amounts of the primers and nucleotides are utilized that the amplification reaction remains within the linear range for a given number of cycles (Fig. 3). Finally, primer pairs have been designed (Table 1) which are capable of differentiating all five receptor subtype mRNAs (Fig. 4). Although internal standards have not yet been created for the other mRNAs (and so absolute comparisons cannot be made), it is obvious that all five subtypes are present within total striatal RNA but are present at differing levels. Not surprisingly, D1 and D2

S.L. Vrana et al. / Molecular Brain Research 34 U995) 127-134

mRNAs are the major species and are predominantly displayed after only 18 amplification cycles in the single tube RT-PCR reaction. In contrast, 22 cycles are required to visualize D3 and D5 and 30 cycles are necessary to produce a minimal D4 signal. Given the differences in signal intensities and the number of amplification cycles, conservative estimates suggest a 103-104-fold difference in abundance between D4 versus D1 or D2 mRNA in the striatum. Internal standards for all of the subtypes will be required to definitively establish quantitative relationships. Whenever low RT-PCR signals are detected, there must be a concern that amplification of a DNA contamination is responsible. This is not an issue in the present case. As demonstrated in Fig. 4, all of the RT-PCR bands are dependent on inclusion of reverse transcriptase (RNA-dependent DNA polymerase). This is true even for the D4 signal which required 30 amplification cycles to be visualized. The primary caveat is that cycle numbers should be minimized because DNA contamination does become apparent under high amplification conditions (data not shown). The routine inclusion of 'minus RT' controls, however, alleviate concerns with genomic DNA contamination. These findings suggest that all five DAR subtype mRNAs are expressed within the striatum. While this is generally accepted for the D1 and D2 genes, there is little definitive data for the other subtypes. It has been reported that the D3 gene expression is very low in striatum [3,21,24,25], a fact which is confirmed in the present study. Recent characterization of D1, D2 and D5 transcription using Northern analysis [13] establishes the presence of D5 mRNA in this tissue. Finally, the present results suggest that D4 mRNA is also present in striatum, albeit at very low levels. While there is some controversy about the expression of D4 in this tissue, the mRNA has previously been reported [28]. Some of the discrepancy may be attributable to the exceedingly rare abundance of this species. It will remain to be seen if this message is actually translated into mature functional receptor protein. In closing, it should be noted that this approach of RT-PCR characterization of mRNA, while it is selective for each subtype, still does not address the levels of functional binding sites. It does, however, permit the characterization of gene expression at the level of mRNA. Overall, quantitative RT-PCR represents a powerful adjunct to the use of traditional Northern blot, RNA dot blot, solution hybridization, and in situ hybridization procedures.

Acknowledgements The authors wish to thank F.J. Monsma (NIH) for use of the D2 dopamine receptor cDNA clone and D.J. Surmeier (University of Tennessee) and S.C. Kumer (Bowman Gray School of Medicine) for valuable scientific

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discussion. This work was supported by USPHS grants DA06634 (K.E.V.), T32DA-07246 (S.L.V.) and DA00230 (S.L.V.).

References [1] Babu, J.S., Kanangat, S. and Rouse, B.T., Limitations and modifications of quantitative polymerase chain reaction, J. Immunol. Methods, 165 (1993) 207-216. [2] Ballagi-Pordany, A., Ballagi-Pordany, A. and Funa, K., Quantitative determination of mRNA phenotypes by the polymerase chain reaction, Anal. Biochem., 196 (1991) 89-94. [3] Bouthenet, M.-L., Souil, E., Martres, M.P., Sokoloff, P., Giros, B. and Schwartz, J.-C., Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: Comparison with dopamine D2 receptor mRNA, Brain Res., 564 (1991) 203-219. [4] Briggs, J.P., Todd-Turla, K., Schnermann, J.B. and Killen, P.D., Approach to the molecular basis of nephron heterogeneity: Application of reverse transcripfion-polymerase chain reaction to dissected tubule segments, Semin. Nephrol., 13 (1993) 2-12. [5] Bunzow, J.R., VanTol, H.H.M., Grandy, D.K., Albert, P., Salon, J., Christie, M., Machida, C.A., Neve, K.A. and Civelli, O., Cloning and expression of a D2 rat dopamine receptor cDNA, Nature, 336 (1988) 783-787. [6] Chomczynski P., A reagent for the single step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples, BioTechniques, 15 (1993) 532-535. [7] Dal Toso, R., Sommer, B., Ewert, M., Herb, A., Prichctt, D., Bach, A., Shivers, B. and Seeburg, P.H., The dopamine D2 receptor: Two molecular forms generated by alternative splicing, EMBO J., 8 (1989) 4025-4034. [8] Gingrich, J. and Caron, M.G., Recent advances in the molecular biology of dopamine receptors, Annu. Rev. Neurosci., 16 (1993) 299-321. [9] Gilliland, G., Pertin, S., Blanchard, K. and Bunn, H.F., Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction, Proc. Natl. Acad. Sci. USA, 87 (1990) 2725-2729. [10] Giros, B., Sokoloff, P., Martres, M.P., Riou, J.F., Emorine, L.J. and Schwartz, J.C., Alternative splicing directs the expression of the two D 2 dopamine receptor isoforms, Nature, 342 (1989) 923-926. [11] Hemsley, A., Anaheim, N., Toney, M.D., Cortopassi, G. and Galas, D.J., A simple method for site-directed mutagenesis using the polymerase chain reaction, Nucleic Acids Res., 17 (1989) 65456551. [12] Itakura, K., Rossi, J.J. and Wallace, R.B., Synthesis and use of synthetic oligonucleotides, Annu. Rev. Biochem., 53 (1984) 323356. [13] Laurier, L.G., O'Dowd, B.F. and George, S.R., Heterogeneous tissue-specific transcription of dopamine receptor subtype messenger RNA in rat brain, Mol. Brain Res., 25 (1994) 344-350. [14] Levesque, G., Lamarche, B., Murthy, M.R., Julien, P., Despres, J.-P. and Deshaies, Y., Determination of changes in specific gene expression by reverse transcription PCR using interspecies mRNA as internal standards, BioTechniques, 17 (1994) 738-741. [15] Monsma, F.J. Jr., McVittie, L.D., Gerfen, C.R., Mahan, L.C. and Sibley, D.R., Multiple D 2 dopamine receptors produced by alternative splicing, Nature, 342 (1989) 926-929. [16] Mueller, P.R., Garrity, P.A. and Wold, B., Ligation-mediated PCR for genomic sequencing and footprinting. In: F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl (Eds.), Current Protocols in Molecular Biology, Vol. 2, John Wiley and Sons, New York, 1994, pp. 15.5.21-15.5.22.

