B~teck Adv.Vol. 11, pp. 13-29,1993 0734-9750/93 $24.

00
in Great Britain. All Rights Reserved. @ 1993Pecgamc~ Press Llxl

REVERSE TRANSCRIPTION-POLYMERASE C H A I N
REACTION: A N OVERVIEW OF THE T E C H N I Q U E
A N D ITS APPLICATIONS

NICK W. OHAN and JOHN J. HEIKKILA

Department of Biology, University of Waterloo, Waterloo,Ontario, N2L 3G1 Canada

Abstract

The polymerase chain reaction (PCR) has galvanized molecular biologists by

virtue of its ability to provide them with large quantities of any desired
fragment (up to l lkb) of DNA. This power combined with its flexibility has
also inspired many useful applications, including new methods of DNA
sequencing, cloning and mutagenesis. One logical variation of PCR has been its
application to the detection and analysis of messenger RNA by the addition of
a reverse transcription step prior to performing PCR. Due to the exquisite
sensitivity of PCR, reverse transcription-PCR (RT-PCR) has been used to
characterize mRNAs previously undetectable by established methods of RNA
analysis such as Northern hybridization and RNase protection assays.
"~urthermore, its capacity as a method of quantitative analysis is currently
,ing developed. RT-PCR has also been used to diagnose the presence of
certain diseases. Recently, RT-PCR has been employed to identify and isolate
genes that are differentially expressed in different cells or environmental
conditions.

Keywords

Reverse transcription, polymerase chain reaction (PCR), RT-PCR, mRNA, cDNA,

primer design, contamination, quantitation

Introduction

PCR allows the sensitive, selective detection and amplification of a target DNA
fragment (43). It involves the initial denaturation of the DNA, followed by the

13
14 N.W. OHAN and J. J. HEIKKILA
annealing of target-specific oligonucleotide primers to each strand of DNA, and
finally the extension of the primers to produce a copy of the selected region.
By r e p e a t i n g this cycle of denaturation, primer annealing and primer
extension, there is an exponential amplification of the target. In theory, after
20 cycles, there could be 220 , or over one million copies of the target produced.
Rapid advances are being made in PCR technology (2,15, 53, 34). An extension
of the technique involves the addition of a reverse transcription step prior to
PCR, allowing one to apply the power of PCR to RNA (Fig. 1) (40,24,27,48). This
procedure has been variously referred to as RT-PCR, RNA phenotyping,
RNA/PCR and message amplification phenotyping (MAPPing). This overview
presents the various components comprising RT-PCR, including methods of
RNA isolation, reverse transcription, PCR reagents, primer design and dealing
with c a r r y - o v e r contamination, among others. Finally, some of the recent
applications of RT-PCR are examined. These include quantitative analysis of
mRNA levels via competitive RT-PCR and a relatively new variation of RT-PCR
which allows the study of differential eukaryotic mRNA transcription.

RNA Isolation

RT-PCR can be performed with relatively small quantities of RNA. Typically,

the reverse transcription of one microgram of total RNA (of which mRNA
comprises less than 5%) provides enough cDNA for ten PCR reactions. As little
as 60 pg of total RNA has been used successfully (48). In the polymerase chain
reaction step less than 100 molecules of cDNA can be detected and amplified
(40). The very selective nature of the reaction allows the amplification to
proceed against a high background of non-target cDNA such that purification
of mRNA, prior to reverse transcription, is not necessary. However, since the
PCR will amplify DNA as readily as cDNA, it is essential to eliminate any DNA
contamination. Designing primers that define a region containing an intron can
obviate this problem (40). Amplified genomic DNA can then be distinguished
from cDNA by virtue of its larger fragment size. Analysis of mRNA encoded by
i n t r o n - l e s s genes requires more e l a b o r a t e means to d i s t i n g u i s h D N A
contamination (see primer design section). Selecting an efficient RNA isolation
protocol can help minimize this problem from the start.

