456 BIOL PSYOtlATRY I~')3;33:456=-4~

Biologica! Stability of mRNA Isolated from Human Postmortem Brain Collections

Sherry Leonard, Judith Logel, Desiree Luthman, Darrell Kirch, and Robert Freedman

Manuel Casanova,

RNA isolated from frozen human postmortem brain tissue was evaluated for its utility in molecular biological studies. Samples varying in postmortem interval, delay period before freezing, and long-term freezer storage were anal.vzed. It was found that storage of human postmortem brain at -70°C for more than 5 years may compromise its use for oligo-dT primed librar)' construction and in vitro expression studies. Although biological competency of the messenger RNA may be affected by long-term freezer storage of human brain, enough fidl-length or partial transcripts remained for amplification by the polymerase chain reaction. We conclude that human postmortem brain collections will continue to be valuable resources for the study of gene expression and isolation of nucleotide sequences.

Key Words: mRNA, postmortem stability, human brain collection, PCR, in vitro translation

Introduction

A number of clinic~d studies have established that there are major genetic int~!uences in the development of severe psychiatric disorders such as schizophrenia (Kety et al ! 0-/~. ~,.c.. . . . . . A G-" . . . . . . , aQc,, ~..:_ ,^=: . . . . "ph • / I , .~ ' , ~ v t ~ . ~ J U ~ LLi l t~I ~ L ~ . . . . . . : -" 2 ~ . , , , ) ) . L l l a l l i l l g ~ l 1 3 1 1 t l l l U l a r -

macological manipulation in animals have suggested sev- eral biochemical ~ypotheses for this disease (Adler et al 1986; Port et al 1991), but it has been difficult to confirm such evidence in humans. The investigation of involve- ment of specific gene systems, such as those for neuro-

From the Departments of Pharmacology (SL, RF) and Psychiatry (RF), University of Colorado Health Sciences Center and Veterans Admimstration Medical Center (SL, JL. DL, RFL Denver, Colorado; Clinical Brain Disorders Branch (MC), National Institute of Mental Health, Washington, D.C.; and the Division of Clinical Research (DK), National Institute of Mental Health, Rockvili~, Maryland.

Address reprint requests to S. Leonard, Ph.D., Department of Pharmacology, University. of Colorado Health Sciences Center and Veteran's Administration Medical Center U.C.H.S.C., Box C-268-71, 4200 E. 9th Street, Denver, CO 80262.

Received January. 21, 1992: revised December 12, 1992.

transmitter receptors and growth factor families, in the etiology of schizophrenia can be advanced by the use of molecular biological techniques on human brain tissue, where questions can be answered with regard to both expression of candidate genes and polymorphism in their nucleotide sequence. Unlike neurobiological studies in an- imals, human brain tissue is only rarely available from biopsy, and the major source of material for molecular studies is restricted to brain taken at autopsy. The local availability of human postmortem brain to many investi- gators is often limited. In addition, autopsy material is not uniform, and variations in drug regimens and agonal state of the patient must be considered in the evaluation of results. Such heterogeneity in experimental material may necessitate the analysis of large numbers of samples to obtain statistically meaningful results.

An important source for this valuable tissue lies in sev- eral brain collections sponsored by the National Institute of Mental Health and similar agencies in other countries. Many of these facilities have been ab.le to collect human

:~ 1993 Society of Biological Psychiatry 0006-3223/93/$06.00

Molecular Studies on Human Postmortem Brain BIOL PSYCHIATRY 457 1993;33:456--466

postmortem brain from deceased subjects with different types of mental illness, as well as from control subjects, and thus constitute a potential resource for the molecular neurobiologist. Previous studies have examined the effect of postmortem interval on mRNA isolation (S.A. Johnson et al 1986; Perrett et al 1988) but have not addressed how dissection time or long-term freezer storage of human brain tissue affects the integrity of mRNA and its use for the multiple molecular biological studies necessary to char- acterize the expression of a candidate gene. These are parameters, which, if important, could be addressed in the choice of samples for a given experiment. Our current study evaluates the recovery and quality of RNA isolated from prefrontal cortex obtained from the brain collection of the Section on Neuropathology in the Clinical Brain Disorders Branch at the National Institute of Mental Health (NIMH) in Washington, D.C., as well as brain tissue ob- tained from autopsies performed at our home institution.

Postmortem interval, incubation time on ice for dis- section, and freezer-storage periods were examined for their effects on isolation of nucleotide sequence and for biological activity of the mRNA. The methods used in our analyses---northern blot, in vitro translation, first-strand cDNA synthesis, and the polymerase chain reaction ad- dressed both the size and biological integrity of the mRNA transcripts that could be isolated from the various samples. Our results indicate that tissue stored in human postmortem brain collections is suitable for RNA isolation and the study of fibroblast growth factor gene expression using the poly- merase chain reaction; however, prolonged freezer storage and extended dissection times may compromise use of tissue when the mRNA is to be used for in vitro expression studies or oligo-dT primed library construction.

