Molecular Diagnosis of Infectious Diseases

Prévia do material em texto

1 
Identification of Novel Diagnostic Markers 
by Differential Display 
Peng Liang, Feilan Wang, Weimin Zhu, Robert P. O’Connell, 
and Lidia Averboukh 
1. Introduction 
Accurate and early diagnosis of a disease state such as a viral infection, or in 
a more complicated situation cancer, means live saving because proper medi- 
cal interventions can be applied in a timely manner before it is too late to treat 
the disease. Thus it is crucial that good diagnostic markers for any relevant 
diseases can be obtained. The markers can be in the form of DNA such as yirai 
integration or chromosomal DNA aberrations including deletions, transloca- 
tions, and point mutations. As a result, these genetic abnormalities, in turn, 
may lead to altered gene expressions, such as new genes being turned on. 
Therefore, the markers can also be in the form of mRNAs or their protein prod- 
ucts. DNA-based diagnosis is now done mostly with amplification technology 
breakthroughs such as polymerase chain reaction (PCR). However, protein- 
based diagnosis such as a blood antibody test for a disease specific antigen 
(e.g., HIV and HPV virus infections; prostate-specific antigen for prostate can- 
cer) are more accurate, convenient, and noninvasive. Traditionally, a good 
diagnostic marker for an infectious disease can be obtained by identifying the 
etiological agent such as a vnus or a bacterium, which may not be always easy. 
But for noninfectious diseases such as cancer, a good marker may be even 
harder to come by because the alteration is more subtle and difficult to detect. 
In this chapter we present a methodology known as differential display (1) 
that is particularly useful in findmg diagnostic markers for pathological pro- 
cesses in which altered gene expression plays a role. Examples are given for 
the applications of the method to identify a viral infection and a candidate 
secretory marker for oncogenic YLZS mutation. 
From Methods m Molecular Medrone, Vol 13 Molecular Dfagnoski of lnfectrous Dmases 
Edlted by U Relschl Humana Press Inc , Totowa, NJ 
3 
4 Llang et al 
The general strategy for differential display is outlined m Fig. 1. Thts method 
depends on combmatlon of three techniques brought together by one concept. 
1 Reverse transcriptlon from anchored oligo-dT primers to subdwde total mRNA 
population, 
2 Choice of arbitrary primers for settmg the number of amplifiable cDNAs, each 
corresponding to part of a mRNA 3’ termml, and 
3 Denaturmg polyacrylamlde gels for high resolution separation of amplified 
cDNA fragments 
By changing primers systematlcally from both dlrectlons, most mRNA 3’ 
termml m a cell may be displayed without any prior knowledge about their 
sequence mformatlon (2) Side-by-side comparison of mRNAs from drfferent 
samples (e.g., normal versus abnormal or infected versus noninfected) would 
allow genes uniquely expressed m an affected sample to be detected, isolated, 
and used as a marker for disease ldentlficatlon. 
The objective 1s to obtain a cDNA tag of a few hundred bases, that is suffi- 
ciently long to uniquely identify a mRNA and yet short enough to be separated 
from others by size at high resolution by denaturing polyacrylamlde. Pairs of 
primers are selected so that each will amplify cDNA from about 50-100 mRNAs, 
because this number 1s optimal for display m one lane of the gel. In the original 
scheme of differential display, 12 two-base anchored ohgo-dT primers were used 
to subdivide the mRNAs during the reverse transcription reaction (I) Two-base 
anchored primers that degenerate at the penultimate base, were since mtroduced 
to overcome the redundancy of their prtmmgs and simplify the reverse transcnp- 
tlon reactions (3). More recently one-base anchored oligo-dT primers were 
described for the same purpose (4). The method described in this chapter is 
largely adapted from the protocol of the RNAmapTM kit from GenHunter Corpo- 
ration (Nashville, TN), based on the use of degenerate two-base anchored pnm- 
ers. However, the method 1s the same no matter what type of anchored primers 
are used and, therefore, the protocol could be adopted for different primer variations. 
2. Materials 
1 5X Reverse transcrlptlon buffer 125 mA4 Tns-HCl, pH 8.3, 188 mA4 KCI, 
7 5 M&l,, 50 mMDTT 
2 100 U/mL MMLV reverse transcriptase 
3 250 @dNTP 
4. 10 @4TlzMG (see Note 1) 
5 10 @4T12MA (see Note 1) 
6 10 @4T12MT (see Note 1) 
7. 10 @I TlzMC (see Note 1) 
8 1 OX PCR buffer 100 mMTns-HCl, pH 8 4,500 mMKCl,l5 mM MgCl,, 0 0 1% 
gelatin 
Different/al Display 5 
G---An 
. ..------ _ -----. 
T---An 
Cm--An 
I Reverse Transcription 5'-TTTTTTTTTTTTMG-3'(T12MG) 
dNTPs 
MMLV Reverse Transcriptase 
Cm--An 
rc GMTTTTTTTTTTTT 
I 
5'-AGCCAGCGAA-3(AP-1 Primer) 
II. PCR Amplification 5'-TTTTTTTTTTTTMG-3' (T12MG) 
dNTPs 
a-[35S-dATP) 
AmpliTaq DNA pOlymerase 
AGCCAGCG; 
GMTTTTTTTTTTTT 
AGCCAGCGA' 
GMTTTTTTTTTTTT 
AGCCAGCGAA 
GMTTTTTTTTTTTT 
I:: 3enaturmg Polyacrylamide Gel 
RNA Sample' X Y 
I- 
-- Negative Electrode (-) 
-- 
-+ 
-- 
-- 
Positive Electrode(t) 
Fig. 1. SchematIc representation of differential display method 
6 Liang et al. 
9 25 @4dNTP. 
10 2 pA4 Arbitrary 1Omers with 50-70% GC content. 
11, 10 mg/mL Glycogen. 
12 Autoclaved distilled water. 
13. Loading dye: 0.1% xylene cyanole FF, 0.1% bromophenol blue, 10 mM EDTA, 
95% formamrde. 
14. 10 U/pL RNase-free DNase I. 
15 Thermocycler 
16. 5 U/pL AmphTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). 
17. a-[35S]dATP (> 1,000 Ci/mmol) or a-[33P]dATP (2,000 Wnrnol) (DuPont- NEN). 
3. Methods 
3.1. DNase I Treatment of Total RNA 
Removal of all contaminating chromosomal DNA from the RNA sample is 
essential before carrymg out differential display (see Note 2). 
1 Incubate 10-100 pg of total cellular RNA with 10 U of DNase I (RNAse free) m 
10 mMTrrs-HCl, pH 8.3, 50 mMKC1, 1.5 mMMgC1, for 30 mm at 37°C. 
2. Inacttvate DNase I by addmg an equal volume of phenolchloroform (3: 1) to the 
sample. Mix by vortexmg and leave the sample on Ice for 10 mm 
3 Centrrfuge the sample for 5 mm at 4°C m a Eppendorf centrifuge. Save the super- 
natant and ethanol precipitate the RNA by adding 3 vol of ethanol in the presence 
of 0 3MNaOAC After incubation at -8O’C for 30 mm, pellet the RNA by centri- 
fuging at 4’C for 10 mm Rinse the RNA pellet with 0 5 mL of 70% ethanol 
(made with DEPC-H,O) and redissolve the RNA in 20 $ of DEPC-treated Hz0 
4 Measure the RNA concentration at ODsbO with a spectrophotometer by diluting 
1 pL of the RNA sample m 1 mL of H20. Check the integrity of the RNA samples 
before and after cleaning with DNase I by runnmg l-3 pg of each RNA on a 1% 
agarose gel with 7% formaldehyde. Store the RNA sample at a concentration 
higher then 1 pg/pL at -80°C before usmg for dtfferentlal display 
3.2. Reverse Transcription of mRNA 
The success of the differential display technique depends on the integrity of 
the RNA and that it is free of chromosomal DNA contaminatton. 
1. Set up four reverse transcrrption reactions for each RNA sample in four PCR 
tubes (0.5 mL size), each containmg one of the four different T12MN anchored 
primers, m a final volume of 20 pL: 9.4 pL dH,O, 4 pL 5X RT buffer, 1.6 pL 
dNTP (250 ClM) 2 pL (0.1 clg/$ DNA-free total RNA) mRNA, 2 pL T,*MN (10 pA4) 
2. Program your thermocycler to 65°C 37°C for 5 min, 95°C for 60 min, 4’C for 5 min. 
3. After 10 min 1 pL MMLV RT is added to each tube at 37°C to mniate the reverse 
transcrtption reaction. At the end of the incubation, spm the tube briefly to col- 
lect condensatton. 
4 Set tubes on ice for PCR or store at -7O’C for later use 
Differential Display 7 
3.3. PCR Amplification and Labeling of cDNAs 
1. Set up a 20-@, PCR reaction for each primer set combmatron using the followmg 
formula for afinal volume of 20 pL (see Note 3): 9.2 pL dH20, 2 pL 10X PCR buffer, 
16 pL dNTP(25 tu2/i), 2 pL AP-pnmer (2 CUM), 2 pL Tt,MN (10 p&I), 2 pL (wnh the 
same T,JviN used for the PCR) RT-mix from Step 1,1 & (or 0 2 $ of a-[33P]dATP, 
2000 Ci/mmol) a[35S]-dATP (1200 Ct/mmol), 0.2 pL AmphTaq (Perkin-Elmer). 
2. Mix well by prpetmg up and down. Add 25 pL mineral or1 if the thermocycler 
requires tt PCR to 94°C 40°C for 30 s, 72°C for 2 mm, 30 s, 40 cycles at 72°C 
for 30 s, and 4°C 5 min 
3. Followmg PCR, mcubate 3 5 & of each sample plus 2 $ of loading dye at 8O’C 
for 2 mm immediately before loading onto a 6% DNA sequencing gel. 
4. Electrophorese for about 3 h at 60 W constant power until the xylene dye IS 10 cm 
from the bottom. The DNA sequencing gel should be dried without fixing with 
methanol/acettc acrd. 
3.4. Recovery and Reamplification of cDNA Probes 
1 Orient the autoradiogram and dried gel with radtoacttve mk or needle punches. 
2. After developing the film (overnight to 72-h exposure), ahgn the autoradiogram 
with the drred gel. Locate bands of interest either by marking wrth a clean pencil 
or cutting through the film (handle the dried gel with gloves and save tt between 
two sheets of clean paper) Cut out the bands of interest with a clean razor blade. 
