Semi-quantitative reverse-transcriptase polymerase chain reaction: an approach for the measurement of target gene expression in human brain

Brain Research Protocols 4 Ž1999. 132–139 www.elsevier.comrlocaterbres

Protocol

Semi-quantitative reverse-transcriptase polymerase chain reaction: an approach for the measurement of target gene expression in human brain Li Chen a , David M. Segal b , Deborah C. Mash

a,c,)

a

Department of Neurology, UniÕersity of Miami School of Medicine, 1501 NW 9th AÕenue, Miami, FL 33136, USA Department of Psychiatry and BehaÕioral Sciences, UniÕersity of Miami School of Medicine, Miami, FL 33136, USA Department of Molecular and Cellular Pharmacology, UniÕersity of Miami School of Medicine, Miami, FL 33136, USA

b c

Accepted 5 February 1999

Abstract Polymerase chain reaction ŽPCR. is a very powerful tool for qualitative evaluation of nucleic acids due to its high efficiency and convenience. Together with the reverse transcription ŽRT. reaction, the PCR method has been widely applied to the quantitative measurement of DNA and RNA messages. Since RT-PCR is much more sensitive than all of the traditional methods for quantification of mRNA, including Northern blot, ribonuclease protection, RNA blot, and solution hybridization assays, it is the method of choice for quantitative analyses of low abundance mRNA messages. However, because of the exponential nature of the PCR amplification, RT-PCR quantitation may be problematic, giving false estimates of the abundance of the target messages. By using the constitutively expressed ‘housekeeping’ gene cyclophilin as a reference gene to normalize mRNA levels, and by taking data from the exponential phase of the PCR amplification, we have developed a rapid and reliable semi-quantitative measurement of the relative abundance of dopamine transporter ŽDAT. mRNA. The semi-quantitative PCR method has been applied to illustrate its use for the measurement of DAT mRNA in post mortem human brain. q 1999 Elsevier Science B.V. All rights reserved. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Signal transduction: gene expression Keywords: Polymerase chain reaction ŽPCR.; Reverse transcription ŽRT.; Dopamine transporter ŽDAT.; Human brain

1. Type of research The present method is suitable for the following studies: Ø Semi-quantitation of low abundance DNA or mRNA messages. Ø Transcriptional regulation of target gene expression.

2. Time required Ø RNA extraction: 3 h. Ø RT-PCR reaction: 5 h.

) Corresponding author. Department of Neurology, University of Miami School of Medicine, 1501 NW 9th Avenue, Miami, FL 33136, USA. Fax: q1-305-243-3649; E-mail: [email protected]

Ø Polyacrylamiderurea gel electrophoresis: 2 h. Ø Synthesis of radioactive molecular marker: 3 h. Ø Detection of radioactive PCR products on PhosphoImager: 5–7 days. Ø Densitometric analysis: 1 h.

3. Materials 3.1. Special equipment Thermolyne Amplitron II was from BarnsteadrThermolyne ŽDubuque, IA, USA.; Spectronic 1001 plus spectrophotometer was from Omilton Roy ŽRochester, NY, USA.; LKB 1211 Rackbeta liquid scintillation counter was from Wallac ŽGaithersburg, MD, USA.; Brinkmann homogenizer PT 10r35 and eppendorf centrifuge 5415 were from

1385-299Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 9 . 0 0 0 0 9 - 4

L. Chen et al.r Brain Research Protocols 4 (1999) 132–139

Brinkmann Instruments ŽWestbury, NY, USA.; Sorvall superspeed RC 2-B centrifuge was from Dupont Instruments ŽNewtown, CT, USA.; Electrophoresis apparatus was from BioRad ŽHercules, CA, USA.; Nitrocellulose filters GFrC was from Whatman Intl. ŽMaidstone, UK.; PhosphoImagere:SF Žmodel 455. and ImageQuante Žsoftware version 3.3. were from Molecular Dynamics ŽSunnyvale, CA, USA.. 3.2. Chemicals and reagents Superscript II preamplification system for first strand cDNA synthesis, oligonucleotide primers, Taq DNA polymerase, dNTPs, T7 RNA polymerase, RNasin, RNase free RQ1 DNase were from Gibco BRL Life Technologies; RNA century marker plus template was from Ambion ŽAustin, TX, USA.; w a-32 Px-dCTP Ž3000 Cirmmol, 10 mCirml. was from Dupont NEN ŽBoston, MA, USA.; sodium lauryl sarcosinate, guanidinium thiocyanate, phenol, isoamyl alcohol, chloroform, formamide, xylene cyanol, sodium pyrophosphate were from Sigma ŽSt. Louis, MO, USA.. 3.3. Brain tissue preparation Post-mortem human brain specimens were obtained at routine autopsy from drug-free, age-matched control subjects Ž N s 8; 32.1 " 2.8 years; six males; two females.. The brain was photographed and cut into 1.5 cm coronal slabs. We fabricated a plexiglass holder with guide plates at 1 cm intervals to facilitate whole-brain sectioning. The anterior and posterior aspects of each block were photographed to record sulcal patterns and structural landmarks. The coronal blocks were rapidly frozen in 2-methylbutane on dry ice at y308C and were subsequently stored at y708C. The post mortem interval ranged between 8 to 27 h Žaverage 15.8 " 2.3, mean " S.E... The substantia nigra was dissected from cryopreserved coronal block specimens taken at a level through the posterior commisure, the medial and lateral geniculate bodies, and the red nucleus. A series of three small lateral to medial punches Ž200 mg wet weight. were pooled from the pars compacta of the A9 dopaminergic cell group.