Materials and Methods

Human Postmortem Brain Tissue

The parameters for all tissue samples used in this study are listed in Table 1. Two experimental groups of post- mortem brain tissue were provided by the Section on Neu- ropathology, Clinical Brain Disorders Branch, of the NIMH, located in the Neuroscience Center at Saint Elizabeth's in Washington, D.C. In one group, the whole brain had been removed and placed on wet ice. One hemisphere was placed in formalin,, and the other was coronally sectioned, frozen on dry ice, and placed in storage at -70°C. Medical rec- ords were reviewed for each case, and the brain was ex- amined by both a forensic pathologist and a neuropath- ologist for gross and microscopic abnormalities to rule out central nervous system pathology. Prefrontal cortex, in- cluding both white and gray matter, from six human brains obtained at autopsy following various postmortem inter-

vals [long term storage (LTS) samples 1-6] was obtained from the NIMH. The tissue was from age-matched men, with suicide as the cause of death, although mode of su- icide differed. Another uncontrolled variable was toxicol- ogy. Two of the brains were from carbon monoxide poi- soning victims, and one was from a man with high levels of controlled substances in his bloodstream at the time of death. These brains had been stored at - 70°C in separate freezers between 4 and 6 years when the samples were shipped to us on dry ice. RNA was isolated and assayed within 2 months following shipment.

An additional set of human prefrontal cortex samples was prepared specifically for the dissection-time delay (DTD) study and received from the same NIMH collection facil- ity. A single prefrontal cortex was dissected from a brain with a postmortem interval of 40.75 hr and divided into several uniform l-g samples from the same region, which were placed on wet ice. The specimens were frozen in liquid nitrogen after 1.25-, 3.75-, 5.0-, and 6-hr intervals (NIMH, samples 7-10) and stored at -70°C for less than 3 months before assay. The toxicology results on this tissue were 0.11% ethanol and 0.04 mg% cocaine. Cause of death was listed as a blunt-force injury. RNA from these samples was isolated and assayed within 3 months follow- ing collection.

Three other locally collected postmortem brain samples were included in the analysis. These brains were obtained at autopsy from the Denver Veterans Administration Med- ical Center (DVAMC, samples 11-13) following the post- mortem intervals indicated in Table 1. Hippocampus, a region of special interest for our schizophrenia research, was dissected from central, l-cm coronal slices and frozen in liquid nitrogen within 30 min following brain removal. The coronal slices were frozen, after dissection, between plexiglass sheets in dry-ice snow. All brain tissue was stored at -70°C; material used in this study had been stored less than 2 months before assay. The cause of death,

. . . . ~ ' ~ . t - I ~ " ~ n c w ~ age, and postmortem interval were va~mt,~ in these ~,~,,- pies, and no toxicology was performed because death was due to natural causes.

Materials

Oligo-dT cellulose was obtained from Sigma Chemical Co. (St. Louis, MO) and Duropore microfilters from Mil- lipore. RNase-free DNase and placental RAlase inhibitor were obtained from Boehringer-Mannheim. Random hex- amer oligonucleotides and radionuclides were from Amer- sham. Moloney murine leukemia virus (M-MLV) reverse transcriptase was purchased from Bethesda Research Lab- oratories, and polymerase chain reaction (PCR) reagents, including Taq polymerase, were obtained from Perkin-

458 BIOL PSYCHIATRY 1993;33:456-466

S. Leonard et al

Table I. Parameters for Human Postmortem Brain Samples"

Source and PM! Age Autops) sample no. (hr) (yr) Gender Cause of death Toxicology report date

NIMH I 7 29 M Suicide (fall) PCP 0.01 mg%, 2/16/86

morphine 0.007 rag%, cocaine 0.05 mg%, cannabis positive

2 14 29 M Suicide (fall) Negative 2/27/85 3 21 29 M Suicide (hanging) Negative 7/30/85 4 30.5 30 M Suicide (CO 90% CO 2/27/85

poisoning) 5 40.5 27 M Suicide Negative 9/26/87

(exsanguination) 6 42.25 28 M Suicide (CO 60% CO 6/i/85

poisoning) 7/10 40.75 32 M Blunt-force Ethanol 0. I 1%, cocaine ,5/4/90

injury 0.04 mg%

DVAMC 11 i 8 39 M Kidney failure ND 5/!/90 12 3 75 F Myocardial ND 6/12/90

infarction 13 3 60 F Multiple ND 6/15190

myeloma

~NIMH, National Institute of Mental Health; DVAMC. Denver Veterans Administration Medical Center; PMI, postmortem interval; M, male; F, female; ND, not determined.