3. Soak the gel slice along with the 3Mpaper m 100 pL dHzO or TE buffer for IO mm 
Boil the tube wrth tightly closed cap for 1.5 min Spin for 2 mm to collect conden- 
satron and pellet the gel and paper debris. 
4. Transfer the supernatant to a new microfuge tube Add 10 & of 3M NaOAC, 5 pL 
of glycogen (10 mg/mL) and 450 pL of 100% ethanol Let sit for 30 mm on dry 
ice or m a -80°C freezer. Spin for 10 mm at 4°C to pellet DNA 
5 Remove supernatant and rmse the pellet with 200 @ ice-cold 85% ethanol. Spm 
briefly and remove the residual ethanol Dissolve the pellet with 10 pL of dHzO 
and use 4 pL for reamplification Save the rest at -2O’C in case of mishaps 
6 Reampltfication should be carried out using the same prrmer set and PCR condi- 
ttons except the dNTP concentrations are at 20 @4 (use 250 w dNTP stock) 
mstead of 2-4 @4 and no isotopes added A 40 pL final volume for each primer 
set combmatton 1s recommended: 20.4 pL dH*O, 4 pL 10X PCR buffer, 3.2 pL, 
dNTP (250 @I), 4 pL. AP-primer (2 @I), 4 & T12MN (10 p&I), 4 pL. cDNA 
template from step 8, 0.4 pL AmpbTaq (Perkm-Elmer) 
7 Remove 30 pL of PCR sample and run on a 1.5-2% agarose gel stained wrth 
ethrdmm bromide. Save the remaining PCR samples at -20°C for subclonmg. 
Check to see if the size of your reamphfied PCR products are consistent with 
theu size on the DNA sequencing gel 
8. Extract the reamphfied cDNA fragments from the agarose gel with a Qraex kit 
(Qragen) Perform Northern blot analysis of cDNA probes obtained from differential 
display following the standard procedure (5). It 1s recommended that the washing 
temperature does not exceed 55°C as these cDNA probes are short (1 O&500 bp) 
8 Liang et al 
3.5. Subcloning and Sequencing of Cloned cDNA Probes 
After confirmation by Northern blot analysts, reamplified cDNA probes 
could be cloned into various plasmrd vectors kits such as pCR-TRAPTM posl- 
trve-selectton cloning vector (GenHunter) or TA cloning ktt (Invitrogene, 
San Diego, CA) and then subjected to DNA sequence analysts. 
3.6. Isolation of the FuN-Length cDNA, Expression 
of Recombinant Proteins and Antibody Preparation 
As you can see, differential display is a method to identify and isolate probes 
to drfferentrally expressed genes. With the 3’ termmt of cDNA obtained by the 
method, the correspondmg full length cDNAs can be tsolated by screenmg a 
cDNA library, the encoded proteins can be expressed and used to prepare anti- 
body, all followmg the standard procedures (4) 
3.7. Specific Applications of Differential Display 
Smce Its descrtptron, drfferenttal display methodology has been used to suc- 
cessfully identtfy a number of genes that are potentially important not only for 
the understanding of a disease but also for their dragnosrs. These include the 
rdenttficatton of a macrophage lectm as a candidate marker for rejection fol- 
lowing heart transplantation (6), cyclin G as a marker for tumor suppressor gene 
p53 mutation (7), and Mob-l as a candtdate marker for oncogemc ras mutation 
(81 Sections 3.7.1. and 3.7.2. contam examples how these markers could be 
Identified. 
3.7.1. Identification of Viral infections by Differential Display 
NIH3T3 cells were compared with its spontaneously transformed derrvatrve 
NIH3T3-T by differential display m an attempt to understand the nature of the 
transformatton of the parental cell lme (see Fig. 2A) When the TlzMC primer 
was used m combinatron with five arbitrary decamers (AP-6 to AP-10, 
GenHunter) to amplify the mRNAs, the cDNA patterns displayed between the 
nontransformed cells and the transformed cells looked very similar, except 
a band amplified with the AP-9 primer appeared to be strtkmgly different 
(Fig. 2A; mdtcated by an arrowbar). The band was recovered and amplified as 
a probe for Northern blot analysis (see Fig. 2B). The result confirmed that this 
gene was indeed only expressed in the transformed cell DNA sequence analy- 
SIS indicates that the gene matches the cDNA of gag gene from murine leuke- 
mta vnus. This result suggests that the cause of cell transformatron may be the 
infection of the murme retrovirus. The gag gene therefore represents an excel- 
lent molecular marker for drfferentratmg the transformed cells from the normal 
parental cell line m this case. 
Differential Display 9 
3684 
Fig. 2. Identification of a viral infection by differential display. (A) Total RNA 
samples from NIH3T3 cells (lanes 1, 3, 5, 7, 9) and its spontaneously transformed 
derivative NIH3T3-T (lanes 2,4,6,8, 10) were compared by differential display using 
the T12MC anchored primers and 5 arbitrary decamers, AP-6 to 10 (GenHunter). 
Primer sequences are AP-6 (5’-GCAATCGATG-3’), AP-7 (5’- CCGAAGGAAT-3’), 
AP-8 (5’-GGATTGTGCG-3’), AP-9 (5’-CGTGGCAATA-3’), and AP-10 (5’-TAG- 
CAAG-TGC-3’). The arrowbar indicates gag, an cDNA dramatically overexpressed 
in the transformed cells (lane 8). (B) Northern blot analysis of gag in NIH3T3 (lane 1) 
and its transformed derivative NIH3T3-T (lane 2). The same blot was reprobed with 
36B4 as a control for equal RNA loading. 
3.7.2. Identification of a Secretory Marker 
for Oncogenic H-ras Mutation 
One of the earliest and most potent oncogenes identified in human cancer 
has been the mutant ras (9), and the most commonly found mutations in human 
cancer are in the ~53 tumor-suppressor gene. Mutations in both YUS and p53 
were found at high incidence in neoplasia of colorectal, bladder, pancreatic, 
and subgroups of certain lung cancers. The finding that mutated YUS and p53 
cooperate in cell transformation of rodent primary embryo fibroblasts, makes 
the system an excellent model for studying molecular mechanisms of cancer 
IO 
(I&11). The early detection of mutations in ras or ~53 genes (mostly single 
base substitutions) at DNA level are still difficult and invasive, given the fact 
that these proteins are not secreted. As discussed, ideally a good diagnostic 
marker would be detectable through nonmvasive procedure such as a blood or 
urine test (no surgery needed). So the identification of any secretory proteins 
encoded by genes activated by mutation m oncogene ras or tumor-suppressor 
gene ~53 would provide candidate tumor markers that could be potentially 
detected from body fluids such as blood. 
In an attempt to identify genes that are differentially expressed as a result of 
cell transformation caused by the cooperation of mutant H-ras and p53 genes, 
differential display was applied systematically to compare patterns of mRNA 
expression from nearly isogenic cells plus and minus mutatedH-ras and p53 
genes These cells are normal rat embryo fibroblast (REF) cells and their 
derivatives that were doubly transformed by oncogenic H-ras and either a 
nontemperature sensitive (Tl 01-4 cell line) or temperature sensitive mutant 
~53 (Al-5 cell line) grown at nonpermtssive (mutant conformation) and per- 
missive temperatures (wild-type conformation) (II). One cDNA probe ampli- 
fied with T,,MA and AP-2 primers, designated mob-l (8), was detected by 
differential display to be reproducibly seen only m the transformed cells 
(Fig. 3A). Its expression did not appear to be affected by the status of ~53 
protein. The 240-bp cDNA fragment was recovered from the dried denaturing 
polyacrylamide gel and reamplified using the correspondmg pair of primers. 
The reamplified cDNA was used as a probe m a Northern blot analysis to con- 
firm the differential expression of a 1.2 kb mRNA only m the transformed cells 
(see Fig. 3B). The promoter analysis confirms the mob- 1 gene is activated tran- 
scriptionally be ras mutation. The full length mob- 1 cDNA was isolated from a 
cDNA library and completely sequenced. The predicted protein encodes a 
secreted polypeptide (with signal peptide) of 8 kDa (8). 
As differential display is now widely used for the identification and isola- 
tion of differentially expressed genes due to its simplicity, sensitivity, and ver- 
satility, one should be reminded that the method is unlikely able to detect 
mutations at the DNA level directly. For diseases caused by single gene muta- 
tions that have clear genetic component, chromosome mapping of the mutation 
locus should be a method of choice. Differential display is unlikely to detect 
such mutations unless the mutated gene affects other gene expressions as m the 
case of p53 or ras mutations. It should be emphasized that the method is only a 
simple screenmg tool and it is by no means a fool-proof method if precautions 
are not taken seriously (see Notes 4 and 5). As such, neither every dtfference in 
the pattern of displayed DNA may represent a true differential gene expres- 
sion, nor would every differentially expressed gene be obviously relevant to 
the system being studied 
Differential Display 11 
B 
12345 
Fig. 3. Differential display and Northern blot confirmation of mob-l gene expres- 
sion in primary REF cells versus their derivatives doubly transformed by mutant 
H-ras and ~53. (A) Total RNAs from normal REF cells (lane 1) and its derivatives 
transformed by mutant H-ras and ~53, T 10 l-4 (lane 2) and A l-5 which contains 
a temperature-sensitive mutant p53 grown at nonpermissive and permissive tempera- 
tures for 24 h (lanes 3 and 4) were compared by differential display. T,,MA was used 
as an anchored oligo-dT primer and AP-2 (5’-GACCGCTTGT-3’) was used as a 
arbitrary primer. A band, named mob-l (indicated by the arrow) was differentially 
detected. (B) Northern blot confirmation of differential expression of the mob- 1 gene 
in the transformed cells TlOl-4 and Al-5 at nonpermissive and permissive tempera- 
tures (lanes 3 to 5, respectively) but not in the REF and immortalized Rat1 cells (lanes 
1 and 2, respectively). Twenty micrograms of total RNA from each cell line was ana- 
lyzed with the 240 bp mob- 1 cDNA probe obtained by differential display. The lower 
panel is a negative of ethidium bromide staining of total RNA samples as control for 
equal loading. 