4. Detailed procedure The semi-quantitative RT-PCR approach shown here is modified from the common co-amplification method, which was first developed by Chelly et al. w5x. The method shown here uses separate amplification of a marker gene along with the target of interest. The constitutively expressed housekeeping gene cyclophilin was used to normalize the level of DAT mRNA. This approach afforded a direct

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comparison of DAT mRNA levels in reference to cyclophilin expression to be made across individual control subjects. 4.1. Total RNA extraction from human brain tissue Total RNA was prepared from cryopreserved human brain specimen w7,20x. Using oligoŽdT.12 – 18 primers, mRNAs in 5 mg of total RNA was specifically reverse transcribed and later amplified for studying the relative mRNA levels in different samples. To ensure the lack of contamination of the RNA preparation with genomic DNA, it is recommended to pretreat the RNA samples with RNase-free DNase I. The detailed procedure involves the following steps. Ža. Weigh the frozen brain tissue punches and prepare the homogenization solution, which contained 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% Žvrv. sarcosyl and 0.72% Žvrv. b-mercaptoethanol. Žb. Add 10 volumes Žml. of the homogenization solution Žvrv. to the brain tissue according to the weight of the brain sample Žmg. and homogenize with a Brinkmann polytron homogenizer for 15 s at a setting of 2.5. Žc. Mix the solution thoroughly by inverting the tube 10 times after the addition of each of the following reagents: 0.1 volume Žof the tissue weight. of 3 M sodium acetate, pH 5.2, 1 volume Žof the wet brain tissue weight. of water saturated phenol, and 0.2 volume of chloroform:isoamyl alcohol Ž49:1. mixture. Vortex the mix vigorously for 10 s and chill on ice for 15 min. Žd. Separate the organic and aqueous phases by centrifugation ŽSorvall HS-4 rotor, 5.75 cm radius. at 6500 rpm at 48C for 20 min. Transfer the RNA-containing aqueous phase carefully into a fresh centrifuge tube, and precipitate the RNA by addition of an equal volume of cold isopropanol. Store sample at y808C for at least 1 h and then centrifuge ŽSorvall SS-34 rotor. at 15,000 rpm, 48C for 20 min to pellet RNA. Že. Aspirate the supernatant and dissolve the RNA pellet in 0.15–0.2 ml of the homogenization solution, and transfer it to a 1.5 ml Eppendorf tube. Precipitate RNA again by the addition of 2.5 volumes Žvrv. of cold ethanol and chill at y808C for at least 1 h. Centrifuge the solution for 20 min at 48C in a Brinkmann eppendorf centrifuge Žmodel 5415. at maximum speed Ž14,000 = g . and wash the pellet with 1 ml of cold 75% ethanol. Žf. Briefly dry the pellet and dissolve in 10 ml of Tris EDTA buffer Ž10 mM Tris, pH 8.0, 1 mM EDTA., followed by addition of 1 ml of RNase-free RQ1 DNase for digestion at 37FC for 1 h. Add 40 ml of Tris EDTA buffer and extract with equal volume of phenol:chloroform:isoamyl alcohol Ž25:24:1. twice. Precipitate RNA by adding 5 ml of 3 M ammonium acetate ŽpH 5.2., 125 ml of ethanol and store at y808C for 1 h. Žg. Centrifuge for 10 min at the speed of 14,000 = g in a microcentrifuge, followed by one wash with 1 ml of 70%