The most commonly employed method of RNA isolation is the guanidine

thiocyanate/ cesium chloride procedure (GuSCN/CsC1) (8). In this protocol,
tissue is first homogenized in 4M guanidine thiocyanate which is a strong
RNase inhibitor. RNases are probably the main source of failure when isolating
RNA and appropriate precautions must be taken to eliminate RNase activity
(32). The sample is then layered on a cesium chloride cushion and the RNA is
 REVERSE TRANSCRIFHON-PCR

TOTAL RNA REVERSE

ISOLATION TRANSCRIBE

0
Guanidine
p-
A M V - R T OR MmLV-RT
Oligo (dT)/Random
p- c D N A for MR

Yhioeyanate/CsCl
Hexamers/SpeciflcPrimers
Method

I~ O L ~ S E
CHAINREACTION I
I
CYCLE 1 r''~
i
I
CYCLE2 CYCLE 3
DENATURATION 3' TTT5': 3' TTT5' 3T TTT5'
~NA 5'-- TTT3,
15'-- rrTylI
'Long'Product 5'-- TTT 3'
3,__..-........~,5,

PRIMER 3' i TTT5'

3' m TTT5'
MgNEALING 3' 1 TTT5, ', 5 ' ~ T T 3,

,L
5 ~ T T 3, 5 ' ~ T T 3,
3,1--......,,~ 5,

3 ' ~ T T 5'
PRIMER 3' + - - I t : : t : : ~ T T 5' 3 ' ~ T T 5' 5' -~T'r'rrr=r'ffTT 3,
EXTENSION 5'~;x~'T, TT3'
!
i 5' ~ ] ~ : G ~ I - T T T 3,
*
0 ......... ,

3 ' ~ 5 '
Repeat

I 5' Primer~GEND 3' Primer 1 20 - 40

ElecU'ophoresis + cycles

,+++"+
+,r~or ,++==+l
3,~+ j
o °o= PCR Products:
- verify l e n g t h
more

I----I
- Southems
- Restriction digest
- Sequ~ce

FIGURE 1: A SUMMARY OF RT-PCR. In the reverse

transcription-polymerase chain reaction, the target sequence first
appears in its proper length at the end of the second cycle (**).
This fragment is amplified exponentially. The 'long' product is
amplified linearly and will not appear on the gel.
 16 N.W. OHAN and I. J. HEIKKILA
physically separated from protein and DNA by ultracentrifugation. Subsequent
treatment with R N a s e - f r e e DNase can remove any r e s i d u a l DNA. A
microadaptation of the procedure uses a micro-ultracentrifuge, permitting the
isolation of RNA in a single day (40). Another modification of the technique
e m p l o y s p h e n o l - c h l o r o f o r m extractions to eliminate the ultracentrifugation
step (9). RNA isolation kits are also commercially available.

Reverse Transcription

The design of the reverse transcription reaction is quite straight-forward (32).

One need only decide which primer and reverse transcriptase should be
u t i l i z e d . Reverse transcription requires a DNA primer to initiate the reaction.
Three types of primers are available: a target specific primer, oligo (dT), and
random hexanucleotides. The latter two are recommended for RT-PCR (24,48).
Oligo(dT) provides cDNA up to 2-3 kilobases (kb) in length from all
polyadenylated RNAs (48). Random hexanucleotides will generate cDNA from
all RNA and is especially useful for RNA longer than 2-3 kb, which might not
be fully reverse transcribed using oligo(dT). The target specific primer, usually
the 3'-PCR primer, is not often used, because optimal concentrations must be
experimentally determined in each specific instance (48).