12 Liang et al 
4. Notes 
1 In TlzMN primers, “M” represents a degenerate base with an equal mixture of G, 
A, or C (3) Although some of the materials may be purchased from various ven- 
dors, most of them can be obtained m kit form or mdlvldually from GenHunter 
2 Total RNA Isolated by any method frequently has a different degree of chromo- 
somal DNA contammatlon Without its removal by DNse I treatment, chromo- 
somal DNA will be amplified along with cDNA made by reverse transcrlptlon 
Different degrees of DNA contammatlon m samples being compared is a fre- 
quent cause of high background, false posltlves that cannot be confirmed by 
Northern blot analysis, or failure of detecting signals on Northern blot as 95% 
chromosomal DNA do not encode mRNAs It is recommended that total RNA be 
always treated with DNase I before bemg used for differential display 
3 Make core mixes as much as possible to avoid plpetmg errors, otherwlse, It would 
be difficult to accurately pipet 0 2 pL of AmphTaq For example, aliquot RT-mix 
and AP-primer separately, but make 10 times of the PCR core mix. This core mix 
can be used m combmatlon with five different AP-primers for a pan of RNAs to 
be compared. 
4. One should make an effort to ensure the umformlty of the samples being com- 
pared When RNAs isolated from tissue specimens contammg mixed cell types 
are being compared, verlficatlon of dlfferentlal gene expression should be car- 
ried out also at the cellular level 
5. It 1s fair to say that finding dlfferentlally expressed genes IS no longer rate-llmlt- 
mg with differential display method However, one of the most commonly 
ignored aspect m using differential display 1s a poor experlmental design. Given 
the versatility of dlfferentlal display, instead of comparmg just two samples, one 
should think about the posslbillty of comparing multiple RNA samples simulta- 
neously so adequate experlmental control or mltial characterlzatlon of the gene 
being isolated are bmlt-in to mmlmize lsolatmg false positives or trivlal genes 
This 1s particularly true when most of the slgmficant genes Isolated with this 
method had elegant experimental designs. 
Acknowledgments 
The authors dedicate this chapter to Arthur B. Pardee for his mspiratlon, 
guidance, and constant support. We also thank GenHunter Corporation for the 
permission to adapt the protocol from Its dlfferentlal display kits. 
References 
1, Llang, P and Pardee, A B. (1992) Differential display of eukaryotlc messenger 
RNA by means of the polymerase cham reaction. Science 257,967-97 1 
2. Llang, P., Averboukh, L , and Pardee, A B (1994) Method of differential dls- 
play. Methods Mel Genetm 5, 3-16 
3. Liang, P , Averboukh, L., and Pardee, A. B (1993) Distribution and clonmg of 
eukaryotlc mRNAs by means of differential display. refinements and optlmiza- 
tlon. Nucleic Acids Res 21, 3269-3275 
Differential Display 13 
4. Llang, P., Zhu, W , Zhang, X , Guo, Z , O’Connell, R P , Averboukh, L , Wang, 
F., and Pardee, A B (1994) Dlfferentlal display usmg one-base anchored ollgo- 
dT primers. Nucleic Acids Res 22, 5763-5764. 
5. Ausubel, F., Brent, R , Kingston, R E , Moore, D. D , Seidman, J G , Smith, J 
A, and Struhl, K (1988) Current Protocols zn Molecular Biology, Greene and 
Wiley, New York. 
6 Russell, M., Utans, U , Wallace, A F., Llang, P., Arcecl, M J , Karnovsky, M J , 
Wyner, L. R , Yamashlta, Y., and Tarn, C. (1994) Identlficatlon and upregulatlon 
of galactose/N-acetylgalactosamme macrophage lectm m rat cardiac allografts 
with arteriosclerosis J Clan Invest 94, 722-730 
7 Okamato, K and Beach, D. (1994) Cyclin G 1s a transcrlptlon target of the ~53 
tumor suppressor protein. EMBO J 13,48 16-4822 
8 Llang, P , Averboukh, L , Zhu, W , and Pardee A B (1994) Ras activation of 
genes Mob-l as a model. Proc Nat1 Acad Scz USA 91, 12,515-12,519. 
9 Shih, C and Weinberg, R A (1982) Isolation of a transformmg sequence from a 
human bladder carcinoma cell lme Cell 29, 16 l-l 69 
10 Land, H , Parada, L F , and Weinberg, R A (1983) Tumorlgemc converslon of 
prrmary embryo fibroblasts requires at least two cooperatmg oncogenes Nature 
304,596-602 
11 Martmez, J., Georgoff, I , Martinez, J , and Levme, A J (199 1) Cellular locahza- 
tion and cell cycle regulation by a temperature-sensitive ~53 protein. Genes Dev 
5, 152-159 
2 
lmmunoprecipitation 
Kari Johansen and Lennart Svensson 
1. Introduction 
Immunoprecipitationallows the investigator to detect and quantitate anti- 
gens in a mixture of proteins or characterize a specific antibody response to 
already well-characterized proteins. Addition of antibodies to proteins, usually 
radiolabeled, allows formation of antigen-antibody complexes. After separa- 
tion from contaminating proteins, the complexes are disassociated and the pro- 
teins of interest are separated by sodium dodecyl sulphate-polyacrylamide gel 
electrophoresis (SDS-PAGE). Size and quantity of proteins may then be ana- 
lyzed either by autoradiography or a gel scanning procedure. Immunoprecipi- 
tation is extremely sensitive and may detect very small amounts of radiolabeled 
protein (detection level -100 pg protein or 100 cpm/protem). Unlabeled 
proteins may be used if other sensitive detection methods are utilized, e.g., 
enzymatic activity assays or Western blotting. The advantage of the immuno- 
precipitation technique vs nnmunoblotting is the possibilrty to analyze the 
immune response to proteins expressed in their native conformation. Radioim- 
munoprecipitation assay (RIPA) is used routinely for the detection of viral pro- 
teins, characterization of monoclonal and polyclonal antibody preparations, 
and determination of the specificity of the immune response to various 
pathogens (l-3). 
The major steps m mnnunoprecipitatlon are: 
1. Labeling of proteins expressed by mammalian cells; 
2. Lys~s of the cells, 
3 Addition of antibodies to lysed cells and formation of antigen-antibody complexes, 
4 Purification of the specific immune complexes; and 
5 Analysis of the immunoprecipitated proteins 
From Methods m Molecular Medune, Vol 13 Molecular Dlagnosm of Infectlow Diseases 
Edtted by U Reschl Humana Press Inc , Totowa. NJ 
15 
16 Johansen and Svensson 
7.1. Metabolic Labeling of Proteins 
Several techniques are available for labeling proteins. Usually, radiolabeled 
essential ammo acids, such as 35S-labeled methiomne and 35S-labeled cysteme, 
are used to label newly synthesized protems expressed m mammalian cells, 
either naturally or after transfection or infection 35S emits a weak P-radiation 
easily detected either by autoradiography or gel scanning and has a half-life of 
87.1 d. Although most strams of yeasts and prokaryotes synthesize methtonine 
and cysteme (I), 35S-labeled sulphate is used as the primary metabolic precur- 
sor to label protems in these orgamsms. 
Other possibilities for labelmg are biotmylatron or iodmation of, e.g., sur- 
face proteins or secreted protems. The biotmylated proteins may be mnnuno- 
precipitated by avidm bound to agarose beads (Vector, Burlmgame, CA), which 
presents as an interestmg alternative to the use of radioactive isotopes. 
1.2. Lysis of Cells 
Lysis of the cells is the most crucial part of mrmunoprecipitation and several 
techniques may be utilized. The aim is usually to solubihze the target antigen 
m an immunoreactive, undegraded, and btologically active form. When choos- 
ing a lysis method, a good strategy is to start with a crude lysis buffer and if the 
antigen is released, then step back, remove and alter the buffer composition 
until conditions have been optimized. Variables that have been found to influ- 
ence the efficiency of solubilization are the iomc strength; salt concentration 
and pH of the lysis buffer; the presence of divalent cations, cofactors, and sta- 
bilizing ligands, and the concentration and type of detergent used (ionic, zwit- 
teriomc, or nomomc). Among the common detergents, SDS denatures protems, 
whereas most other detergents do not. Antigen release may be tested by 
immunoblottmg of the lysate and the remammg cell debris. In general, deter- 
gent lysis with a single or triple detergent lysis buffer is usually advised as a 
start. Repeated freezing and thawing of lysates may lead to excessive pro- 
teolytic degradation. 
Most soluble nuclear and cytoplasmic proteins may be solubilized by lys~s 
buffers contammg the nomomc detergents Nomdet P-40 (NP-40), Triton X- 100 
or the zwittenomc detergent 3-[(3-Cholamidopropyl)dimethylammon~a]- 1 -pro- 
pane-sulfonate (CHAPS) (Boehrmger-Mannhelm, Mannheim, Germany) con- 
tammg a relatively high salt concentration (0.5M NaCl or 0.6M KCl), low salt 
concentration, or no salt (Table 1). Possible ranges for the lysis buffers include 
nomonic detergent concentrations between 0.1 and 2%, ionic detergent con- 
centrattons between 0 01 and 0.5%, salt concentrations between 0 and lA4, 
divalent cation concentrations between 0 and 10 mM, EDTA concentrations 
between 0 and 5 mM, and pH values between 6 0 and 9 0. The addition of 
RNases and DNases as well as protease mhibitors may protect the target antl- 
lmmunoprecipitation 17 
Table 1 
Detergents Often Used in Lysis Buffer 
Detergent 
Ionic detergents 
Deoxychohc acid, 
sodium salt 
SDS 
Zwitterionic detergents 
CHAPS 
Noniomc detergents 
Nomdet P40 (NP-40) 
TritonB X- 100 
n-Octylglucoslde 
Ability to disperse Denatures 
protem aggregates proteins 
High No 
High Yes 
High No 
Low No 
Low No 
Low No 
Working 
concentrations 
0. l-l 0 mg/mg 
membrane lipid 
> 10 mgimg protein 
6.5-13 mM 
l-l 0 mA4IL 
l-5 mM 
46 mhf 
gen (Table 2). The susceptibility to proteases varies greatly with cell-surface 
proteins and secreted proteins generally bemg more resistant than cytoplasmic 
proteins. Therefore, it IS advlsed to work on ice and include a protease mhlbltor 
m the lysls buffer. Proteases are divided mto five classes according to their 
mechanism of catalysis: serine proteases, cysteine proteases, aspartlc proteases, 
metalloproteases, as well as enzymes wrth unknown reaction sites (4) Among 
the two most commonly used protease inhlbltors, aprotmm 1s a serene protease 
inhibitor and phenylmethylsulfonyl fluoride (PMSF) IS a serine and cysteme 
protease inhibitor (5), 
Unlike other compartments m the cell where protein folding occurs, the 
endoplasmic reticulum is oxidizing and therefore promotes the formation of 
disulfide bonds. Although the cytosol of mtact cells IS reduced, and therefore 
prevents formation of dlsulfide bonds, disruption of cells without previous 
treatment with alkylating agents such as N-ethylmalermlde (NEM), ~111 mcor- 
rectly introduce disulfide bonds in cytosolic proteins (6). To avold artificial 
disulfide bond formation during cell lysls, cells should be washed with ice- 
cold phosphate-buffered saline (PBS) containing NEM and lysed m lysls buffer 
containing NEM. 