Elmer/Cetus. NuSieve GTG and Seakem GTG agarose were obtained from FMC. The cDNA probe for human 13- actin (Ponte et al 1983) was obtained from Dr. Sandra Martin, Department of Molecular and Structural Biology, University of Colorado Health Sciences Center, Denver, Colorado. The basic fibroblast growth factor (Kurokawa et al 1988) and fibroblast growth factor receptor (D.E. Johnson et al 1990) cDNA clones for verifying PCR prod- ucts were gifts of Dr. Lars Olson, Karolinska Institute, Stockholm, Sweden, and Dr. L.T. Williams, Department of Medicine, University of California, San Francisco, Cal- itomia, respectively. Oiigonucieotide primers for I ~ R were synthesized on an Applied Biosystems PCR/Mate oligon- ucleotide synthesizer and purified over OPC cartridges (Perkin-Elmer/Cetus).

Isolation of Total RNA and Polyadenylic Acid RNA

Total RNA was prepared from approximately 1 g of prefrontal cortex by the method of Chomczynski and Sacchi (1987), with the addition of an RNase-free DNase treatment and two phenol/chloroform extractions prior to the final ethanol precipitation. Polyadenylic acid ~_NA ([poly A+]-RNA) was prepared by a single-batch ad- sorption of up to 500 ~g of the total RNA onto 50 mg of oligo-dT cellulose, followed by washes and elation through 0.45-p.m Duropore microfilters (Millipore), which

can be centrifuged in a microfuge (Badley et al 1988). RNA was quantitated, and purity was estimated by spectrophotometric readings at 26t) and 280 nm (Sam- brook et al 1989).

Northern Blot Analysis of mRNA

mRNA was electrophoresed through 1% agarose gels con- taining formaldehyde, and the denatured mRNA was trans- ferred to Hybond nylon membrane (Arnersham) for north- ern blots (Sambrook et al 1989). A cDNA probe for human ~-actin was prepared by excision of a BamHI fragment from the cDNA clone pHF[3A (Ponte et al 1983) and ran- dom-primed labeling (Feinberg and Vogelstein 1983) of the fragment with [a-3Zp]deoxycytosine triphosphate (dCTP) (Amersham). Hybridization and washes were as described previously (Sambrook et al 1989). Briefly, the [3-actin probe was used at 106 cp~ru'ml in hybridization buffer con- taining 6 x (SSC) (1 x SSC is 0.15 mol/L NaCi, 0.015 mol/L sodium citrate); 5 x Denhardts' [50 x Denhardts' is 1% Ficoll (Pharmacia), 1% poiyvinylpyrroiidone, 1% bovine serum albumin]; 0.5% sodium dodecyl sulfate (SDS), 100 p,g/ml salmon sperm DNA, and 50% formamide. Hy- bridization was at 42°C for 16 hr. The filters were washed twice in 2 x SSC and0 .1% SDS for 15 mina t room temperature, once in 0. l SSC and 0.1% SDS for 15 rain at room temperature, and once in 0.1 SSC and 0.1% SDS

Molecular Studies on Human Postmortem Brain BlOt. PSYCHIATRY 459 1993;33:456-- 466

for 30 min at 55°C. The damp filters were then covered with Saran wrap and exposed to Kodak XAR-5 film.

In Vitro Translation

In vitro translation of RNA was performed with a rabbit reticulocyte kit purchased from New England Nuclear (NEN). Total RNA (5 I~g) was heated to 65*C for 5 min and then translated in a 25-1xl reaction containing 25 p, Ci of l~SSl-methionine (NEN), 2 U of placental RNase in- hibitor (Boehringer-Mannheim), and the manufacturer's buffer adjusted to 100 mmol/L potassium acetate and 1.3 mmol/L magnesium acetate. Optimal salt and RNA con- centrations were determined in preliminary experiments using total human RNA as template. Additional amino acids minus methionine (Amersham) were also added to 1 raM, and the reaction was initiated by addition of retic- ulocyte lysate. A no-RNA control was included in each experiment. Tobacco mosaic vLrus (0.15 p,g) and total yeast RNA (1 p,g) were translated in separate reactions as positive controls. The translations were incubated at 37°C for 60 min. Two microliters of each reaction was precip- itated with trichloracetic acid (TCA) to determine the ra- dioactivity incorporated into protein. An equal number of counts incorporated into TCA-precipitable material were loaded cnte a 5%-15% linear polyacrylamide gradient gel with appropriate molecular weight standards. The gel was dried and exposed to XAR film overnight.

First-Strand eDNA Synthesis From Total RNA

First-strand eDNA was synthesized from 10 lxg of total RNA, using a eDNA synthesis kit purchased from Amer- sham, with 2 IxCi of [ot-32p]-dCTP added per reaction. Oligo-dT was used as primer, and reactions were run at 42°C for I hr, as specified by the manufacturer. Labeled eDNA was resolved on an alkaline agarose gel according to Sambt'ook et al {1989). The gel was dried and exposed to XAR film.

