Post-mortem human brain specimens were obtained at

routine autopsy from drug-free, age-matched control subjects Ž . Ns8; 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 average 15.8 Ž . "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.
ŽA series of three small lateral to medial punches 200 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. 5 . The method shown w x
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
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 7,20 . Using oligo dT primers, mR- w x Ž .12 – 18
NAs 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.
Ž . a Weigh the frozen brain tissue punches and prepare
the homogenization solution, which contained 4 M guanidium thiocyanate, 25 mM sodium citrate, 0.5% v Ž . rv
sarcosyl and 0.72% v Ž . rv b-mercaptoethanol.
Ž . Ž . b Add 10 volumes ml of the homogenization solution v Ž . rv to the brain tissue according to the weight
of
the brain sample mg and homogenize with a Brinkmann Ž .
polytron homogenizer for 15 s at a setting of 2.5.
Ž . c Mix the solution thoroughly by inverting the tube 10
times after the addition of each of the following reagents:
0.1 volume of the tissue weight of 3 M sodium acetate, Ž .
pH 5.2, 1 volume of the wet brain tissue weight of water Ž .
saturated phenol, and 0.2 volume of chloroform:isoamyl
alcohol 49:1 mixture. Vortex the mix vigorously for 10 s Ž .
and chill on ice for 15 min.
Ž . d Separate the organic and aqueous phases by centrifugation Sorvall HS-4 rotor, 5.75 cm radius at 6500 Ž .
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 Sorvall SS-34 rotor at 15,000 rpm, Ž .
48C for 20 min to pellet RNA.
Ž . e 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 v Ž . rv 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
Ž . Ž . model 5415 at maximum speed 14,000=g and wash
the pellet with 1 ml of cold 75% ethanol.
Ž . f Briefly dry the pellet and dissolve in 10 ml of Tris
EDTA buffer 10 mM Tris, pH 8.0, 1 mM EDTA , Ž .
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 25:24:1 twice. Precipitate RNA by Ž
.
adding 5 ml of 3 M ammonium acetate pH 5.2 , 125 Ž . ml of
ethanol and store at y808C for 1 h.
Ž . g Centrifuge for 10 min at the speed of 14,000=g in
a microcentrifuge, followed by one wash with 1 ml of 70%
134 L. Chen et al.rBrain Research Protocols 4 1999 132–139 ( )
ethanol. Briefly dry the RNA pellet under vacuum. Resuspend the pellet in 20–30 ml of Tris–EDTA pH 8.0 , and
Ž.
quantitation by absorbance Ž . A reading at 260 nm and 280
nm. The final preparation should be free of protein with
the A260 280 rA 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.
Ž . Ž . a Add 1 ml of oligo dT primer 100 pmol to 5 12 – 18
mg of total RNA sample and make the final volume to 12
ml by addition of diethylpyrocarbonate DEPC treated Ž .
H O. Incubate at 70 2 8C for 10 min, followed by chill on ice
for at least 1 min.
Ž . b Prepare a master reaction mix according to the total
number of reactions Ž . n , including one no-RT reaction and
Žone control RT reaction use 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 dNTP mix and Ž . n=2 ml
of 0.1 M dithiothreitol DTT . Add 7 Ž . ml of the above
mixture to each sample and mix the solution well, followed by a brief centrifugation step.
Ž . c Incubation of the above solution for 5 min at 428C,
and then add 1 ml of superscript II Reverse Transcriptase
to each tube for the no RT control, substitute 1 Ž ml of
DEPC-treated H O . Incubate for 50 min at 42 2 . 8C 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.
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
subjects. DAT and cyclophilin primers were created using
PrimerDesign software version 1.10, Marburg, Germany Ž .
with similar GC content 50–60% and melting tempera- Ž .
ture Ž . Tm 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 wee synthesized by Gibco BRL Life Technologies according to manufacturer’s instructions Table 1 . Ž .
For the amplification of DAT and cyclophilin mRNAs, the
steps are as follows.
Ž . a Serial dilutions were prepared for each cDNA sample obtained from the first strand cDNA synthesis, i.e.,
two-fold dilution 2 Ž . = , 5=, 10=, 20=, 50=, 100=
and 200=. For PCR standard, cDNA was pooled from at
least three human brain samples for PCR amplification.
Ž . Ž b Preparation of the master PCR reaction mix for 50
PCR reactions was follows: 3.6 ml of DEPC H O, 500 . 2 ml
of 10=PCR buffer, 300 ml of 25 mM MgCl , 100 2 ml of
10 mM dNTPs, 100 ml of each 5 mM DAT primer or 50 Ž
ml of each 5 mM cyclophilin primer , 50 . ml of 1:4 diluted
wa- P -dCTP. Aliquot 98 32 x 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 2.5 unit into each Ž .
tube.
Ž . c 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 radioacti˝e marker for gel electrophoresis
A radioactive marker was synthesized in vitro using
Century Marker Template Plus Ambion as described by Ž .
the following steps:
Ž . a Mix 6.9 ml of DEPC H O, 0.5 2 ml of century
marker, 1.6 ml of 100 mM DTT, 4 ml of 2.5 mM
Table 1 A,G,UTP, 1 ml of 0.4 mM CTP, 4 ml of wa- S -CTP 32 x
Ž . Ž vacuum dried , 4 ml of 5= reaction buffer from
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
Ž . 25:24:1 .
Ž . b 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.
Ž . c 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 5 ml per Ž
filter , and one wash with 95% ethanol 5 ml per filter . . Ž .
Measure the radioactivity of the air-dried filters with scintillation spectrometry to calculate the specific activity
of
the marker cpm Ž . rml of stock and use 10 cpm for each 5
marker lane in polyacrylamiderurea gel electrophoresis to
obtain the molecular size of the amplified fragments, DAT
and cyclophilin.
4.5. Polyacrylamiderurea gel electrophoresis and densitometry
Separation of the PCR products and densitometry were
performed as follows:
Ž . a 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 pH 5.2 , 100 Ž . ml of
ethanol and precipitate at 808C for at least 1 h.
Ž . b 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 80% formamide, 0.1% Ž
xylene cyanol, 0.1% Bromophenol blue and 2 mM EDTA,
pH 8.0 , followed by heat denaturation at 90 . 8C for 5 mins
and then chill on ice.
Ž . c 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