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ethanol. Briefly dry the RNA pellet under vacuum. Resuspend the pellet in 20–30 ml of Tris–EDTA ŽpH 8.0., and quantitation by absorbance Ž A. reading at 260 nm and 280 nm. The final preparation should be free of protein with the A 260rA 280 ratios across runs at 1.8 or higher. 4.2. First strand cDNA synthesis The first strand synthesis was accomplished by using the Superscript II Preamplification System for First Strand cDNA Synthesis kit from Gibco BRL. The cDNA protocol includes the following steps. Ža. Add 1 ml of oligo dT12 – 18 primer Ž100 pmol. to 5 mg of total RNA sample and make the final volume to 12 ml by addition of diethylpyrocarbonate ŽDEPC. treated H 2 O. Incubate at 708C for 10 min, followed by chill on ice for at least 1 min. Žb. Prepare a master reaction mix according to the total number of reactions Ž n., including one no-RT reaction and one control RT reaction Žuse a known RNA template provided in the kit.. Therefore, the mix contains n = 2 ml of 10 = PCR buffer, n = 2 ml of 25 mM MgCl 2 , n = 1 ml of deoxynucleoside triphosphate ŽdNTP. mix and n = 2 ml of 0.1 M dithiothreitol ŽDTT.. Add 7 ml of the above mixture to each sample and mix the solution well, followed by a brief centrifugation step. Žc. Incubation of the above solution for 5 min at 428C, and then add 1 ml of superscript II Reverse Transcriptase to each tube Žfor the no RT control, substitute 1 ml of DEPC-treated H 2 O.. Incubate for 50 min at 428C and then terminate the reaction by incubation at 708C for 15 min, followed by chilling on ice. Briefly centrifuge and then add 1 ml of RNase H to remove the residual RNAs and incubate for 20 min at 378C. Store the samples at y208C.

subjects. DAT and cyclophilin primers were created using PrimerDesign Žsoftware version 1.10, Marburg, Germany. with similar GC content Ž50–60%. and melting temperature ŽTm 55–608C.. The specificity of the primers was later confirmed by southern hybridization with an in vitro synthesized radiolabeled human DAT or cyclophilin probe for the amplified fragments, respectively. These primers do not span any intron regions since they anneal to the cDNAs that were reverse transcribed from mRNAs. The primers were synthesized by Gibco BRL Life Technologies according to manufacturer’s instructions ŽTable 1.. For the amplification of DAT and cyclophilin mRNAs, the steps are as follows. Ža. Serial dilutions were prepared for each cDNA sample obtained from the first strand cDNA synthesis, i.e., two-fold dilution Ž2 = ., 5 = , 10 = , 20 = , 50 = , 100 = and 200 = . For PCR standard, cDNA was pooled from at least three human brain samples for PCR amplification. Žb. Preparation of the master PCR reaction mix Žfor 50 PCR reactions. was follows: 3.6 ml of DEPC H 2 O, 500 ml of 10 = PCR buffer, 300 ml of 25 mM MgCl 2 , 100 ml of 10 mM dNTPs, 100 ml of each 5 mM DAT primer Žor 50 ml of each 5 mM cyclophilin primer., 50 ml of 1:4 diluted w a-32 Px-dCTP. Aliquot 98 ml of the master mix into each tube and add 2 ml of cDNA sample either from the original first strand cDNA synthesis reaction or from the respective serial dilutions. Heat at 948C for 5 min and then add 0.5 ml of Taq DNA polymerase Ž2.5 unit. into each tube. Žc. PCR amplification was programmed as follows: 948C for 30 s, 608C for 1 min and 728C for 2 min. After 30 cycles for DAT amplification and 25 cycles for cyclophilin, PCR reactions were further incubated at 728C for 10 min and then chilled at 48C. 4.4. Preparation of a radioactiÕe marker for gel electrophoresis

4.3. Polymerase chain reaction The protocol described here is provided as an example of a general method and uses the constitutively expressed cyclophilin mRNA as an internal control of the semiquantitative PCR to measure DAT mRNA in human substantia nigra that was regionally dissected from post mortem brains of drug-free and age-matched male and female

A radioactive marker was synthesized in vitro using Century Marker Template Plus ŽAmbion. as described by the following steps: Ža. Mix 6.9 ml of DEPC H 2 O, 0.5 ml of century marker, 1.6 ml of 100 mM DTT, 4 ml of 2.5 mM