1.3. Formation of Antigen-Antibody Complexes 
1.3.7. Direct lmmunoprecipitation 
The antibody or antigen preparation to be tested by immunopreclpitatlon IS 
now allowed to react with ahquots of the cell lysate or a specific antibody The 
antibody-antigen complex IS then precipitated by one of several methods. 
Depending on the antlbody to be preclpltated, staphylococcal protem A or 
18 
Table 2 
JohansenandSvensson 
Protease Inhibitors Often Used in Lysis Buffers 
Inhrbttor Specificity Solubrlity/stabthty 
Startmg 
concentrations 
Anttpam- Trypsm, papain, 
drhydrochlortde plasmin 
Aprotmm Plasmm, trypsm, 
chymotrypsm 
Chymostatm a, p, y, &Chymo- 
trypsm 
E-64 Papain, 
cathepsm B, L 
Leupeptin Trypsm, 
papain, plasmm, 
cathepsm B 
PMSF Chymotrypsm, 
trypsm, 
thrombm, 
papain 
Soluble m H,O, 50 pg/mL 
methanol DMSO 
Dilute soluttons 
stable 1 mo at -20°C 
Soluble m H,O, 0.06-2 pg/mL 
aqueous buffers 
Dilute solutrons 
stable 6 mo at -20°C 
Soluble m glactal 0 1 ng/mL 
acetic acid, DMSO 
Dilute solutrons 
stable 1 mo at -2O’C 
Soluble m a 1.1 0.5-10 pg/mL 
mixture (v/v) of 
ethanol and water 
Soluble m H,O 0.5 pg/mL 
Dilute solutions 
stable 6 mo 
at -20°C 
Soluble m 2-propanol 17-l 70 pg/mL 
Stock soluttons > 100 mM (0 l-l n&Q 
PMSF m 100% 2-propanol 
stable at room 
temperature>l yr 
streptococcal protein G bound to Sepharose beads or Jacalin, a lectin purified 
from jack-fruit, bound to agarose beads may be used 
Protem A consists of a smgle polypeptlde chain with two accessible high- 
affimty binding sites for the Fc region of IgG and secondary binding sites for 
the Fab region. The protein A antibody complexes disassociate at low pH, boil- 
mg, and/or the addition of reducing reagents. Protein A binds a relatively broad 
spectrum of antibodies and antibody subclasses from different species (Table 3). 
One milliliter of protein A-Sepharose beads can adsorb 1 O-20 mg of IgG. The 
specificity of antibody capture applications can be manipulated by adJusting 
the pH and iomc strength of the bmdmg and elution buffers (7-9). 
Native and recombmantly engineered protem G bmd specifically to more 
species of immunoglobulms than does protein A (Table 3). The recombmant 
lmmunoprecipitatlon 79 
Table 3 
Specificity of Protein A, Protein G, and Jacalin 
for lmmunoglobulins from Various Speciesatb 
Antibodies Protein A Protein G type 2 Jacahn 
Human IgG 1 ++ ++ - 
Human IgG 2 ++ ++ 
Human IgG 3 - ++ - 
Human IgG 4 ++ ++ - 
Human IgA 1 + ++ 
Human IgA 2 + - 9 
Rabbit ++ ++ 
cow + ++ 
Horse - 4-t 
Goat + ++ 
Guinea pig ++ ++ 
Sheep - ++ 
Dog i-i- ++ 
Pig ++ ++ 
Mouse IgG -I- ++ 
Mouse IgA 9 9 - 
Rat - + 
OReproduced with the permlsslon of Pharmacla Blotech from the data file 
for GammaBmdTM G, type 2 
hBmdmg capacity ++, strong, +, mtermedlate, -, weak 
form does not crossreact with IgM, IgE, IgA, IgD, or serum albumin as protein 
A may do (10-12). 
Jacalin, purified from the jack-fruit Artrocarpus integrifolia, bound to aga- 
rose beads, has recently been mtroduced for the purification of human mono- 
meric immunoglobulin A, subclass 1 (Table 3) (I 3,14). Jacalin IS a lectm 
composed of four identical subunits of approx 10,000 Da each. This glycoprotem 
appears to bind only O-glycosidically linked oligosaccharides, preferrmg the 
structure galactosyl @- 1,3) N-acetylgalactoseamine. The binding capacity of 
the jacalin-agarose beads is 3-4 mg monomeric IgA/mL of gel (Vector). Immu- 
noglobulm A may be eluted m a biologically active form by O.&I4 o-galactose 
m 175 mM Tris-HCl (pH 7.5). 
7.32. indirect lmmunoprecipitation 
If your antibody does not sufficiently bind m any of the aforementioned 
systems, another possibility is to utilize an antt-immunoglobulm antibody 
already bound to, e.g., protein A or G. 
20 Johansen and Svensson 
Table 4 
Polyacrylamide Gel Mixtures 
Separation gel0 
40% Acrylamide 
2% bzs 
8X 1MTris @H 8 8) 
dH,O 
10% Amps 
10% SDS 
TEMED 
6% 75% 
6mL 
324mL 
49mL 
2522 mL 
230 pL 
0.4 mL 
33 N- 
742mL 
4mL 
49mL 
22 77 mL 
230 pL 
0.4 mL 
33 N- 
10% 
10mL 
5 3 mL 
49mL 
19mL 
138 p.L 
0.4 mL 
14 d- 
11% 
11 mL 
588mL 
49mL 
175mL 
138 pL 
0.4 mL 
14 r-1L 
15% 
15 mL 
10mL 
4.9 mL 
948mL 
138 pL 
04mL 
14 N- 
Stackmg gelb 
40% Acrylamrde 
2% bu 
8X lMTris(pH68) 
dH20 
10% Amps 
10% SDS 
TEMED 
35% 
13 mL 
1 mL 
19mL 
11 mL 
110 pL 
150 pL 
20 M- 
4 5% 
168mL 
09mL 
19mL 
105mL 
100 pL 
150 pL 
20 r-IL 
OTotal volume 40 mL 
“Total volume 15 mL 
1.4. SDS-PAGE 
The unmunoprecipttated proteins may be analyzed by electrophoresrs m 
polyacrylamrde gels either under reducing or nonreducmg condmons (25,16). 
SDS, a strong aniomc detergent, IS often used in combinatton with a reducmg 
agent and heat to drsassociate the proteins before loading on to the gel. SDS 
bound to denatured proteins will give the protein a negative charge. The amount 
of SDS bound to proteins is usually proportional to the size of the polypepttde 
so that the formed complexes of SDS-polypeptide ~111 migrate through the gel 
accordmg to size. Mol-wt markers included in each run will help to calculate 
the estimated molecular weights of unknown polypeptides. N- and U-linked 
glycosylation will decrease the electrophoretic mobility through the gel If pro- 
teins are separated under nonreducmg conditrons the electrophorettc mobility 
will be changed as compared to separation under reducing condmons 
Polyacrylamide gels are chains of polymertzed acrylamlde that are cross- 
linked by NJ’-methyleneblsacrylamtde to form pores through which the 
polypeptrdes must pass. By varying the concentration of polyacrylamide the 
effective range of separation of polypeptides may be changed (Table 4). 
Imtially introduced by Ornstem and Davis (17, I??), discontmnous buffer 
systems are commonly used. The buffer in the reservoirs differs m pH and 
lmmunoprecipitation 21 
romc strength from the buffer used to cast the gel. SDS added m all buffers and 
the gel, introduced initially by Leammli (19), will bind to all denatured pro- 
teins and give them a negative charge. 
1.5. Detection and Analysis of lmmunoprecipitated Proteins 
Although gel scanners are slowly moving into many research laboratories, 
most researchers are still usmg autoradiography because of the hrgh costs of 
gel scanners. The great advantage of gel scanners 1s the shortening of the time 
before results are obtained. With the gel scanner, results are available rmmedt- 
ately while autoradrography results are available within days or weeks. In auto- 
radiography, permanent images are produced on photographic film applied to 
a dried gel. The P-particles emitted from the labeled proteins in the gel will 
interact with the stlver halide crystals m the emulsion of the film. The film 
exposure should take place m a light-proof cassette at -70°C in order to stabr- 
hze the silver atoms and tons that form the image of the radtoactive source. 
The autoradiographrc images may be amplified by fluorescent chemtcals 
which emit photons when they encounter a smgle quantum of radiation and 
this may increase the detectron level 1 O-fold. Several commercial preparations 
are available but in most cases 1M sodium saltcylate (pH 6.0) treatment for 
30 mm ~111 serve the purpose. 
2. Materials 
2.1. Metabolic Labeling of Proteins 
1. Monolayer cultures approximately subconfluent to confluent or suspension cultures 
2. Methionme-free and/or cysteine-free medium. 
3. 35S methionme and/or 35S cysteme The rate of synthesis and the half-life of the 
protein of interest as well as the number of cells to be labeled affect the time 
required as well as the mtensny of labeling. Labeling for 2-4 h with 100-400 uCi 
of 35S-labeled ammo acids is commonly used, preferably at the time of maximum 
protein synthesis m the cells (see Note 1) 
2.2. Lysis of Cells 
Choose one of the following lysrs buffers (Z-6) (see Note 3): 
1. Triple-detergent lysis buffer. 50 mM Tris-HCI (pH 8 0), 150 mh4 NaCl, 0.02% 
sodium azide, 0.1% SDS, 1% NP-40,0.5% sodmm deoxycholate. 
2. Smgle-detergent Iysls buffer 50 mM Tris-HCl (pH 8 0), 150 miM NaCl, 0 02% 
sodium azide, 1% Triton X-100 or NP-40. 