Reverse Transcriptase/PCR Analysis of mRNA

The reverse transcriptase (RT)/PCR assay was peiformed by reverse transcribing the mRNA in a sample of total RNA, using random hexamers as primers and M-MLV RT, and then amplifying the eDNA with gene-specific oligonucleotide primers and Taq polymerase in the same tube using the PCR in a Perkin-Elmer thermal cycler (Ka- wasaki and Wang 1989), with some modifications.

Briefly, the RT step contained 1 mmol/L dNTPs, 0.5 U/Ixl RNase inhibitor, 0.01 mol/L dithiothreitol, 2.5 U/~I M-MLV RT, 1 x RT buffer from the manufacturer, 300 p.mol random hexamers, and 1 p,g total RNA in a final

volume of 40 Ixl. The RNA was incubated at 65°C for 5 min, quick-chilled on ice, and added to the RT reaction mix. Incubation at room temperature for I0 min to allow annealing of the random hexamers was followed by 60 min at 37°C. The RT was heat inactivated at 99°C for 5 rain, and 60 Ixl of PCR reaction mix containing 1 × PCR buffer (provided in the Perkin-Elmer Cetus GeneAmp kit), upstream and downstream primers to a final concentration of I I~mol/L each, MgCI2 to 2 mmol/L, 3 gCi [et-32pi- dCTP, and 2.5 U Taq polymerase were added. The tubes were placed in a Perkin-Elmer thermal cycler with the following program: 1 min at 94°C; 2 min at 60°C; 3 min at 72°C; 30 cycles. Aliquots of 10 p.l were electrophoresed through 4% agarose (3% NuSieve GTG/1% Seakem GTG) containing ethidium bromide. The gel was photographed, dried onto DE81 paper, and autoradiographed.

Primers for the PCR were designed using a software program that allows the selection of primers with varying G/C content, melting point, and designated length (Lowe et al 1990). Nucleotide sequence for the test genes basic fibroblast growth factor (bFGF), fibroblast growth factor receptor FGP, and adenosine phosphoribosyltransferase (APF'.T) was obtained from GenBank (Bilofsky et al 1986) through Intelligenetics, Inc. (Mountain View, CA). PCR products derived from human RNA were identified in pre- liminary results as being gene specific by Southern blot hybridization with eDNA probes for bFGF (Kurokawa et al 1988) and FGF r (D.E. Johnson et al 1990) and an in- ternal oligonucleotide for APRT.

Resu|ts

Isolation of Total RNA and [Poly A +]-RNA from Human Postmortem Brain

Total RNA and [poly A ÷ ]-RNA were prepared from ap- proximateiy 1 g of tissue from each of the samples, as described in Materials and Methods. The yields of RNA • ,,,a ,~D~,~ e,-,~m the ,,,~,-i,~,,~ ~amples, calculated from spectrophotometric readings at 260 nm, are shown in Table 2. The average amount of total RNA that we obtained from 1 g of postmortem human brain was 611 -_. 69 (SD) ixg/g, and the mean for all the mRNA preparations was 29.6 +-. 9.0 ~g/g of tissue. The yields of mRNA in this study, isolated from one pass over oligo-dT cellulose, were approximately 3%-5% of the total RNA. An analysis of variance (ANOVA) did not indicate any significant dif- ferences in micrograms of total RNA, mRNA, or mRNA fraction of total PNA among any of the sample groups.

Northern Blot Analysis of mRNA

The integrity of the isolated RNA for use m molecular biological studies was assessed for several different uses, northern blot, in vitro translation, eDNA synthesis using

4 ~ BIOL PSYCHIATRY S. Leonard et al 1993;33:456--466

Table 2. Total RNA and [Poly A ÷ ]-RNA Yield From Tissue Samples a

p,g/g of tissue

Source and Total mRNA/ sample no. PMI (hr) FTD (ha') RNA mRNA total RNA

NIMH I 7.00 0.50 667 31.5 b 0.047 2 14.00 0.50 463 22.4 c 0.048

3 21.00 0.50 588 14.4 c 0.024

4 30.50 0.50 591 31.4 ~ 0.053 5 40.50 0.50 558 28.1 0.050

6 42.25 0.50 605 31.6 0.052 7 40.75 1.25 683 440 0.064 8 40.75 3.75 618 36.0 0.058 9 40.75 5.00 715 44.2 0.062

10 40.75 6.00 711 32.7 0.046

DVAMC 11 18.00 0.40 530 18.8 0.035 12 3.00 0.50 612 34.0 0.056

13 3.00_ 0.25 601 16.2 0.027

~Total RNA and [poly A ~ ]-RNA were isolated from approximately 1 g of all the tissue samples, as described in Materials and Methods. The mean total RNA yield for all samples was 611 __ 69 I.tg~g of tissue; the mean for [poly A ÷ ]-RNA was 29.6 - 9.0 p.g/g. PMI, postmortem interval; FTD, freeze-time delay.