Table 1 Primers for RT-PCR Amplication Target Gene DAT Cyclophilin a

Primer Sequencea

Gene position

X

X

5 -TCCGGCTTCGTCGTCTTCTC-3 X X 5 -GATGTCGTCGCTGAACTGCC-3 X X 5 -TCCTAAAGCATACGGGTCCTGGCAT-3 X X 5 -CGCTCCATGGCCTCCACAATATTCA-3 X

nta 1078–1524

Amplicon b

446 bp

nta 266–431c

165 bp X

The top primers of each primer pair are the sense 5 primers and the bottom primers of each primer pair are the antisense 3 primers. Ref. w9x, Giros et al. Ž1992.. c Ref. w10x, Haendler et al. Ž1987.. b

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A,G,UTP, 1 ml of 0.4 mM CTP, 4 ml of w a-32 Sx-CTP Žvacuum dried., 4 ml of 5 = reaction buffer Žfrom Promega., 1 ml of RNasin, and 1 ml of T7 RNA polymerase and incubate at 378C for 1 h. Then, add 1 ml of RQ1 DNase to the mix and incubate at 378C for an additional 15 min. Finally, add 80 ml of Tris–EDTA to bring the volume up to 100 ml, followed by extraction with 100 ml of phenol:chloroform:isoamyl alcohol Ž25:24:1.. Žb. Precipitate the aqueous layer with 1 ml of 25 mgrml yeast tRNA, 20 ml of 3 M ammonium acetate and 300 ml of ice-cold ethanol for 1 h or more at y808C. Pellet the RNA and dissolve in 100 ml of Tris–EDTA and repeat the above phenol extraction and ammonium acetate precipitation steps. Clean the pellet with 1 ml of 75% ethanol once and resuspend the final pellet in 20 ml of Tris–EDTA. Žc. Take 1 ml of the above product to dilute 50 fold and spot on GFrC filters, followed by two washes with ice-cold 20 mM sodium pyrophosphate, 5% TCA solution Ž5 ml per filter., and one wash with 95% ethanol Ž5 ml per filter.. Measure the radioactivity of the air-dried filters with scintillation spectrometry to calculate the specific activity of the marker Žcpmrml of stock. and use 10 5 cpm for each marker lane in polyacrylamiderurea gel electrophoresis to obtain the molecular size of the amplified fragments, DAT and cyclophilin. 4.5. Polyacrylamider urea gel electrophoresis and densitometry

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subjects. In this study, the internal control is the mRNA of the housekeeping gene cyclophilin. Total RNA was extracted from human post mortem substantia nigra by the single-step method developed by Chomczynski and Sacci w7x. Oligo ŽdT.12 – 18 primer hybridized to the 3X poly ŽA. tails which are found in the vast majority of eukaryotic mRNAs. First strand cDNA reaction was carried out by using Superscript II reverse-transcriptase Žfrom Gibco BRL. to catalyze the synthesis of full-length cDNA from mRNArprimer mixture. After the first strand cDNA synthesis was complete, all RNAs, including RNA in the cDNA:RNA hybrid, were removed by digestion with RNase H in order to ensure the specificity of the PCR amplification from target cDNA. During PCR amplification, each pair of primers Žfor either DAT or cyclophilin. specifically annealed to their target gene. Using the free dNTPs and the radioactive 32 P-dCTP that were provided in the reaction, the fragment between these two primers was then synthesized according to the template sequence and amplified exponentially after a number of PCR cycles Ž30 cycles for DAT and 25 cycles for cyclophilin.. Thus, the amplified fragment provided a strong enough signal on polyacrylamiderurea gel for visualization. A schematic RT-PCR protocol is shown in Fig. 1. Three cases were randomly chosen to determine the range of linearity of the DNA amplification. The amplification of the serial dilutions from this pooled sample of post mortem human brain gave the standard curve, which showed the linear range at the exponential phase of the

Separation of the PCR products and densitometry were performed as follows: Ža. Take 40 ml from the 100 ml PCR reaction product into a 1.5 ml eppendorf tube and add 1 ml of 20 mgrml glycogen, 5 ml of sodium acetate ŽpH 5.2., 100 ml of ethanol and precipitate at 808C for at least 1 h. Žb. Centrifuge in the microcentrifuge at the maximum speed for 15 min to pellet the DNA and dissolve the pellet in 10 ml of gel loading buffer Ž80% formamide, 0.1% xylene cyanol, 0.1% Bromophenol blue and 2 mM EDTA, pH 8.0., followed by heat denaturation at 908C for 5 mins and then chill on ice. Žc. Load the samples on 5% polyacrylamider8 M urea gel for separation of the PCR products. Fix the gel in 10% acetic acid, 30% methanol for 30 min and vacuum dry the gel for 1 h. Expose the gel to the PhosphoImager for 5–7 days and perform densitometric analysis by the ImageQuant program.