3. High salt lysis buffer I: 50 mMHEPES (pH 7 0), 500 mMNaC1, 1% NP-40 
4. High salt lysis buffer II* 10 mM Tris-HCl (pH 7.8), 150 mMNaCl,600 mM KCI, 
5 mM EDTA, 2% Triton X- 100 
5. No salt lysis buffer 50 mA4 HEPES (pH 7.0), 1% NP-40 
22 Johansen and Svensson 
6. Very gentle single detergent buffer. 50 m&I HEPES (pH 7 5), 200 mMNaCI,2% 
CHAPS. 
7 Protease inhrbrtors are often included in lysrs buffers. Aprotinm at a concentratron of 
1 pg/mL and PMSF at a concentratron of 100 pg/mL are regularly used (see Note 5) 
Many other protease mhibrtors are available and the need for addmonal mhibrtors 
must be established m each new system For further mformation, see Table 2 
8 To avoid artrficral drsulfide bond formation during cell lys~s cells should be 
washed with me-cold PBS contammg 20 mMNEM and lysed m lysrs buffer con- 
taming 20 mMNEM 
2.3. Formation of Antigen-Antibody Complexes 
1 Radiolabeledcell lysate (see Note 6) 
2 Monoclonal or polyclonal antibody for immunoprecrprtatron 
3 Protem A or G bound to Sepharose beads (Pharmacra Brotech, Prscataway, NJ) 
or Jacalm-agarose (Vector) 
4. RIPA-buffer: 2% (v/v) Triton X-100, 150 mA4NaCl,600 mA4 KCl, 5 mM drsodium 
EDTA, 3 WPMSF, 1 pg/mL aprotmm, and 20 mMNEM m 10 mMTris-HCl, pH 7.8 
5. Low-salt washing buffer. 10 mMTrrs-HCI (pH S.O), 150 mMNaC1 
6 Reducmg sample buffer: 3% SDS, 3% 2-mercaptoethanol, 0.1% EDTA, 10% (v/v) 
glycerol in 62 n-&f Trrs-phosphate, 0.02% bromphenol blue, pH 6 8. A 2X stock 
solutron can be stored at room temperature 
7 Nonreducing sample buffer 3% SDS, 0.1% EDTA, 10% (v/v) glycerol m 62 mM 
Tris-phosphate, 0 02% bromphenol blue, pH 6.8 
2.4. SDS-PAGE 
1. Acrylamide and N, N’-methylene bzs-acrylamrde. A stock solutron containing 
40% (w/v) acrylamrde and 2% (w/v) bzsacrylamrde should be stored m the dark 
at 4°C (see Notes 8 and 9). 
2. 10% SDS: Store at room temperature 
3. Trrs buffers prepared from Trrs base. Stock solutrons should be prepared of 8X 
1MTrrs buffer (pH 8.8) and 8X 1MTris buffer (pH 6.8) 
4 10% Ammonium persulfate. To be prepared fresh every second day. Ammomum 
persulfate provides the free radicals that drive the polymerrzatron 
5. TEMED (N,N,N’,N’-tetramethylethylenedlamine). TEMED accelerates the poly- 
merization by catalyzing the formatron of free radicals from ammomum persulfate 
6. Trrs-glycme electrophoresrs buffer: 25 mM Tris base, 250 mA4 glycine, pH 8.3, 
0 1% SDS A stock solution of 1 OX wrthout the SDS can be stored at room tem- 
perature. Add SDS right before the run of the gel. 
7. Gel apparatus. 
2.5. Defection and Analysis of lmmunoprecipitated Proteins 
1. FIX 10% (v/v) glacial acetic acid and 35% (v/v) methanol m deromzed water. 
2. IA4 Sodmm sahcylate (pH 6 0) m deionized water (Sigma, St Lotus, MO) (see 
Note 10). 
lmmunoprecipitation 23 
3. Whatman 3-mm paper 
4. Gel dryer (see Notes 11 and 12). 
5. Light proof cassette (see Note 13) 
6. X-ray film (Kodak Xomat, Eastman Kodak, Rochester, NY). 
7. Developer (X-ray developer LX 24, Eastman Kodak). 
8 Fixattve for X-ray film (X-ray, Eastman Kodak) 
9 Dark room 
3. Methods 
3.1, Metabolic Labeling of Proteins 
1. Wash monolayers twice with PBS or wash and centrifuge cells m suspension 
twice, and add methionine and/or cysteme deficient medmm (wtthout fetal calf 
serum) prewarmed to the appropriate temperature. 
2. Incubate the cells for 2&60 min to deplete the intracellular pools of methionme 
and/or cysteine. 
3. Replace the ammo acid deficient medium with methtonme and/or cysteme free 
medium including 35S-labeled ammo acids Incubate for the desired pertod of 
time (see Note 1). Keep the volume of medium down to increase the concentra- 
tion of radtolabeled ammo acids. Suggested volumes for adherent cells are: l-2 mL 
for a 25-cm2 flask, l-2 mL for a 90-mm Petri dish, 250-500 & for a 60-mm 
Petri dish, 100-200 JJL for a 30-mm Petri dish Cells growing m suspension 
should be resuspended at a concentration of 107/mL (see Note 2). 
4 If the antigen of mterest is a secreted antigen, the radioactive supernatant is saved 
for tmmunopreciprtatton If the antigen of Interest accumulates mtracellularly, 
the radioactive supernatant 1s discarded m the radioacttve waste 
3.2. Lysis of Cells 
1. Wash the cells twice with ice cold PBS containing 20 mA4 NEM, dram the last 
PBS with a Pasteur pipet and add the lysts buffer of choice (2-3 mL to a 25-cm2 
flask) (see Note 3). Let monolayers solubtlize for 30 mm on me 
2. To clear the lysate from cell debrts, centrtfuge for 10 mm at 12,000g in a 
microfuge Before storage of the labeled antigen at -70°C ahquot the antigen to 
avoid repeated freezing and thawmg (see Note 4). 
3. Check efficiency of metabolic labeling by running an SDS-PAGE and 
autoradtography 
3.3. Formation of Antigen-Antibody Complexes 
1 Mix appropriate amounts of the cell lysate (usually 5-l 00 pL) with the monoclonal 
or polyclonal antibody and dilute to 500 pL m RIPA buffer and incubate at 4°C 
overmght. Aim at a sufficient amount of anttbody to prectpttate all of the target 
antigen Several factors, such as concentration of antigen and titer and avidtty of 
the antrbody, will affect the amount of antibody to be used Start the immu- 
noprecipitation by titrating the antibody against a fixed amount of target antigen. 
24 Johansen and Svensson 
Complete immunoprecipttation 1s usually obtamed by 0 5 pL--5 uL of 
polyclonal annserum, 5-100 pL of hybrtdoma ttssue culture medium, or 0 l-l .O pL 
of ascmc fluid (see Note 6) 
2. If your antibody does not bmd effictently to protein A, G, or jacalm, add a second 
anttbody that IS dtrected agamst your primary anttbody and bmds strongly to one 
of these protems, and incubate at 4°C for 1 h The amount of antt-immunoglobu- 
lm antibody must be titrated against a fixed amount of antigen-primary antibody 
complex to exclude reacttvtty between secondary antibody and target antigen 
3 Add 25-l 00 pL of protein A-lprotem G-Sepharose beads or jacalm-agarose beads 
to the antigen-antibody mixture and Incubate on a rocker for 1 h at 4°C or at 
room temperature. 
4 Centrifuge the newly formed immune complexes at 12,OOOg for 30 s and 
remove the supematant. Wash the complexes to remove nonspectlically adsorbed 
protems at least four times wtth 1 mL of RIPA buffer and resuspend the beads 
with careful vortexing between washes The last wash should always be per- 
formed with the low-salt washmg buffer Take care to remove the last traces of 
the final wash. 
5 To disassociate the nnmune complexes, add 40 $ of sample buffer and mcubate 
at 100°C for 2-3 mm (see Note 7) Centrifuge the samples for 20 s at 12,000g 
m a mtcrofuge and save the supernatants for SDS-PAGE Samples can be fro- 
zen at -20°C before analysts by SDS-PAGE 
3.4. SDS-PAGE 
1 
2 
3 
4 
5 
6 
7 
8. 
9. 
10 
Assemble the electrophorests apparatus 
Prepare the separation gel at the desired concentration m an Erlenmeyer flask, 
and degas. Add TEMED last, smce polymerizatton ~111 start tmmedlately (see 
Notes 8 and 9) 
Pour the acrylamtde solutton between the glass plates Leave space for the stack- 
mg gel Carefully overlay the separation gel with isobutanol to prevent oxygen 
dtffuston mto the gel and mhibttton of polymertzation 
When polymerizatton of the separation gel IS complete, pour off the tsobutanol 
overlay and rinse carefully with water Drain with an edge of a paper towel 
Prepare the stackmg gel at the desired concentration 
Pour the stacking gel and insert a TeflonTM comb mnnediately. Let polymerize 
for 30-60 mm 
After polymertzation of the stacking gel, remove the Teflon comb, mount the gel 
m the apparatus, and pour the Trts-glycme electrophorests buffer All air bubbles 
trapped at the bottom of the gel must be removed 
MIX the samples with sample buffer and Incubate at 100°C for 3 min. 
Load the samples with a Hamilton syrmge Wash the syringe between apphca- 
tton of samples with buffer from the lower buffer reservoir. 
Attach apparatus to electric power. To start, apply a voltage of 8 V/cm gel, and 
when samples are moving mto the separation gel, increase the voltage to 15 V/cm 
and run the gel until the bromphenol blue leaves the gel at the bottom, 
lmmunoprecipitation 25 
1 I. Turn off the electric power and remove the gel with the two glass plates. Separate 
the glass plates and cut a corner of the gel to guide further interpretation. 
12. Fix the gel. 
3.5. Detection and Analysis of lmmunoprecipitated Proteins 
Autoradlography: 
1 After washing the fixed gel twice m deionized water soak the gel in 50-100 mL 
of 1M sodium salicylate m delomzed water for 30 mm on a rocker (see Note 10). 
2 Dry the gel onto a 3-mm Whatman paper, presoaked m water, m a gel dryer at 
6OT for 2-6h (see Note 11). 