bAverage of ~plicate preparations. "Average of duplicate preparations.

oligo-dT primers, and the PCR. We first de,.'ermined whether an abundant transcript such as [3-actin could be detected on a northern blot of mRNA isolated from these samples• Messenger RNA (3 ~g in each lane) from both the LTS and DTD sample groups was resolved on formaldehyde gels and blotted to nylon membranes, as described in Ma- terials and Methods. Staining of a nondenaturing minigel of the LTS samples with ethidium bromide suggested some degradation of the 28S and 18S ribosomal RNA bands (not shown). The probe used for the northern blot hybridization was a fragment excised from the plasmid pHFI3A (Ponte et al 1983) containing sequences complementary to the 3'- untranslated region of human 13-actin mRNA, labeled with [ o t - 3 2 p l - d C T P by a r a n d n m - p r l m f - d m m h n d tn,-~hrin-,~r-

Mannheim). Figure la and b show the results of exposing the hybridized filters to film• A band at 2. l kb, the correct size for the human 13-actin transcript, can be seen on both northern blots• Although the same amount of mRNA was loaded for each sample, there was considerable variation in the amount of 13-actin in both LTS and DTD sample groups. Tailing below the major band in several of the LTS samples suggested that these specimens also con- tained a considerable amount of degraded 13-actin mRNA but that the degradation did not correlate with increasing postmortem interval.

Because northern blots may be insensitive to small structural changes that could affect biological activity, we evaluated the effects of LTS and DTD on both in vitro

translation and first-strand cDNA synthesis primed with oligo-dT.

In Vitro Translation

Both a 5'-terminal 7-methylguanosine (m7G) cap (Ven Murthy 1982) and a 3'-terminal polyadenylic acid [poly A + ] tail (Munroe and Jacobson 1990) have been shown to be necessary for efficient translation of eukaryotic mRNA. Loss of either or both of these structures through mRNA degradation could affect the translatability of mRNA iso- lated from postmortem brain.

Intact mRNA, isolated from human postmortem brain, • ~ r ~ l . , . , , - ~ I - ~ , - ~ . ~ l ~ l - ~ , - I " can be In , , u , . , u m , ~ , a t ~ u i ~ t o proteins over a large range

of molecular weights, with some larger than 90,000 Da (Sajdel-Sulkowska et al 1983). In vitro translation products were generated from total RNA isolated from both the LTS and DTD samples, as well as RNA isolated from locally collected postmortem brain (DVAMC), as de- scribed in Materials and Methods•

Stimulation of [35S]methionine incorporation into TCA- precipitable material was twofold over the no-RNA control for the DTD and DVAMC human samples and fourfold for rat, using 5 Ixg of total RNA in each assay. The stim- ulation of [35S]methionine incorporation into the LTS sam- ples, however, was barely above that found in the control, where no RNA was added.

Translation products, resolved on a polyacrylamide gel,

Molecular Studies on Human Postmortem Brain BIOL PSYCHIATRY 461 1993;33:456-466

A. P o s t m o r t e m In te rva l B. Dissect ion Delay

0 0 0 m m It)

Actin 2.1kb

m m o o c~ h, o o

Figure 1. Northern blot analysis of mRNA from human postmortem brain. [Poly A+I-RNA (3 Izg each lane) from the LTS samples (A) or FTD samples (B) were eleclxophoresed on formalde- hyde gels and blotted to nylon mem- branes, as described in Materials and Methods. The blots were hybridized with an lot3zP]-dCTP-labeled human 13-actin cDNA probe.

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Figure 2. In vitro translation of total RNA isolated from human brain. Total RNA (5 l~g) isolated from the LTS, DTD, and locally collected samples (DVAMC) was translated in vitro in a rabbit reticulocyte system, as described in Materials and Methods. Controls included were translation of total RNA from yeast and tobacco mosaic virus (TMV) mRNA and a translation reaction with no RNA added. Incorporation of [35S]methionine was measured by TCA precipitation. Equivalent amounts (40,000 cpm each) of the labeled translation products were resolved on 5%-15% polyacrylamide gradient gels, and the gels were dried and exposed to X-OMAT film (Kodak). [~4C]-molecular weight standards were resolved in the same gels, and the sizes (in kilodaltons) are indicated. (A) Translation products derived from the LTS samples of 7.0-, 14.0-, 21.0-, ~ .5 - , and 42.25-hr postmortem intervals (PMI). (B) LTS RNA samples from the shortest (7.0 hr) and longest (42.25 hr) PMIs were freshly prepared and translated again along with the dissection time

delay (DTD) samples and samples " tl i-13) from three postmortem human brains collected at the Denver Veterans Administration

Medical Center (DVAMC).

