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2.13.1. Method
1 Fix sections, and carry out tmmunohlstochemistry as above, having filter-steril-
ized all reagents usmg 0 2-pm filters (Sartorms, Gottmgen, Germany). Reagents
should contam 1 U/pL ribonuclease mhtbttor and 1 mM DTT
2. After tmmunohtstochemtstry, wash secttons m 0.1% Tnton X- 100 in PBS for 10 mm
at room temperature to mmimize nonspecific binding of probe and to increase
hybrtdlzatlon efficiency on unmunohtstochemlcally stained cells (7)
3 Sections are then subjected to ISH usmg 1251-labeled RNA probe as above.
4. Counterstain: Because nuclear emulsion is rapidly dtssolved in low pH, a brief
immersion of l&30 s m fast hematoxylm (7) IS sufficient Eosm staining 1s not
used due to the presence of DAB precipitate

3. Notes
1 As a general rule, the optimal preservation of antigens IS attamed by fixing for the
minimum time compattble with preservation of morphology, which we find 1s60
mm m fresh PLP m the case of mouse spmal gangha Many laboratories make use
of central tissue embedding services. However, tf this procedure 1s adopted, tt 1s
worthwhtle checkmg whether a formaldehyde fixation step 1s mcluded, since this
˜111have the effect of greatly increasing the time spent m fixative
2. In the case of severe background problems, it may be necessary to Increase the
concentratton of serum m the blocking solution, conversely, tf background 1sfound
to be very low, then satisfactory blockmg may be achieved m 10% calf serum
3. As an example, we have obtained good results with Dako polyclonal antisera to
HSV, diluted 1 m 50, followed by swine-antirabbit antisera at 1 in 25 dilution
followed with rabbit peroxtdase-anttperoxrdase at 1 m 100 dilution
4. Increased background can result from using Impure reagents at this pomt: tt 1s
recommended that analytical-grade (AR) reagents be used throughout
Assays for HSV Gene Express/on 353

5. If excessive background IS encountered, it may be useful to repeat the acetylation
step Acetic anhydride has a very short half-life and must be made up freshly.
6. Calculation of T,,,* the temperature (T,,,) at which 50% of double-stranded RNA
hybrids will dissociate m hquid mto smgle-stranded molecules is defined by the
equation:
T,,, (RNA/RNA “C) = 79.8 + 18.5 log,s[Na+] - (0.35 x %formamide) +
584(G+C)+ 11 S(G+C)*
where G + C is (%G + C)/lOO (24,2.5).