3 Place the gel together with an X-ray film m a light-excluding X-ray film 
casette at -7O’C and expose the film for an appropriate time (days-weeks) 
(see Note 12) 
4 Develop the film in an automatic X-ray film processor or manually for 5 mm 
each as follows (see Note 13). developer, water bath, fixative, and running water. 
4. Notes 
4.1. Metabolic Labeling of Proteins 
1 When radlolabelmg cells for longer than 6 h, all the radlolabeled ammo acids 
may be consumed It 1s therefore sometimes necessary to add unlabeled ammo 
acids, I.e., methlonme and/or cysteme as shortage of amino acids may cause an 
interruption in protein synthesis 
2. If small volumes are used during labeling, keep the flask on a slow rocker or 
shake the dishes every 15 mm to ensure that the cells do not dry 
4.2. Lysis of Cells 
3 To optimize the extracting conditions, try a stronger lysls buffer on the centri- 
fuged cell debris than the buffer you initially used for lysls of the cells Then test 
by lmmunoblottmg whether or not most of the labeled proteins were extracted 
with the lysis buffer initially used. 
4 To remove aggregates of cytoskeleton elements after thawing of lysate, centn- 
fuge samples at 12.OOOg for 5 min. 
5. Caution: PMSF 1s extremely destructive to mucous membranes of the resplra- 
tory tract, the eyes and skin Also, the other protease inhibitors are toxic 
4.3. Formation of Immune Complexes 
6. To avoid nonspecific bmdmg of complexes to the tube wall, use good quality 
tubes (e.g., Eppendorf, Hamburg, Germany) or pre-coat the tubes with 0.5% 
bovine serum albumin for 15 mm. 
7. Before heating samples to lOO’C, use a needle to make a small hole m the tube 
cap This will prevent the building of excess pressure in the tube and the tube cap 
will remain closed during the heating step 
26 Johansen and Svensson 
4.4. SDS-PAGE 
8 Caution: Acrylamlde and brs acrylamide are neurotoxlc and may be absorbed 
through the skin Polyacrylamlde 1s considered nontoxlc but may contain 
unpolymerized material 
9 When preparmg and handling gels, use gloves to avold exposure to unpolymer- 
lzed polyacrylamtde and radIoIsotopes 
4.5. Detection and Analysis of lmmunoprecipitated Proteins 
10. Caution: Salicylate may ehclt allergic reacttons and IS readily absorbed through the 
skm As an altematlve use commercial fluorescents available from several compames 
11 Shrinkage, dlstortlon, and cracking of the gel are common problems encountered 
when trying to dry gels To avold shrinkage and distorslon, dry the gel onto a 
3-mm Whatman paper (presoaked m water) Make sure there are no air bubbles 
between the gel and the paper before startmg the gel dryer To avoid cracking do 
not turn off the gel dryer or break the vacuum before the gel 1s completely dry If 
possible, use thm gels (0 75 mm), smce crackmg 1s more common with thicker 
gels containing larger amounts of polyacrylamlde. 
12 Take care to prewarm the cassette to room temperature for 15 min before devel- 
oping the film, smce moisture mslde the cassette will destroy the emulsion. 
13 The new time-savmg gel scanners provide an alternatlve to autoradlography 
References 
1 Sambrook, J , Frltsch E F , and Mamatls, T. (eds. ) (1989) Molecular Clonzng A 
Laboratory Manual (2nd ed ), Cold Spring Harbor Laboratory Press, Cold Sprmg 
Harbor, NY 
2 Harlow, E and Lane, D (1988) Antzbodzes* A Laboratory Manual Cold Spring 
Harbor Laboratory Press, Cold Spring Harbor, NY 
3. Burleson, F G , Chambers, T. M , and Wledbrauk, D L (eds ) (1992) Vzrology A 
Laboratory Manual Academic, San Diego, CA 
4 Barrett, A. J and Salvesen, G (eds.) (1986) Protemase lnhzbztors Research Mono- 
graphs m Cell and Tzssue Physzology, vol. 12. Elsevler, Amsterdam, The Netherlands 
5. James, G. T (1978) Inactivation of the protease mhlbltor phenylmethylsulfonyl 
fluoride in buffers Anal. Blochem 86,574-579 
6. Hammond, C. and Helenms, A (1994) Quality control m the secretory pathway 
of a mlsfolded viral membrane glycoprotein mvolves cyclmg between the ER, 
mtermedlate compartment, and Golgl apparatus J Cell Bzol 126,41-52 
7. HJelm, H , HJelm, K , and SJoqutst, J. (1972) Protem A from StaphyZococcus 
aureus Its Isolation by affimty chromatography and its use as an lmmunosorbent 
for Isolation of immunoglobulins FEBS Lett 28,73-76 
8 Sjodahl, J. (1977) Structural studies on the four repetitive Fc-bindmg regions m 
protein A from Staphylococcus aureus Eur J Blochem 78,47 l-490 
9 Goudswaard, J , van der Donk, J A., Noordzij, A., van Dam, R. H , and Vaerman, 
J-P (1978) Protein A reactlvlty of various mammalian immunoglobulins &and 
J Immunol 8,21-28 
lmmunoprecipltation 27 
10 Bjorck, L. and Kronvall, G (1984) Purificatton and some properties of strepto- 
coccal protein G* a novel IgG-bmdmg reagent J Immunol 133,969-974 
11. Akerstrom, B , Brodin, T , Reis, K., and Bjbrck, L (1985) Protein G: a powerful 
tool for binding and detectton of monoclonal and polyclonal antibodies J 
Immunol 135,2589-2592. 
12. Fahnestock, S. R., Alexander, P , Nagle, J., Ftlpula, D (1986) Gene for an immu- 
noglobulm-binding protein from a group G streptococcus J Bacterial 167(3), 
870-880 
13 Roque-Barreira, M C and Campos-Neto, A (1985) Jacalm: an IgA-bmdmg lec- 
tm J ImmunoE. 134, 1740-1743 
14. Johansen, K., Granqvtst, L., Karlen, K., Stmtzmg, G., Uhnoo, I , and Svensson, L 
(1994) Serum IgA mnnune response to individual rotavirus polypepttdes m young 
children with rotavirus mfection. Arch Vu-01 138, 247-259. 
15. Studter, F. W (1973) Analysis of bacteriophage T7 early RNAs and proteins on 
slab gels. J Mel Blol 79, 237-248 
16. Hames, B. D. and Rickwood, D. (eds.) (198 1) Gel Electrophoresis of Protezns* A 
Practzcal Approach IRL, Oxford, UK 
I7 Omstein, L (1964) Disc electrophoresls-I Background and theory Ann NY Acad 
Scz 121,321-349 
18. Davis, B J (1964) Disc-electrophoresis II. Method and apphcation to human 
serum proteins. Ann NY Acad Scz 121,404-427 
19. Laemmh, U. K. (1970) Cleavage of structural proteins during the assembly of the 
head of bacteriophage T4. Nature 227,68@-685 
3 
RTPCR Methods and Applications 
Paul D. Siebert 
1. Introduction and Overview 
Control of gene transcription, the process in which a gene’s DNA sequence 
serves as a template for mRNA synthesis, plays a critical role m the multistep 
process that regulates gene expression. Gene transcrtption levels wtthm a cell 
change in response to a wide variety of signals that occur during cell develop- 
ment, differentiation, and normal physiologtcal function. Changes m transcrip- 
tion levels also occur m response to disease and other factors. In turn, these 
changes m transcription levels cause variations m the steady-state levels of 
mdivtdual mRNAs. Thus, analysis of specific mRNA levels 1s vital m a broad 
range of research areas. 
Tradmonally, levels of mdividual mRNAs have been analyzed by proce- 
dures such as Northern blots, RNA dot/slot blots, nuclease protection assays, 
and uz situ hybridization The polymerase chain reaction (PCR) technique pro- 
vides another method of mRNA analysis. This PCR-based method has been 
variously termed RNA-PCR (I), RT-PCR (2), RNA phenotypmg (.3j, and 
Message Amphfication Phenotypmg (MAPPmg) (4). In this chapter, the term 
RT-PCR 1s used throughout. 
The RT-PCR method has become increasmgly popular for analysis of gene 
transcripts, primarily because it IS highly sensitive and rapid. A flow chart 
illustrating the RT-PCR process is shown m Fig. 1, RNA is first isolated from 
tissues or cells and then used as a template for reverse transcription to compli- 
mentary DNA (cDNA). The cDNA m turn is used as the template for PCR, 
using primers designed to amplify a selected cDNA region Followmg PCR, 
the product IS typrcally analyzed by agarose gel electrophoresisThe identity 
of the amplified cDNA is confirmed by the size of the PCR product (its “phe- 
notype”), which is predicted from the cDNA nucleotide sequence. The PCR 
From Methods m Molecular Medmne, Vol 13 Molecular Diagnosis of Infectious D/s-eases 
Edlted by U Relschl Humana Press Inc , Totowa, NJ 
29 
30 Siebert 
RNA 
i*olot~on 
1 
PCR 
1 
1 
validation of 
PCR products 
1 
quontitotion 
(optionoIl 
Fig. 1. Schematic diagram of the RT-PCR method. 
product can be further validated by restriction digestion, hybridization, or 
nucleotide sequencing. 
The level of expression of the genes under study can be roughly estimated by 
knowing the amount of RNA used for the synthesis of cDNA, the amount of 
cDNA used for PCR, and the number of PCR cycles needed to generate a visible 
band on an agarose gel. More precise quantitation of individual mRNA levels 
can be achieved by careful consideration of amplification efficiencies and yields. 
RT-PCR 31 
This chapter begms by reviewing the steps mvolved m RT-PCR, mcludmg 
RNA isolatton, cDNA synthesis, and PCR amplificatton. Several apphcattons 
of RT-PCR, mcludmg detection of gene transcrtpts from small amounts of 
RNA, simultaneous analysis of multiple gene transcripts, and detection of alter- 
nate sphcmg of gene transcripts are then covered and selected protocols are 
provided. Thts discussion 1s by no means intended to be complete, and readers 
are directed to an excellent review on the biochemistry of PCR by Bloch (5). 
1.1. RT-PCR Method 
1.1.1. RNA lsola tion 
High-quality RNA is important for the success of RT-PCR analysts The 
RNA must not be degraded by rtbonucleases, as determined by the intactness 
of ribosomal (rRNA) bands, and contaminating genomic DNA must be 
removed. The most common and consistently successful methods for isolating 
pure, intact total RNA are modifications of the original guamdinium thiocyan- 
ate method of Chirgwm et al. (6). 