462 BlOL PSYCHIATRY S. Leonard et al 1993;33:456-466

are shown in Figure 2. As can be seen in Figure 2a, the abundance of proteins resulting from translation of mRNA present in the LTS samples is very low compared with translation products in a control RNA. The in vitro trans- lation experiment was repeated on separate RNA prepa- rations of the LTS samples with the shortest and longest postmortem intervals. Total RNAs (5 I~g) from the DTD samples and from freshly collected brain tissue (DVAMC) were also translated. Figure 2b shows that mRNA from the LTS samples does not translate as well as does the mRNA from either the DTD specimens or the freshly col- lected DVAMC brain samples.

Translation of the DTD samples in Figure 2b also sug- gests that translation of the mRNA is affected if the tissue is allowed to remain for more than 3 hr on ice before freezing, as evidenced by a decrease in products at the later times.

Among the samples that translated well, considerable heterogeneity in the protein products was noted. The trans- lated products in specific bands appear to have variable intensity among the samples, suggesting either differences in expression of proteins in the individual brains or a poly- morphism resulting in a difference in size or amount of a specific protein; however, all RNA samples isolated from tissue frozen within approximately 1 hr of brain removal and stored for less than 1 year were translatable into a wide range of protein species.

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First-Strand cDNA Synthesis

We further addressed the integrity of the 3'-ends of the mRNA by comparing the first-strand cDNAs, which could be synthesized using an e_,ligo-dT primer and RT, from our RNA sample population. Oligo-dT, often used as a primer of cDNA synthesis for cloning (Sambrook et al 1989), was annealed to 5 ~g of total RNA isolated from the shortest and longest postmortem interval LTS and shortest and longest time DTD samples, two DVAMC samples (11 and 12), and RNA isolated from rat brain. First-strand cDNA was synthesized as described in Materials and Methods in the presence of [ot-32p]-dCTP, and the products were re- solved on an alkaline agarose gel. An autoradiogram of the dried gel (Figure 3) shows that whereas RT of the rat brain and DVAMC human postmortem brain RNA sam- ples, using an oligo-dT primer, resulted in a normal sized distribution of cDNA products, the LTS samples did not. Reduction in amount of cDNA synthesized from the LTS samples may mean loss of sufficient [poly A ÷] sequence for oligo-dT priming from many mRNAs. A decrease in oize but _not in apparent amount of cDNA synthesized Crnm the 6-hr DTD sample compared with the 1.25-hr sample was also noted, suggesting that there may have been some degradation in the mRNA at the later time.

Figure 3. First-strand cDNA synthesis from total RNA samples. Total RNA (5 I.tg) was annealed to oligo-dT primers and reverse transcribed in the presence of [ct-32]-dCTP, as described in Ma- terials and Methods. The products were resolved on an alkaline agarose gel that was dried and exposed to film. Prodat:ts derived from the LTS samples with 7.00 and 42.25-hr PMIs are shown in the left two lanes. Controls include total RNA isolated from rat brain and a first-strand synthesis with no RNA. (Abbreviations as in Figure 2.)

Multiplex PCR Analysis of mRNA

Although loss of the 5'-mTG or the 3'-[pc!y A +] tract may affect use of the mRNA for in vitro expression studies or construction of directional cDNA libraries, this mRNA could be used for studying gene expression by the PCR. PCR can be used to amplify nucleotide sequences from only a few copies of mRNA and requires only that the short sequence to be amplified is present. We used a mul- tiplex RT/PCR to amplify cDNA fragments reverse tran- scribed from three low-abundance transcripts in the LTS RNA samples that had appeared to be at least partially compromised by northern blot, in vitro translation, and

Molecular Studies on Human Postmortem Brain BIOL PSYCHIATRY 463 1993;33:456--466

first-strand cDNA synthesis analyses. The mRNA was first reverse transcribed into cDNA using random hexamers as primers, rather than oligo-dT, to optimize isolation of eDNA sequences from mRNA with degraded [poly A +] tails. In the same tube, eDNAs for the three unique transcripts were then amplified with the PCR, using gene-speeific primers in the presence of [a-azpI-dCTP, as described in Materials and Methods. Primers were designed for human APRT (Broderick et al 1987), bFGF (Kurokawa et al 1988), and the FGF r (D.E. Johnson et al 1990) using a computer program (Lowe et al 1990). APRT is a constitutively ex- pressed housekeeping gene; bFGF and FGF' are of interest in our studies on growth factor levels in schizophrenia. All are low-abundance mRNAs (Broderick et al 1987; Gospodarowicz et al 1987). An important consideration was that the selected PCR primer sequences lie on either side of an intron. If any DNA contamination was present in the RNA preparations, PCR products that contained the intron would be larger than the expected sizes from the mature mRNA. Figure 4 shows the multiplex RT/PCR products generated from 1 ixg of the total RNA isolated from the LTS samples at six different postmortem inter- vals. The sizes of the amplified fragments were exactly as predicted from mRNA sequence data, indicating that the products were specific for mRNA and not due to DNA contamination in the samples. Signals derived from all three of the primer sets used suggest that RNA isolated from brains with long postmortem intervals and extended freezer storage times is still of sufficient integrity to gen- erate a specific product in a mulitplex PCR assay when random hexamers are used to prime eDNA synthesis.