References
1, Forrester, A , Farrell, H., Wilkmson, G , Kaye, J., Davis-Poynter, N., and Mmson,
T. (1992) Construction and properties of a mutant of herpes simplex vu-us type 1
with glycoprotem H coding sequences deleted. J Vzrol. 66,341438
2 Dobson, A. T., Margohs, T P., Sederati, F , Stevens, J G , and Feldman, L T
(1990) A latent, nonpathogenic HSV-1 -derived vector stably expresses beta-galac-
tosidase in mouse neurons Neuron 5,353-360
3 Lokensgaard, J R , Bloom, D C , Dobson, A T , and Feldman, L T (1994)
Long-term promoter activity during herpes simplex vtrus latency. J Vzrol 68,
71487158
4 Arthur, J., Efstathiou, S , and Simmons, A. (1993) Intranuclear foci containing
low abundance herpes simplex virus latency-associated transcripts vtsuahsed by
nonisotopic in situ hybridizatron J Gen Vwol 74, 1363-1370.
5. Speck, P G. and Simmons, A. (1991) Divergent molecular pathways of produc-
tive and latent infection with a vnulent strain of herpes simplex virus type 1. J
Vu-o1 65,400 l-4005
6 Rogers, A. W. (1979) Techmques of Autoradiography, 3rd ed ElsevietYNorth-
Holland Biomedical Press, Amsterdam.
7. Gowans, E J , Jilbert, A. R., and Burrell, C. J. (1989) Detection of specific DNA
and RNA sequences m tissues and cells by in sztu hybridization, m Nuclerc Aczd
Probes (Symons, R. H , ed.), CRC, Boca Raton, FL.
8. McLean, I. W and Nakane, P. K. (1974) Periodate-lysine-paraformaldehyde fixa-
tive. A new fixattve for immunoelectron microscopy. J. Hzstochem Cytochem
22,1077-1083
9 McAllister, H. A. and Rock, D L. (1985) Comparative usefulness of tissue fixa-
ttves for zn situ viral nucleic acid hybridization. J. Hwtochem Cytochem 33,
1026-1032.
10. Moench, T R , Gendelman, H. E., Clements, J. E , Narayan, O., and Griffin, D E.
(1985) Efficiency of zn situ hybridization as a function of probe size and fixation
technique. J. Viral Methods 11, 119-130.
11. Puchtler, H and McLean, S. N. (1985) On the chemistry of formaldehyde fixation
and tts effects on tmmunohtstochemical reactions. Hzstochemutry 82, 201-204.
12. Maples, J. A. (1985) A method for the covalent attachment cells to glass slides
of
for use in immunohtstochemistry assays Am J Clan Pathol. 83, 356-363
Speck and Efs ta thiou
354
13 Angerer, L M , Stoler, M H , and Angerer R. C (1987) In Sztu hybrtdtzatron wtth
RNA probes* an annotated recipe, m In Situ Hybrzdizatzon Appllcatlons to Neu-
robzology (Valentino, K. L., Eberwme, J H., and Barchas, J. D., eds.), Oxford
University Press, New York, pp 42-70
14. Amersham Life Science Catalogue (1995) Amersham International plc, Little
Chalfont, pp. 98-12 1,
15 Sambrook, J , Fritsch, E F , and Mania&, T (1989) Molecular Conrng A Laboratory
Manual, 2nd ed Cold Sprmg Harbor Laboratory, Cold Sprmg Harbor, NY.
16. Moriarty, G. C., Moriarty, C M , and Sternberger, L A (1973) Ultrastructural
mununocytochemrstry by unlabeled antibodies and the peroxidase-antiperoxidase
complex (PAP) A technique more sensitive than radtotmmunoassay J
Hlstochem Cytochem 21,825-833.
17. Sternberger, L. A (1979) Immunochemutry, 2nd ed John Wiley, New York
18 Boemsch, T (1980) Reference Guide Series 1 PAP/Immunoperoxtdase Dako
Corporatton, Santa Barbara, CA.
19 Blum, H E., Haase, A T , and Vyas, G N (1984) Molecular pathogenests of
hepatitis vnus B mfectton. simultaneous detection of viral DNA and antigens m
paraffin-embedded liver sections Lancet 2(8406), 77 l-775
20 Brahic, M , Haase, A T., and Cash, E (1984) Simultaneous zn sztu detection of
viral RNA and antigens. Proc Nat1 Acad Scz USA 81, 5445-5448.
21. Gendelman, H. E., Moench, T. R., Narayan, O., Grtffin, D E., and Clements, J. E.
(1985) A double labeling technique for performing immunocytochemtstry and znsztu
hybndrzatron m vtrus infected cell cultures and tissues. J Vwol Methods 11,93-103
22 Speck, P G and Sunmons, A (1992) Synchronous appearance of anttgen-post-
tive and latently mfected neurons m spinal ganglia of mice infected wtth a vnu-
lent strain of herpes simplex vu-us. J Gen Vzrol 73, 1281-1285.
23 Rex, M. and Scottmg, P. J. (1994) Simultaneous detection of RNA and protein in
tissue sections by nonradioacttve rn sztu hybrtdtzatton followed by tmmunohis-
tochemistry. Bzochemzca (Boehrmger Mannhetm) 3,24-26.
24 Memkoth, J. and Wahl, G (1984) Hybridization of nucleic acids nnmobthzed on
solid supports Anal Biochem 138,267-284.
25 Bodkin, D K and Knudson, D L. (1985) Assessment of sequence relatedness of
double-stranded RNA genes by RNA-RNA blot hybridization. J Vzrol Methods
10,45-52
24
Analysis of HSV-DNA and RNA
Using the Polymerase Chain Reaction
Ramesh Ramakrishnan, David J. Fink,and Myron Levine


1. Introduction
The polymerase chain reaction (PCR) technique 1sa sensitive method for detec-
tion of nucleic acids that can be used to detect herpes simplex vnus (HSV)-DNA
and RNA in tissuesampleswith greater sensitivity than hybridization with specific
probes (1,2). In its most basic form, PCR involves multiple cycles of denaturation
of DNA, annealing with specific primers and replication of specific DNA using a
thermostable DNA polymerase like Tuq polymerase, resulting m amphfication of
a specific DNA sequence.Reverse transcriptase polymerase chain reaction (RT-
PCR) employs a preliminary reverse transcription stepof RNA, using either a spe-
cific 3™ or an oligo (dT) primer, to produce complementary DNA (cDNA), followed
by PCR using primers specific for the transcriptof interest.In its standardapplication,
PCR offers qualitative information regarding the presence or absence of target
sequences, hasbeen usedto analyzelatently infected ganglia and brain for HSV-
and
DNA (6,842) andRNA (6-l 0,12).As describedin the followmg, with the inclusion of
mutated templatesas internal standards, PCR can be usedto determine a quantitative
estimate the numberof HSV genomesand transcriptsin tissueextracts.Histologi-
of
cally, in situ hybridization (ISH) can be used to detect HSV-DNA and RNA m
specific cells in the nervous system (3-5), although it has not been successfully
applied to detect HSV genomes during latency (6,7). PCR methods can be applied
to tissue sections(in situ PCR), making it possible to identify individual cells har-
boring HSV genomes,even during latency (9,23).
7.7. Quantitative DNA-PCR
Several methods have been used to adapt PCR to quantify specific DNA or
RNA sequences (14,2.5). One ofthe simplest methods to quantitate latent HSV-
From Methods m Molecular Medicme, Vol 10 Herpes Simplex Wrus Protocols
Edited by S M Brown and A R MacLean Humana Press Inc , Totowa, NJ