In the original method (6), tissues or cells are disrupted in high concentra- 
tions of guamdmium thiocyanate to rapidly macttvate ribonucleases The 
resulting lysate is layered over a CsCl cushion and spun in an ultracentrifuge. 
The RNA forms a pellet at the bottom of the tube, while protein and DNA 
remain m or above the CsCl cushion. 
A modified guanidimum thiocyanate method that does not require an ultra- 
centrifuge mvolves co-extraction with phenol at reduced pH to remove protein 
and DNA (7). This is often the method of choice when multiple RNA extrac- 
tions are performed. Both of these methods are rapid, efficient, and work well 
for many tissues and cell types. The molecular cloning manual by Sambrook et 
al. (8) also contains useful mformatron on how to isolate and handle RNA 
properly. Additionally, several companies offer kits for RNA isolation. 
When isolating RNA from small amounts of tissue or cells, a carrier nucleic 
acid such as tRNA (4) or polyinosmic acid (9) should be added at the begin- 
ning of the extraction to facilitate handling of the RNA and to improve yields. 
To ensure optimal RT-PCR, all RNA preparations should be examined by dena- 
turing agarose gel electrophoresis. If the RNA is intact, eukaryotic RNA will 
exhibit clear 28s and 18s rRNA bands, with the 28s band about twice as intense 
as the 18s band. 
Isolated RNA can be stored conveniently as an ethanol precipitate at -20°C 
or in aqueous solution at -70°C or below for up to 1 yr without appreciable 
deterioratron. Repeated freeze/thaw cycles should be avoided. Poly (A)+ RNA 
isolated from total RNA by ohgo cellulose chromatography (‘8) can also 
be used for RT-PCR, although this further purification step 1s not necessary. 
32 
/ A. specific Priming ) 
cDNA 
5’ 
Siebert 
1 B. Oligo(dT) Priming) 
CDNA +TTTT 
mRNA AAdA 
cDNA C-4- -4- -4 
Fig. 2. Three methods of priming cDNA. (A) Gene-specific method. An antisense 
gene-specific oligonucleotide is annealed to the mRNA and extended with reverse 
transcriptase. (B) Oligo(dT) method. Oligo(dT) oligonucleotides (with lengths 
between 12 and 18 nucleotides) are annealed to the poly (A)+ tail of mRNA, and the 
entire population of mRNA molecules used as a template for cDNA synthesis. (C) 
Random priming method. Short oligonucleotides (typically hexamers) having all pos- 
sible nucleotides at each position are annealed randomly to the RNA molecules and 
used to prime cDNA synthesis. 
1, 1.2. cDNA Synthesis 
1 .1.2.1. REVERSE TRANSCRIPTION 
The cDNA template for RT-PCR is synthesized from RNA by reverse tran- 
scription. The author has successfully used both avian myoblastosis virus 
(AMV) and Moloney murine leukemia virus (MMLV) reverse transcriptases 
with comparable results. It is important to include human placental ribonu- 
clease inhibitor in the cDNA synthesis reaction to obtain maximum yields. 
1 .1.2.2. cDNA PRIMING 
There are three ways to prime the mRNA for cDNA synthesis (Fig. 2). In the 
first, a 3’ (antisense) gene-specific primer is annealed to the mRNA and 
extended with reverse transcriptase (Fig. 2A). This generates a cDNA template 
for the 5’ (sense) primer, allowing PCR amplification to occur. When priming 
cDNA with a gene-specific primer, a number of experimental parameters may 
need to be optimized, including primer concentration and annealing tempera- 
ture (1). In the second and third methods, the entire population of mRNA mol- 
ecules is first converted into cDNA by priming with either oligo(dT) (Fig. 2B) 
or random hexamers (Fig. 2C). Two gene-specific PCR primers are then added 
for amplification. The latter two methods have been the most successful, which 
is consistent with their prevalence in the literature. 
RT-PCR 33 
In our RT-PCR experiments we typically start with oligo(dT) priming, which 
yields, on average, fewer PCR side products than random priming. There may be 
situations, however, when gene-specific or random priming of cDNA may be 
beneficial. For instance, the reverse transcriptase may fat1 to fully transcribe an 
mRNA template if the 5’ primer is located further than about 3 kb from the poly 
(A)+ tall or if secondary structures exist that impede the reverse transcriptase. 
Several cDNA synthesis reaction mixes have been designed for compatibil- 
ity with the PCR reaction mix (3). This allows cDNA synthesis and PCR ampb- 
fication to be carried out in the same tube. It is better, however, to synthesize 
cDNA in one 20-pL reaction (see Section 3.1.) and then use a 2-3-a ahquot 
of the cDNA m each PCR amplification tube. In this way, variability in the 
cDNA synthesis within each PCR reaction tube is avoided. This is particularly 
important when a panel of gene-specific primers are to be used. 
1.1.2.3. USE OF POLY (A)+ RNA 
The use of poly (A)+ RNA as a template for cDNA has also been examined. 
As a result of the enrichment for mRNA, a much smaller volume of the reverse 
transcription reaction IS necessary. In one case, only 1% of the cDNA synthe- 
sized from 1 pg of poly (A)+ RNA was necessary to achieve results comparable 
to those obtained with total RNA. The use of total RNA, however, can be advan- 
tageous when comparing cDNA derived from several RNA preparations, since 
one less step (with its potential vanability) is necessary to obtain the cDNA template. 
1.1.3. PCR Amplification 
1 .l 3.1 PCR PRIMER DESIGN 
This discussion of primer design is confined to perfect sequence primers, 
i.e., those that exactly match the cDNA template. The location of the primer 
template within the cDNA sequence is important for several reasons. First, it 
defines the length of the PCR product. Choose primer template locations that 
yield products between 300-l ,000 bp in length. Products smaller than 300 bp 
require special agarose gel formulations for good resolution and may be 
obscured by primers and primerartifacts. Products larger than 1,000 bp are less 
efficiently amplified, owing to limitations in enzyme processivity. For example, 
Taq DNA polymerase is not very processive (5) and the enzyme tends to fall 
off the template during long extensions. With the recent development of long- 
distance PCR (10,11), these limits may be less severe. 
Second, when the cDNA has been primed with ohgo( the prtmer loca- 
tion defines the dtstance that the cDNA must be extended from the 3’ end of the 
mRNA to provide the 5’ primer template. Because reverse transcriptase has 
difficulty transcribing long templates, choose 5’ primer regions not further than 
2 kb from the 3’ end of the mRNA. 
34 Siebert 
Third, primer locations can be designed to distmguish between PCR prod- 
ucts derived from cDNA and products derived from contammatmg genomtc 
DNA. Choose primer sequences that are located on separate exons, so that 
PCR products derived from genomtc DNA will be longer. 
Typically, PCR primers should be between 22 and 30 nucleottdes long and 
have an A/T content about equal to the G/C content, so that the optimal anneal- 
ing temperature of both primers is similar. Avoid using primer sequences that 
can form stable inter- or intrastrand base pairmg It is particularly Important 
that the 3’ ends of the prtmers not be complementary to each other, which would 
lower the effective concentration of primers (I, 12). In addition, primers must 
not have extensive homology to other transcripts, or else more than one PCR 
product can be generated. 
Several computer software programs have been developed to facilitate primer 
design (13) and some are available commercially Even with these guidelines, 
successful PCR primer design 1s empirical, and more than one primer set 
may need to be tested before a good combmatton IS found Fatlure of the 
primer(s) to work correctly is indicated by no bands on the agarose gel (no 
product being made) or multiple bands when only one is expected (nonspe- 
cific amplification). 
1 13 2.PCR PARAMETERS 
Basic PCR components include reaction buffer, dNTPs, primers, cDNA tem- 
plate, and a thermostable DNA polymerase. The composmon of the buffer (e.g., 
MgCl* and KCl) and the concentration of the dNTPs will vary, dependmg on 
the type of enzyme used and, to some extent, the cDNA template and prtmers. 
Use reaction components recommended for the recombinant AmphTuq@ DNA 
polymerase (Perkm Elmer-Cetus, Norwalk, CT). A MgC& concentration of 
1 5 mM is usually satisfactory for most PCR, although some titration 1s occa- 
sionally necessary. Working portions of the reaction components are stored at 
-20°C and should be discarded after thawing about 10 times. 
Thermal cycling parameters (i.e., times and temperatures for denaturation, 
annealing, and extension) may vary depending on the type of thermocycler 
used Typically, denaturation 1s performed at 94°C for 1 mm or less, and the 
polymerase extension step 1s performed at 72°C for two min or less. Perhaps 
the most critical cycle parameter is the prtmer-annealmg temperature. An excel- 
lent discussion of primer length and annealing temperature is provided m ref. 14. 
In order to examme multiple gene transcripts stmultaneously in the same 
thermocycler, cycle parameters need to be opttmized to achieve adequate 
amplification of all cDNAs. Comparisons of expresston of different transcrtpts 
are valid only tf the efficiency of each PCR reaction does not plateau. The 
number of PCR cycles, therefore, should be kept to a minimum. 
RT-PCR 35 
1 1.3 3. HOTSTART PCR 
Even after optimizing primer design and annealing temperatures PCR reac- 
tions may still generate drmerized prrmer-amplified fragments (“primer 
dime?) as well as larger nonspectfic products. The nonspecttic fragments can 
vary m size and yreld and are primer dependent. Such nonspecific fragments 
reduce the yield of desned specific fragments through competttion with the 
specific target in the reaction. Furthermore, nonspectfic products that are 
approximately the same stze as the specific product can cause confusron when 
interpreting results. 
Nonspecific products are thought to originate from DNA polymerase cata- 
lyzed extenston of 3’ ends of primers partially annealed to nonspectfic sites on 
the template DNA under the low-stringency conditions of ambient tempera- 
ture. Efficiencies of thermostable DNA polymerases are greatly reduced at 
ambtent temperature relative to their peak effictencies at higher temperatures. 