Discussion

The extension of experimentation in molecular neurobiol- ogy from animals to humans generally requires the use of human postmortem brain, a tissue of limited availability to many investigators. Several facilities, both in the United

States and abroad, are maintaining and expanding large collections of postmortem brain, particularly tissue from subjects diagnosed with various mental illnesses. Although postmortem brain is routinely stored below -70°C, most postmortem brain collections are compiled over many years, and storage time could be a factor in the integrity of mRNA isolated from these tissues. Additionally, transportation and dissection of specific regions of the brain often require that coronal slices be kept on ice for more than 1 hr fol- lowing autopsy. To assess the possibility of using tissue from existing brain collections for molecular biological studies on growth factors in schizophrenia and to extend our information concerning the stability of RNA in human postmortem brain, we evaluated the isolation and quality of RNA prepared from one of the larger brain collections in the United States, located at the NIMH in Washington, D.C. We found that although the apparent amount of RNA that can be isolated from freezer-stored tissue does not vary significantly, specific structures in the RNA may de- teriorate over time. Although freezer storage for more than 5 years may result in a loss of biological activity in specific mRNAs, this brain tissue remains valuable for northern blot analysis, nucleic acid sequence determination, and evaluation of gene expression by the PCR.

The amount of RNA isolated per gram from human postmorte.-a tissue (approximately 600 tLg/g) was less than the amount we routinely obtain from rodent brains, either directly af-ter decapitation or a_ft_er _freezing in liquid nitro- gen (approximately 1000 p.g/g), confirming the observa- tions of other groups (Sajdei-Sulkowska et al 1983; S.A. Johnson et al 1986; Perrett et al 1988). The additional manipulation of human brain tisme may well be a contri- buting factor; owing to the size and convenience in dis- section of rodent brain, very httle handling is necessary (S.A. Johnson et al 1986). The human brain, however, is usually we~.ghed, examined for gross pathology, and sliced and dissected before freezing. Such additional manipula- tion may increase the risk of re, leasing fibonucleases; how-

FGF r -

7.0

b-FGF-

APRT-

Postmortem Interval (hours)

14.0 21.0 30.5 40.5 42.3 bp

_416

-176

_118

Figure 4. PCR products for adenosine phoshoribosytransferase (APRT), basic fibroblast growth factor (b-FGFr), and fibroblast growth factor receptor (FGF r) derived from RNA isolated from the hu- man LTS samples. Total RNA from the six LTS samples, varying 7.00 to 42.25 hr in postmortem interval, was assayed using RT/PCR, incorporating [a-32P] - dCTP, as described in Materials and Methods. The products were resolved on a 4% agarose gel that was dried and exposed to film.

4 ~ BIOL PSYCHIATRY It~3;33:456--466

S. Leonard et al

ever, one report of RNA yield from a h-man brain sample taken at biopsy was in the same range of 600 p,g/g of tissue (S.A. Johnson et al 1986), suggesting that rather than loss due to manipulation, the total amount of RNA in human brain may be less per gram of tissue than in rodents. There were no significant differences in the amount of total RNA or mRNA isolated per gram of tissue that could be cor- related with any of the sample groups.

The quality of the RNA isolated from the NIMH brain collection was assessed both for use in gene expression studies and for biological activity. Transcripts for the abun- dantly expressed gene 13-actin were examined in northern blots of both the LTS and DTD specimens. No significant differences could be associated with postmortem interval, although there was considerable heterogeneity among the samples in amount of 13-actin transcript present. A northern blot of the DTD samples likewise revealed some variation in amount but no obvious differences correlating with the shortest and longest intervals; however, some degradation was present, as shown by tailing below the main band in every lane. It should be noted that only one pass over oligo-dT was used in the isolation of mRNA to maximize the use of tissue. There was, therefore, residual ribosomal RNA (rRNA), which may have contributed to the small v&,'iation among samples seen on the northern blots and in the fraction of mRNA isolated from total RNA in the samples (Table 2).