355
Ratnakrishnan, Fink, and Lewne
356

DNA is to amplify known amounts of standard and latently infected gangbomc
DNA in parallel reactions (7, IO). This method is subject to error, because sig-
nificant tube-to-tube variations can occur even when the same dilution of tem-
plate is amplified (1.5). In a second method, a cellular DNA template of known
concentration is amplified along with the target DNA m the same tube (16
18). However, it is known that the kinetics of amplification may vary for dif-
ferent substrates of differing length and/or sequence (15,Z 6).
These limitations are overcome in competitive quantitative PCR (8,9,14), m
which a competitor template is constructed that is identical m sizeand sequence
to the target DNA, except for a single-base mutatron, resulting m erther the
addition or loss of a unique restriction site. The competitor DNA is quantified,
serially diluted, and coamplified with a fixed amount of the target DNA m a
series of tubes, using the same set of primers. After codapllfication, the two
PCR products can be distmguished from each other after appropriate restric-
tion enzyme digestion. The amount of DNA product is proportional to the ml-
tial amount of both DNA templates; the rate of the PCR in both cases is the
same because the conditions of amphfication are identical for the target and
competitor DNA. The relative amount of PCR product is determined for target
as well as competitor by ethidmm bromide staining or radioactive mcorpora-
tton during the assay. The tube in which the target and competitor DNAs are
equivalent allows determination of the amount of the target DNA.
We describe a method to quantitate the number of HSV- 1 genomes in latently
infected nervous tissue by competitive quantitative PCR (8,9). A great degree of
care has to be exercised,since amphficatton can be affected by a number of factors,
including the concentration of the different components of the reaction (Mg,
dNTPs, Tag polymerase, template DNA, primers, and so on), and the temperatures
for annealing and extension. We use a two-step PCR method to elimmate
heterodimer formation (14). At the completion of the inmal PCR, an aliquot is
diluted into a fresh reaction mix, followed by only two additional cycles, using a
radioactive tracer to monitor amphfication. We used the AMBIS radioanalytic im-
aging systemfor radioimaging and direct quantitation of the PCR products, since it
is more sensitive than densrtometric scanning of autoradiographs with a dynamic
range of 5 orders of magnitude, compared to lessthan 3 for autoradiography.
7.2. Quantitative RNA-PCR
Conventional methods such as Northern hybridization or ribonuclease pro-
tection assays are limited when attempting to quantitate low levels of viral
RNA transcripts m nervous tissue during HSV latency. RNA can be analyzed
quantitatively by competitive quantitative RT-PCR, analogous to competitive
quantitative PCR for DNA. The same principles considered for quantitative
DNA-PCR hold for RNA quantitation One strategy (IS) mvolves coamphfi-
Analysis of HSV-DNA and RNA 357