5. Results The semi-quantitative RT-PCR was used to assess DAT mRNA content in human substantia nigra from control

Fig. 1. Reverse-transcriptase polymerase chain reaction ŽRT-PCR.. Target mRNA was reverse transcribed to the first strand cDNA by Suprescript II X reverse-transcriptase ŽRT., using oligo ŽdT. to hybridize to the 3 poly ŽA. tail. Template mRNA was removed by RNase H. The first strand cDNA was then amplified by Taq DNA polymerase with a pair of gene-specific primers.

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malized mRNA levels were reported as cDNArmarker ratios. Because of the inherent variability in human brain due to differences in age, race, gender, and agonal state, DAT mRNA levels may show up to 10-fold differences within the same experimental group. This inherent variability within a group sample is an important determinant for designing a protocol to study DAT mRNA regulation in neuropsychiatric diseases or by exposure to drugs of abuse Že.g., psychostimulants or opiates.. However, the relative amounts of DAT mRNA measured across control subjects with different autolysis times and terminal events were found to be very representative within the chosen age-range with the normalized ratios of DATrcyclophilin demonstrating minimal intra-assay and inter-assay variability Ždata not shown.. Since the amplified fragments contained radioactive nucleotide Ž32 P-dCTP., the dried polyacrylamiderurea gel was viewed on a PhosphoImager, which uses a phosphorescent screen to detect the b particles emitted by the radiolabeled PCR products in the gel. The image of the radioactive bands was developed by scanning the phospho-

Fig. 2. Semi-quantitation of DAT RT-PCR products. ŽA. A representative PCR standard curve showing the linear amplification rate at the exponential phase and the saturation phase. The x axis represents the concentrations of the initial template expressed as a percentage Ž%. of the original template obtained from RT reaction. ŽB. Densitometric analysis of a representative PCR standard obtained PhosphoImager. The concentrations of the initial template were shown by x-fold dilutions of the template. ŽC. Three representative samples whose PCR products ŽDAT. were within the linear range of the PCR standard curve.

PCR amplification and the PCR reaction was approaching the saturation phase at higher initial amounts of the template ŽFigs. 2A and 3A.. A preliminary experiment was done to determine the appropriate cycle numbers for DAT and cyclophilin amplifications Ždata not shown.. Thirty Ž30. cycles of PCR reaction for DAT and 25 cycles for cylophilin were within the exponential phase of their amplifications Ždata not shown.. For PCR amplification, 10% of the RT product Žabout 25 ng. was recommended by the manufacturer of the first strand cDNA synthesis kit, as larger amounts of cDNA may decrease the amount of product synthesis. Several dilutions for each target cDNA sample were also chosen for PCR amplification so that at least three runs were obtained for each individual brain sample within the linear range. Representative data for individual cases were shown in Figs. 2C and 3C. Two DAT primers chosen by the PrimerDesign Žversion 1.10. program annealed to the transmembrane segments 7 and 11, respectively and amplified a 446 bp DAT fragment ŽTable 1, w9x.. A constitutively expressed human brain ‘housekeeping’ gene, cyclophilin, was chosen as the marker gene to normalize the DAT mRNA levels w10x. The nor-

Fig. 3. Semi-quantitation of cyclophilin RT-PCR products. ŽA. A representative PCR standard curve for the housekeeping gene cyclophilin expressed as in the legend for Fig. 2. ŽB. Densitometric analysis of a representative PCR standard obtained from the PhosphoImager. ŽC. Three representative samples whose PCR products Žcyclophilin. were within the linear range of the PCR standard curve.

L. Chen et al.r Brain Research Protocols 4 (1999) 132–139

rescent screen and the radioactive signal was quantified by the ImageQuant program. This imaging system provided a fast, convenient and digitized method for visualizing 32 Plabeled products.