However, enzyme activity at ambient temperatures can be enough to generate 
PCR side products, 
The “hot start” PCR method was developed as a means of reducing the 
amplification of nonspectfic products (1.5,16), The original approach was to 
withhold an essential reagent from the reactron (such as DNA polymerase, 
MgC12, or primers) until the reaction mixture was heated to a high temperature 
(e.g., >55”C). This presumably causes melting of partially annealed 3’ primer 
ends from nonspecific sites, preventing their extension. Another approach to 
the hot start method uses a heat-labile wax or Jelly barrter (15,171 to keep crttt- 
cal reaction components separate until heating permits mtxmg of aqueous com- 
ponents. However, these hot start methods increase the probability of crossover 
contamination whenever the reaction tube must be reopened. Furthermore, 
these methods are cumbersome and time-consummg when working wrth mul- 
tiple samples than with conventional PCR techniques. 
Recently, Kellogg et al. (18) described an improved form of hot start that 
uses a neutralizing monoclonal antibody directed against Taq DNA polymerase 
to facilitate hot start PCR. At ambient temperatures the antibody attaches and 
inactivates the Tag polymerase. During the first denaturation step the antibody 
dtssociates and denatures reversing the enzyme inhibitton. PCR amphficatron 
can then proceed specifically. This is the most convement hot start method 
because the antibody can be premixed with the polymerase. Also, no reaction 
tubes need to be reopened so the possibility of crossover contaminatton 1s vir- 
tually eliminated. 
1.1.3.4. DNA CONTAMINATION 
The abihty of PCR to amplify DNA sequences by over six orders of magni- 
tude means that any nucleic acid contammatton poses a sertous problem 
36 Siebert 
whether it comes from external sources such as pipet tips, hands, or reagents, 
or from internal sources such as contaminatmg genomic DNA. 
In RT-PCR, both nucleic acid and ribonuclease contamination must be con- 
trolled. As a general rule, gloves should always be worn and changed fre- 
quently, and semi-sterile technique should be adopted Water used m RNA 
extraction solutrons and m reverse transcriptase reactions should be treated 
with diethylpyrocarbonate. Water used for PCR amphfication should be filter- 
sterilized, since recirculating water in standard autoclaves can be contammated 
with nucleic acids. Many of these precautions are common practice to avoid 
ribonuclease contammation when handlmg RNA. 
Carryover contamination of PCR products from previous amplifications 
must also be mmimized. It is a good habtt to handle pre- and post-PCR solu- 
tions with separate, dedicated prpetors. Special aerosol-free pipet tips are now 
available from several manufacturers. Whenever possible, perform pre- and 
post-PCR procedures m separate laboratory areas. 
Several chemtcal means to eliminate the problem of PCR product carry-over have 
been devtsed. In one (19), dUTP is substituted for dTTP m all amplification expen- 
ments. Each reaction mixture is treated prior to cyclmg with uracil N-glycosylase, 
which cleaves any dUTP-contammg nucleic acids carried over from the previous 
experiment and thereby prevents then use as PCR templates. Physical treatment, 
such as ultraviolet light irradiation, has also been describedto decontammate 
reagents for PCR (2 71, although thts method is only efficient for large PCR targets. 
1 1.3 5. GENOMIC DNA CONTAMINATION 
Another potential problem during RT-PCR is genomic DNA contammation 
m the RNA preparation. This is particularly relevant when the target mRNA is 
expressed at low levels, thus requiring large numbers of amplification cycles, 
Although additional purification steps to completely eliminate genomic DNA 
may be impracttcal, there are means to differentlate between amplified cDNA 
and genomtc DNA products. The easiest way is to design the primers such that 
they span one or more mtrons within the gene Thus, PCR products generated 
from contammatmg genomic DNA ~111 be larger than products from cDNA, as 
shown m Fig. 3. This method is termed “mtron-differential RT-PCR” (20). 
If the mtron/exon structure of the gene is not known, or if the gene lacks 
mtrons, there are several methods that can be used to reduce the effect of 
genomic DNA contamination. In one method, a specially designed 3’ primer IS 
used that contains sequences complementary to the last segment of 3’ untranslated 
mRNA sequence mcludmg part of the poly (A)+ tail (21) In this way, only cDNA 
derived from poly (A)+ RNA can serve as a productive template for PCR. 
In another method, called RNA template-specific PCR (22), a special com- 
posite 3’ primer IS used that contams two sections: a 3’ segment and a tagging 
RT-PCR 37 
A 
cDNA PCR Product Genomic DNA PCR Product 
B 
bp 
872 
603 
Fig. 3. Intron-differential RT-PCR. (A) Schematic diagram of the method. If the 
PCR primers are constructed based on exon sequences separated by one or more introns, 
true RT-PCR products will be smaller than PCR products derived from genomic DNA. 
(B) Comparison of PCR-amplified human TNF-l3 from cDNA (lane 2) and genomic 
DNA (lane 3). Lane 1 contains +X174/HaeIII digests as size markers. PCR primers for 
human TNF-l3, which span 2 introns, can distinguish larger genomic contaminants 
from smaller cDNA products having no intronic sequences, 
true RT-PCR products will be smaller than PCR products derived from genomic DNA. 
(B) Comparison of PCR-amplified human TNF-l3 from cDNA (lane 2) and genomic 
DNA (lane 3). Lane 1 contains +X174/HaeIII digests as size markers. PCR primers for 
human TNF-l3, which span 2 introns, can distinguish larger genomic contaminants 
from smaller cDNA products having no intronic sequences, 
sequence. During cDNA synthesis the tagging sequence becomes incorporated 
into the cDNA and not contaminating genomic DNA which is double-stranded. 
A primer specific to the tagging sequence is then used in the PCR amplifica- 
tion. In yet another method the RNA is treated with RNase-free DNase before 
the reverse transcription (23). 
sequence. During cDNA synthesis the tagging sequence becomes incorporated 
into the cDNA and not contaminating genomic DNA which is double-stranded. 
A primer specific to the tagging sequence is then used in the PCR amplifica- 
tion. In yet another method the RNA is treated with RNase-free DNase before 
the reverse transcription (23). 
1.1.4. Verification of RT-PCR Products 1.1.4. Verification of RT-PCR Products 
Before any conclusions can be drawn from RT-PCR experiments based Before any conclusions can be drawn from RT-PCR experiments based 
solely on the generation of a PCR product of predicted size, the identity of the solely on the generation of a PCR product of predicted size, the identity of the 
PCR product must be verified by a second method. This is typically achieved PCR product must be verified by a second method. This is typically achieved 
38 Siebert 
either by partial (or complete) nucleottde sequencing, restrtctton mapping, or 
sequence-specific probe hybrtdizatton. 
1.1.4 1 NUCLEOTIDE SEQUENCING 
Obtaining a nucleotlde sequence IS the most convmcmg verification method, 
although tt IS technically the most demanding and time consummg. The PCR 
product can be cloned and sequenced by standard methods, or single-stranded 
products can be obtained by asymmetric PCR (241, strand separation techniques 
(2.5), or nuclease dtgestion (26). There are also methods available to dnectly 
sequence double-stranded PCR products (2 7,28). 
1 .1.4.2. RESTRICTION MAPPING 
Restrrctton mapping IS often the most convenient verification method, accom- 
plished srmply by noting the presence of one or more characteristic restrictton 
sites situated between the primer templates. Choose an enzyme that cleaves the 
cDNA fragment only once or twice and yields fragments that can be resolved 
from each other on an agarose gel. An example of a restriction analysis of an 
amplified cDNA segment IS shown m Fig. 4. Unpurified PCR-amplified cDNAs 
have been cut with numerous restriction enzymes, which are hsted m Table 1 If 
the reaction buffer must be changed for compatibility with the restrictton enzyme, 
the PCR products can be passed through a spin chromatography column 
1.1.4.3. SEQUENCE-SPECIFIC HYBRIDIZATION 
RT-PCR products can also be verified by hybridtzation of a synthettc ohgo- 
nucleottde probe that recognizes a unique sequence situated between the PCR 
primer templates. Use antisense, synthetic oligonucleotide probes 30 nucle- 
otrdes in length to allow stringent hybridtzatton and washing. Hybridtzatron 
and analysis can be completed m under 24 h. Protocols are provided and exam- 
ples of several representative hybrtdrzattons are shown m Fig. 5. 
Although verification by probe hybridization requires synthesis of a third 
oligonucleotide and a hybridization step, the resulting data can be obtained in 
the form of an autoradiogram, which can be used to quantrtate the amount of 
PCR product by densrtometry and also to differentiate between specific and 
nonspecific PCR products. Further, synthettc oltgonucleotrde probes and strm- 
gent hybridization and washing condtttons can be used to differentiate between 
related gene transcripts, even rf then PCR products are similar in srze. 
1.2. Applications of RT-PCR 
1.21. Detection of Gene Transcripts from Small Amounts of RNA 
Common, tradrtional methods for detectton and analysts of gene transcripts, 
such as Northern blots and RNA dot/slot blots, require amounts at least several 
RT-PCR 39 
A 
po+ 
TNF-a 
B 
bp 
1353 
'E 
603 
310 
Fig. 4. Validation of RT-PCR products by restriction digestion. (A) Schematic dia- 
gram showing the locations of PCR primers and restriction sites. (B) Two human 
cDNAs, TNF-a (lane 2) and IL-P (lane 4) were amplified using RT-PCR Amplimers 
(CLONTECH), and digested with PvuII (lane 3) and Me1 (lane 5) respectively, for 1 h 
at 37’C. PCR products and digests were electrophoresed on 2% agarose in TBE. Lane 1 
contains $X174IHueIII digests as size markers. The fragments generated exactly 
matched those predicted from the restriction map and the locations of the primer tem- 
plates. No purification or exchange of buffers was performed. 
micrograms of total RNA, even when examining gene transcripts expressed at 
high levels. Typically, RNA analyzed by these methods must be enriched for 
mRNA by oligo(dT) cellulose chromatography. RT-PCR not only provides a 
more sensitive method requiring smaller amounts of RNA and less work, but 
in some cases is the only method that can be used. 
For example, the dystrophin gene, defective in patients with muscular dys- 
trophy, is expressed at very low levels (representing only 0.01-0.00 1% of total 
40 Siebert 
Table 1 
Examples of Restriction 
Enzymes Successfully Used 
to Digest PCR Products 
Without Post-PCR Purification 
Restriction Examples of 
enzyme PCR products cleaved 
BamHi 
EcoRI 
HhaI 
NcoI 
NdeI 
PVUII 
XbaI 
PstI 
Sac1 
MnSOD 
IL-3, IL-4 
CD4 
IL-2R 
IFN-y 
IL-l p, IL-7, TNF-a, 
TNF-P 
IL-6 
tPA, P-Actln 
P-Actm