cation of known amounts of a DNA template along with the target cDNA, using
the same set of primers. The amount of RNA can be underestimated by this
method, since it assumesthat the efficiency of reverse transcription is lOO%,
whereas it can actually range from 590% (19). Another method (20) mvolves
reverse transcription of the target RNA along with a synthetic RNA standard,
followed by coamplilication of the cDNAs. The standard has flanking sequences
complementary to the same primers as the target mRNA, but possessesa differ-
ent internal sequence so that PCR products of different sizesare obtained. This
technique might not be quantitative because the kmetics of reverse transcription
reactions mvolvmg RNA of different sizesand sequencesoften varies (21)
During HSV latency, the only viral transcript detected is the latency-associated
transcript or LAT (522). We describe a method to quantitate the amount of LAT-
RNA in latently infected nervous tissueusing competitive quantitative RT-PCR (8,9).
First, we constructamutant competitor RNA molecule identical to the LAT message
to be amplified, except for the loss of a unique BsaHI site. We have bypassedthe
need for cloning into a T3 polymerasetranscription vector by directly attachmga T3
polymerase promoter site to our mutant LAT-DNA (8,9). The mutant LAT-RNA is
used to compete with target RNA from latently infected tissuesby competitive RT-
PCR. In this method, both the standardand target LAT moleculesaresubjectedto the
samekinetics of reverse transcription and DNA amplification in the sametube. After
coamplification and digestion with BsaHI, reaction products aredetectedand quanti-
fied as described earlier for the DNA amphfication.
7.3. In Situ PCR
In contrast to solution techniques that measure the total number of mol-
ecules extracted from tissue, zn situ techniques determine which cells in the
tissue contain the target molecules. However, in situ hybridization is limited in
sensitivity when low levels of nucleic acid are involved. In sztu PCR (9,23,23)
can be used to localize DNA m appropriately prepared tissue samples. We
describe an in situ techmque to detect HSV-1 DNA in selected sections from
latently infected rat trigeminal ganglia (9). Specific oligonucleotide primers to
the HSV-1 glycoprotein B (gB) gene and digoxigenin-labeled nucleotides are
used to produce amplified digoxigenin-labeled DNAs znsztu, which are local-
ized using an alkaline phosphatase conjugated antidigoxigenin antibody
detected with the BCIP/NBT reagent system. We have identified neurons in
Infected trigeminal ganglia containing HSV- 1 genomes using this method (9).
2. Materials
2.1. Buffers and Enzymes
1. DNA extractionbuffer: 50 mA4Tris-HCl, pH 8.0,2 mM EDTA, 0.5% Tween-20,
400 mg/mL protemase K
Ramakrishnan, Fink, and Levine
358
2 TRI total RNA isolation reagent (Molecular Research Center, Cincinnati, OH)
3. 10X PCR buffer I: 500 mM KCl, 100 mM Trts-HCl, pH 8 4 at 20°C 15 mA4
MgCl,, 1 mg/mL gelatin
4. PCR assay buffer. 10 pL of 1OX PCR buffer, 4 mL dNTPs (10 mM each), 2 pL of
specific primer pairs, 2 pL Tag polymerase, sterile distilled water to volume Top
the PCR mixture with 50 pL autoclaved mineral oil
5 RT-PCR assay buffer. 10 pL 10X PCR buffer-II (Perkm-Elmer, Norwalk, CT), 2
pL of each dNTPs (10 mA4 each), 5 mA4 MgCl,, 4 mL of LAT primer pans, 8 uL
RNA template extract, 2 pL Taq polymerase, 1000 U MMLV reverse transcrtp-
tase, distilled water to 100 pL and topped off with 50 pL autoclaved mmeral oil
6 lOO-ng/pL Primers
7 Taq polymerase* Any commercial vartety avatlable can be used, but optimal concen-
tration to be used must be first determined by tttratmg wtth appropriate substrate
8 Mmeral 011: Light whtte from Sigma (St. LOUIS, MO)
9. Reverse transcrlptase Either MMLV or AMV
10. DNase I, RNase free.
11 NuSteve GTG agarose (FMC BioProducts, Rockland, ME)
12 T3 RNA polymerase
13 In sztu PCR reaction mix. 1X PCR buffer II, 1 mM MgCl,, 1X digoxigenm DNA
labeling mix, 100 ng of primer mix, 10% glycerol, and 1 pL of Taq polymerase
2.2. Primers
The primer pairs used were:
1 For gB. 5™ primer ATT-CTC-CTC-CGA-CGC-CAT-ATC-CAC-CTT; 3™ primer
AGA-AAG-CCC-CCA-TTG-GCC-AGG-TAG-T
2. For LAT: 5™ primer GAC-AGC-AAA-AAT-CCC-GTC-AG; 3™ primer ACG-
AGG-GAA-AAC-AAT-AAG-GG (6), and
3 For glyceraldehyde phosphate dehydrogenase (GAPDH): 5™ primer ATT-GGG-GGT-
AGG-AAC-ACG-GAA, 3™ primer ACC-CCT-TCA-TTG-ACC-TCA-ACT-A

3. Methods
Any one of several methods can be used to extract DNA and RNA from tissue.
One stmple method for preparing DNA and RNA extracts that we have used to quan-
ttfy DNA and RNA from infected rat htppocampus by competmve quantttattve PCR
(8), is described below The samples are taken from ttssue mounted on glass slides
3.1. Extraction Procedure for DNA
1. Chill IO-pm sections on glass shdes at -20°C.
2. Scrape the region of interest from the brain sections at -20°C wtth a prechdled
sterile razor blade and transfer the tissue to stertle Eppendorf tubes containing
1.O mL of absolute ethanol at room temperature Vortex for 1 min and pellet m a

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