6. Discussion 6.1. Troubleshooting The semi-quantitative PCR provides a rapid method to estimate the relative amounts of message in RNA populations using a known housekeeping gene as an internal standard to normalize the expression levels of the target gene of interest. We have developed a rapid and reliable method for semi-quantitation of the relative abundance of DAT mRNA in human brain post mortem, using the housekeeping gene cyclophilin as the chosen internal control. Because of the low copy number and high inherent variability of DAT mRNA in human brain, it is very difficult to quantitate the DAT mRNA levels. In situ hybridization has been the only method reported to date to assay DAT mRNAs present in human brain w2,11x. However, this method is known to be less sensitive and less accurate than RT-PCR for detecting low abundance targets. In addition, in situ hybridization is more time-consuming and requires more brain tissue than the RT-PCR method. The semi-quantitative RT-PCR method has been applied previously for relative estimates of DAT mRNA levels in rats w16x. This report describes the first application of the semi-quantitative RT-PCR method for measures of DAT mRNAs in post mortem human brain. Because the ratio of the amplified products must reflect the initial ratio of the two messages in the starting material, the semi-quantitative PCR methodology requires a prior determination of PCR conditions ŽcDNA concentration and number of cycles. to ensure that the data are taken from the exponential phase of the amplification of target gene and internal control template. During the exponential phase of the PCR reaction, the amplification rate remains constant because there is a sufficient amount of primers and no inhibitory PCR reaction by-products. Under these conditions, the ratio of the amplified products between any two samples represents the RNA messages in the starting materials. After the exponential phase, the amplification rate decreases dramatically as the PCR reaction reaches the saturation phase. Thus, it is crucial to ensure that the PCR process is kept within the exponential phase of the amplification. To ensure linear reproducibility, a standard curve was included in each batch run of the PCR amplification. The mRNA for the standard curve was pooled from a representative number of human brain cases in order to control for differences in the DAT mRNA levels across subjects. The linear range of the PCR reaction was defined by the PCR products from the serial dilutions of the

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standards in every batch run of the PCR amplification. The initial concentrations of the starting materials and the cycle numbers also must be optimized in order to keep the PCR reaction within the exponential phase to obtain a reliable quantitative evaluation. We determined that 30 cycles for DAT and 25 cycles for cyclophilin was optimal for PCR amplification and densitometric analysis. Serial dilutions of each sample after completion of the RT reaction afforded determination of the optimal template concentrations for each individual sample. The serial dilution approach also saved RT product. The limited product from the first strand cDNA synthesis can be used for multiple assays of different target genes of interest. This approach is especially suited for human brain studies from neuropsychiatric diseases and age-matched control cases, since well-characterized post mortem brain specimens are often very difficult to obtain in large numbers. Before the DAT mRNA levels are compared across individual cases for studies of gene regulation, we have found that it is necessary to normalize the DAT mRNA expression for each case by using an internal standard. However, as we are interested in the relative DAT mRNA levels among different biological samples, it was not necessary to design a synthetic standard to obtain the absolute quantity of mRNA in each brain sample. We have used cyclophilin, a housekeeping gene that is constitutively expressed in neurons, as the internal reference control to measure the relative abundance of the DAT mRNA target gene. The ratio between the DAT mRNA and cyclophilin affords a normalized measure of DAT expression in each case. The DATrcyclophilin ratios were compared across different human brain cases in the same batch to obtain the relative abundance of DAT message for each representative subject. The strategy is illustrated by the following equations: Y s Y0 Ž 1 q E .

n

E is efficiency, n is cycle number, Y is the yield of PCR reaction and Y0 is the initial amount of the amplicon. If efficiencys 100%, then, Y s Y0 Ž2. n. In practical PCR reaction, efficiency is usually less than 100%. If A is target gene and B is the control gene, then A s Ž 1 q Ea . B s Ž 1 q Eb .

na nb

A0 B0

For case No. 1, A1 s Ž 1 q Ea1 . B1 s Ž 1 q Eb1 .

na nb

A 01 B01

For case No. 2, A 2 s Ž 1 q Ea2 . B2 s Ž 1 q Eb 2 .

na nb

A 02 B02

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To compare the relative abundance of the target gene between case 1 and 2, A1

na

Ž 1 q Ea1 . A 01 nb

B1 Ž 1 q Eb1 . B01 s na A2 Ž 1 q Ea2 . A 02 nb B2 Ž 1 q Eb . B 2

02

Since Ea1 s Ea2 s Ea3 s . . . . . . s Ea n and Eb1 s Eb 2 s Eb 3 s . . . . . . s Ebn Then A1