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S.L. Vrana et aL /Molecular Brain Research 34 (1995) 127-134

[17] O'Malley, K.L., Mack, K., Gandelman, K. and Todd, R.D., Organization and expression of the rat D2a receptor gene: Identification of alternative transcripts and a variety donor splice site, Biochemistry, 29 (1990) 1367-1371. [18] Riedy, M.C., Timm, E.A.,Jr. and Stewart, C.C., Quantitative RT-PCR for measuring gene expression, BioTechniques, 18 (1995) 70-76. [19] Robinson, M.O. and Simon, M.I., Determining transcript number using the polymerase chain reaction: Pgk-2, mP2, and PGK-2 transgene mRNA levels during spermatogenesis, Nucleic Acids Res., 19 (1991) 1557-1562. [20] Sambrook, J., Fritsch, E. and Maniatis, T. (Eds.), Molecular Cloning: A Laboratory Manual, 2nd end., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [21] Schwartz, J.-C., Giros, B., Martres, M.-P. and Sokoloff, P., The dopamine receptor family: Molecular biology and pharmacology, Semin. Neurosci., 4 (1992) 99-108. [22] Seeman, P., Dopamine receptor sequences: Therapeutic levels of neuroleptics occupy D2 receptors, clozapine occupies D4, Neuropsychopharmacology, 7 (1992) 261-283. [23] Siebert, P.D. and Larrick, J.W., Competitive PCR, Nature, 359 (1992) 557-560. [24] Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L. and Schwartz, J.-C., Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics, Nature, 350 (1990) 610-614.

[25] Sokoloff, P., Martres, M.-P., Giros, B., Bouthenet, M.-L. and Schwartz, J.-C., The third dopamine receptor (D3) as a novel target for antipsychotics, Biochem. Pharmacol., 43 (1992) 659-665. [26] Tiberi, M., Jarvie, K.R., Silvia, C., Falardeau, P., Gingrich, J., Godinot, N., Bertrand, L., Yang-Feng, T., Fremeau, R.T. and Caron, M.G., Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D l dopamine receptor subtype: Differential expression pattern in rat brain compared with the DIA receptor, Proc. Natl. Acad. Sci. USA, 88 (1991) 7491-7491. [27] Todd-Turla, K.M., Schnermann, J., Fejes-Toth, G., Naray-FejesToth, A., Smart, A., Killen, P.D. and Briggs, J.P., Distribution of mineralocorticoid and glucocorticoid receptor mRNA along the nephron, Am. J. Physiol., 264 (1993) F781-91. [28] Van Tol, H.H., Bunzow, J.R., Guan, H.C., Sunahara, R.K., Seeman, P., Niznik, H.B. and Civelli, O., Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine, Nature, 350 (1991) 610-614. [29] Wadhwani, K.C., Fukuyama, R., Giordano, T., Rapoport, S. and Chandrasekaran, K., Quantitative reverse-transcriptase-polymerase chain reaction of glucose transporter 1 mRNA levels in rat brain microvessels, Anal, Biochem., 215 (1993) 134-141. [30] Wang, A.M., Doyle, M.V. and Mark, D.F., Quantitation of mRNA by the polymerase chain reaction, Proc. Natl. Acad. Sci. USA, 86 (1989) 9717-9721.