In vitro translation of all RNA samples using a rabbit reticulocyte system yielded some interesting results. We f~und that very few polypeptides could be translated from the samples stored for 4-6 years, even tbo,gh the post- mortem intervals (PMIs) ranged from 7.00 to 42.25 hr. On the other hand, the freshly collected tissue from both the DTD (40.75-hr PMI) and DVAMC (3-18-hr PMI) provided RNA that was translatable into many protein species. Thus, it does not seem likely that PMI is a sig- nificant factor in the isolation of intact mRNA from brain tissue collected at autopsy and stored for later use, in agreement with other investigators (Sajdel-Sulkowska et al 1983; S.A. Johnson et ai 1986; Perrett et al i988).

Long-term freezer storage of tissue may, however, be detrimental to some mRNA species. Although isolation of apparent full-length transcripts for abundant mRNAs such as 13-actin may be accomplished from human postmortem brain stored for long periods, our in vitro translation and first-strand cDNA synthesis experiments suggest that many messages may he m o r e diffic',,llt to i s o l a t e a~ intact bio- logical mRNAs from LTS brain. RNA isolated from the stored samples (LTS, samples 1-6) was neither well trans- lated in an in vitro rabbit reticulocyte system nor tran- scribable into a normal range of cDNA by RT and an oligo-dT primer. Although this loss of biological activity may mean a loss of some 5'-cap swactures and/or 3'-[poly

A +] ~acts, our results do not rule out inhibitors of trans- lation and RT that could have copurified with the RNA in the LTS samples.

Although RNA, isolated from human postmortem brain stored for more than 5 years did not seem suitable for in vitro expression or directional ,:loning, we have found it to be usable for northern blot analysis of abundant mRNAs, the isolation of nucleotide sequence, and detection of mRNA expression by the RT/PCR. The latter procedure does not require a full-length mRNA transcript; only a short se- quence must remain intact for random hexamer priming of cDNA synthesis and subsequent amplification of a 100- 500-bp fragment. Oligonucleotide primers can be designed to generate RT/PCR products of specific sizes, enabling the isolation of fragments for several different genes in a single reaction. We selected APRT, bFGF, and FGF r for our low-abundance test genes in the evaluation of brain tissue from the NIMH brain bank because we are interested in the expression of the FGF family and its receptor in schizophrenia. When the samples were compared by mul- tiplex RT/PCR, the results indicated that it is possible to obtain information on expressed gene sequence, even after relatively long PMIs and following storage of frozen brain tissue for up to 6 years.

Our PCR products were generated from cDNA synthe- sized from total RNA with random hexamers as primer instead of oligo-dT, eliminating the requirement for intact [poly A +] tails. This procedure may help to increase the representation of all gene sequences in library construction from human postmortem brain when degradation of [poly A ÷ ] tracts occurs.

Although the degradation of eukaryotic mRNA in hu- man postmortem brain is not well understood, it is likely to involve different rates and mechanisms for rrLRNA spe- cies (Huez et al 1981; Brawerman 1987; Shapiro et ai 1987; Noguchi et al 1991). Although our PCR results show that RNA isolated from human postmortem brain stored for more than 5 years can be used to isolate nucleotide sequences for bFGF, FGF ~, and APRT. a recent report on premortem and postmortem variables for another gene family suggested that freezer storage of even 3 years may be an important factor in the degradation of some mRNAs (Burke et al 1991). Because our study addressed the general in- tegrity of all messages in a relatively small sample pop- ulation, it will be of interest to examine the biological stability of other mRNA species in human postmortem brain tissue stored for many years.

An additional observation in our in vitro translation studies is the heterogeneity in proteins tran,dated from three fresh postmortem brain samples from the DVAMC. These specimens were all from the hippocampus, whereas the NIMH samples were from prefrontal cortex. Although differences in expression might be expected between cor-

Molecular Studies on Human Postmortem Brain BIOL PSYCHIATRY 465 1993;33:456-466

tical and hippocampal tissue, our results suggest that gene expression may vary considerably in the same brain region from different human brains. The DVAMC samples were from subjects differing widely in age and cause of death. Additionally, samples 12 and 13 were from subjects di- agnosed as manic depressive. Gene expression effects of premortem drug treatment and preagonal state as well as freezer storage of the tissue may therefore need to be assessed for each genetic system under study.

In conclusion, our present study showed that human brain from an extensive collection at the NIMH that was stored up to 6 years ai --70"C is suitable foi many mo- lecular biological experiments. With so many variables in human postmortem brain sample populations, diseased and control tissue for experimentation must be carefully matched.

Our results suggest that time required for dissection and lengthy freezer storage are two controllable factors that should be considered in the collection and choice of tissue for molecular neurob:oiogy. Continuing improvements in cryoproteetion of human postmortem brain (Lu and Haber 1992) may also result in extended use of this valuable tissue for RNA studies. Finally, methods such as the RT/PCR reaction, which increase sensitivity of detection of mRNA, should greatly enhance the use of existing and planned human postmortem brain tissue collections for molecular biology.

This work was supported by U.S.P.H.S. grant MH44212 and the De- partment of Veteran's Affairs Medical Research Service.

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