A 01

B1 B01 s A2 A 02 B2

B02

Therefore, the comparison of the DATrcyclophilin ratios across brain cases represents the initial difference of the relative abundance of the target gene across subjects. 6.2. Comparison with alternatiÕe approaches An alternative strategy for measuring mRNA is to co-amplify the target gene with the marker in the same reaction. Although co-amplification of the target gene with the marker gene is easier to perform and it can eliminate some intra-assay variability, it has been reported that coamplification often may cause interference with the amplification of either the target or internal marker or both genes, due to the large difference in the relative abundance, sequence and structure w4,8x. Large differences in levels of mRNA of the target and the marker genes often results in competition for amplification materials between the two templates. Moreover, the marker gene, which is usually more abundant than the target gene, will reach the plateau phase much faster than the target gene. Thus, it may prove difficult to co-amplify both genes and keep the amplification processes within the exponential phase. If this is the case, the targetrmarker ratios obtained from co-amplification will not represent the real difference between the target and the housekeeping genes in the original samples, yielding inaccurate measures of relative abundance of target mRNA among different cases. Our initial attempts of co-amplification of DAT with either actin or cyclophilin gave extra bands on gel electrophoresis in addition to the desired PCR fragments, suggesting there was interference andror competition during PCR amplification of the target and the marker genes. In contrast, separate amplification of the target and the marker genes avoided this problem and was found to be more reliable than the single tube co-amplification.

A number of RT-PCR methods have been developed recently for quantitative evaluation of DNA or RNA levels w1,3,8,12,15,17–19,21,22,24,25x. Although some methods provide quite accurate measurement of the absolute quantity of the nucleotides, they are more complicated and time-consuming as compared with the method reported here for measurements in human brain. The most popular quantitative RT-PCR method, competitive PCR w21,22x, requires a synthetic control sequence which uses the same primers as those for the target gene. In this method, the PCR product from the marker gene Žcompetitor. has to be distinguished from the PCR product of the target gene by gel electrophoresis. Therefore, the competitor is usually designed to have one of the following characteristics: Ž1. a restriction site is added or deleted by site-directed mutagenesis, or Ž2. a short intron is contained in the sequence to generate a genomic DNA PCR product, or Ž3. a non-homologous DNA fragment of the desired size is engineered to use the primers for the template gene. The accurate amount of the competitor has to be determined before it is added to the PCR reaction with the target gene. The amount of competitor DNA yielding equal molar amount of PCR product to that of the target gene gives the initial amount of the target nucleotides. Since all competitive PCR methods require that the target gene and the competitor to be amplified with equal efficiency during the coamplification, it is better to have the synthetic cDNA standard highly homologous to the target sequence so that both amplicons will have the same or very close amplification efficiency w17x. However, high homology between the two sequences may be problematic due to the formation of heterodimers between the native and the mutated amplified fragments during the later amplification cycles w19x. Moreover, the presence of introns in the genomic sequence also may affect quantitation, because of the differences in the amplification efficiencies of genomic and complementary DNAs. Finally, with competitive PCR, the competitor is added only at the PCR stage, without monitoring of the efficiency of the RT process w21x. In the semi-quantitative PCR method shown here, the housekeeping gene is reverse transcribed with the target gene at the same time as the total mRNA extract, ensuring that the marker gene and the target gene have the same RT efficiency. Taken together, the present semi-quantitative RT-PCR method is less problematic and more reliable than competitive PCR, although the absolute quantity of the messages cannot be obtained from semi-quantitation. The results shown here illustrate the reliability and convenience of the semi-quantitative method for measures of mRNA levels among different human brain samples. This method has proven useful for studying the regulation of a target gene in human brain from drug abusers w6x. It has a number of advantages over other RT-PCR methods. First, the reference gene is reverse transcribed together with the target gene. Therefore, the RT efficiency is the same for both the target and the marker gene. Second, the

L. Chen et al.r Brain Research Protocols 4 (1999) 132–139

reference gene is amplified separately from the target gene so that the inherent problems caused by competitive PCR amplification are avoided. Third, no artificial gene is needed to obtain the absolute quantity of mRNA message. Fourth, polyacrylamiderurea gel electrophoresis of radioactive-labeled samples was used to detect the PCR products, which is more sensitive than the agarose gel electrophoresis method. Fifth, the semi-quantitative PCR method is more suitable for comparative studies of DAT gene expression across a large number of brain samples.

w6x

w7x

w8x

w9x

7. Quick procedure U U U

U

Extraction of total RNA from human brain tissue First strand cDNA synthesis PCR amplification and semi-quantitation by densitometry Synthesis of radioactive molecular marker for gel electrophoresis

w10x w11x

w12x

8. Essential literature references

w15x

Refs. w1,4,5,17–19,21,22,24x.

w16x

Acknowledgements

w17x

Supported by a grant from US PHS NIDA ŽDA06227.. w18x

References w1x J. Babu, S. Kanangat, B. Rouse, Limitations and modifications of quantitative polymerase chain reaction Application to measurement of multiple mRNAs present in small amounts of sample RNA, J. Immunol. Methods 165 Ž1993. 207–216. w2x M.J. Bannon, C.J. Whitty, Age-related and regional differences in dopamine transporter mRNA expression in human midbrain, Neurology 48 Ž1997. 969–977. w3x M. Becker-Andre, ´ K. Hahlbrock, Absolute mRNA quantification using the polymerase chain reaction ŽPCR.. A novel approach by a PCR aided transcript titration assay ŽPATTY., Nucl. Acids Res. 17 Ž1989. 9437–9446. w4x V. Braga, S. Gendler, Co-amplification of two cDNAs in RT-PCR can alter the yield of both products, Biotechniques 17 Ž1994. 228–230. w5x J. Chelly, J.C. Kaplan, P. Maire, S. Gautron, A. Kahn, Transcription

w19x

w20x

w21x w22x

w24x

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of the dystrophin gene in human muscle and non-muscle tissues, Nature 333 Ž1988. 858–860. L. Chen, D.M. Segal, C.T. Moraes, D.C. Mash, Transcriptional regulation of dopamine transporter gene expression in the substantia nigra of human cocaine fatalities, Soc. Neurosci. Abstr. 23 Ž1997. 1102. P. Chomczynski, N. Sacci, Single-step method of RNA isolation by acid guanidinium thiocyanate phenol–chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. G. Gilliland, S. Perrin, K. Blanchard, H.F. Bunn, Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction, Proc. Natl. Acad.Sci. 87 Ž1990. 2725–2729. B. Giros, S.E. Mestikawy, N. Godinot, K. Zheng, H. Han, T. Yang-Feng, M.G. Caron, Cloning, pharmacological characterization, and chromosome assignment of the human dopamine transporter, Mol. Pharmacol. 42 Ž1992. 383–390. B. Haendler, R. Hofer-Warbinek, R. Hofer, Complementary DNA for human T-cell cyclophilin, EMBO J. 6 Ž1987. 947–950. J.N. Joyce, G. Smutzer, C.J. Whitty, A. Myers, M.J. Bannon, Differential modification of dopmaine transporter and tyrosine hydroxylase mRNAs in midbrain of subjects with Parkinson’s, Alzheimer’s with parkinsonism, and Alzheimer’s disease, Movement Disorders 12 Ž1997. 885–897. K. Kato, Adaptor-tagged competitive PCR: a novel method for measuring relative gene expression, Nucl. Acids Res. 25 Ž1997. 4694–4696. A. Nicoletti, C. Sassy-Prigent, An alternative quantitative polymerase chain reaction method, Anal. Biochem. 236 Ž1996. 229–241. C. Perrone-Capano, A. Tino, G. Amadoro, R. Pernas-Alonso, U. di Porzio, Dopamine transporter gene expression in rat mesencephalic dopaminergic neurons is increased by direct interaction with target striatal cells in vitro, Brain Res. Mol. Brain Res. 39 Ž1996. 160–166. R. Repp, A. Borkhardt, R. Gossen, J. Kreuder, J. Hammermann, F. Lampert, Consturction of RNA standards for high-resolution automatic product analysis in quantitative competitive RT-PCR, Biotechniques 19 Ž1995. 84–90. M. Riedy, E. Timm, C. Stewart, Quantitative RT-PCR for measuring gene expression, Biotechniques 18 Ž1995. 71–76. S. Santagati, M. Garnier, P. Carlo, E. Violani, G.B. Picotti, A. Maggi, Quantitation of low abundance mRNAs in glial cells using different polymerase chain reaction ŽPCR.-based methods, Brain Res. Protocols 1 Ž1997. 217–223. D.M. Segal, C.T. Moraes, D.C. Mash, Up-regulation of human dopamine D3 receptor mRNA in human cocaine fatalities, Mol. Brain Res. 45 Ž1997. 335–339. P. Siebert, W. Larrick, Competitve PCR, Nature 359 Ž1992. 557– 558. P. Siebert, W. Larrick, PCR MIMMCS: Competitve DNA fragments for use as internal standards in quantitative PCR, Biotechniques 14 Ž1993. 244–249. A. Wang, M. Doyle, D. Mark, Quantitation of mRNA by the polymerase chain reaction, Proc. Natl. Acad. Sci. 86 Ž1989. 9717– 9721. M. Zenilman, W. Graham, K. Tanner, A. Shuldiner, Competitive reverse-transcriptase polymerase chain reaction without an artificial internal standard, Anal. Biochem. 224 Ž1995. 339–346.