A v i a n m y e l o b l a s t o s i s virus (AMV) and Moloney murine l e u k e m i a virus

(MmLV) are commonly used reverse transcriptases (32). The A M V - R T is best
suited to the synthesis of relatively short cDNAs due to its high RNase H
activity. This activity will cleave the extending strand if the A M V - R T pauses
during transcription. This is more likely to occur if a long cDNA is being
synthesized. Also, its high optimal temperature reduces RNA secondary
structure allowing more efficient cDNA synthesis of problematic RNA. MmLV-
RT is better for the synthesis of longer cDNAs due to its low RNase H activity.
In this case, problems with RNA secondary structure can be dealt with using
methyl mercury hydroxide (32).

Finally, a relatively new enzyme which is capable of reverse transcription and

efficient activity as a thermostable DNA polymerase in PCR has been described
(36). The very high optimal temperature of the Tth p o l y m e r a s e should
minimize RNA secondary structure. The Tth p o l y m e r a s e has been reported to
be more efficient and sensitive than typical reverse transcriptases and DNA
polymerases.
 REVERSETRANSCRIPTION-PCR 17

The Polymerase Chain Reaction

Once the cDNA has been synthesized, RT-PCR is subject to the same
parameters as regular 'DNA' PCR. However, there are some RT-PCR-specific
factors which must be addressed with respect to primer design and
contamination problems. The PCR and RT-PCR variables are briefly described
below. These include the concentration of reagents, selection of a DNA
polymerase, the thermal profile, product analysis, primer design and the
prevention of contamination. Once organized, RT-PCR can be completed in a
single day - from reverse transcription to visualization of the PCR products
following electrophoresis.

The PCR Variables

The concentrations of the PCR reagents depend on the DNA polymerase, PCR
primers and target length, and thus must be optimized separately for each
case. General PCR protocols (24) provide a good starting point for optimization
of the reaction.

The original DNA polymerase used in PCR to catalyze the extension of the
primers was the Klenow fragment of E. coli DNA polymerase I (14). The
problem with the Klenow polymerase was that it was denatured at the
beginning of each cycle and thus had to be continually replaced. The discovery
of thermostable DNA polymerases, able to withstand normal denaturing
temperatures, revolutionized the polymerase chain reaction by facilitating its
automation (43). The most commonly used thermostable polymerase is the
Taq DNA polymerase. Other polymerases are also available; for example, there
is the Vent DNA polymerase, which has a 3'-->5' proof-reading exonuclease
activity not possessed by Taq (13), making it more useful in sequencing
reactions because of its higher fidelity. As mentioned before the T t h
polymerase is also available and is capable of both reverse transcription and
PCR (36).

A typical PCR thermal profile begins with a denaturation period of 3-5

minutes. This is then followed by cycles of denaturation, primer annealing and
primer extension (Fig. 1). Denaturation occurs at 95°C for approximately one
minute. Primer annealing temperatures are a function of the primer sequences
(see primer design section). Annealing times are also generally one minute.
Primer extension occurs at 72°C (optimal for Taq) and the time varies
18 N.W. OHAN and J. J. HEIKKILA
according to the length of the target. As a rule of thumb, 1 minute/kilobase
(kb) is followed. Extension times will also vary according to the efficiency of
the thermocycler that is used (20, 52). Following 20-40 of these cycles, a final
extension period at 72°C for 5-10 minutes allows any incompletely extended
fragments to be completed. 'Two-step' PCR is also practiced (20). This involves
the combination of the annealing and extension steps into a single step at the
same temperature. The primers must be designed to have annealing
temperatures near the extension temperature of 72°C. Recently, it has been
recommended that 'hot start' PCR be utilized (15). This involves the addition of
a key reagent (e.g. T a q ) at the high temperatures during the initial
denaturation phase. This seems to reduce non-specific amplification due to
mis-priming as well as minimizing primer-dimer formation (see primer design
section) thereby increasing the overall efficiency. A more sophisticated
approach to achieving a 'hot start' employs a wax barrier to separate the PCR
components until the wax melts, allowing the reagents to mix at high
temperatures (10). The wax also replaces the mineral oil normally present to
prevent evaporation.

PCR products can be visualized after electrophoresis using either 1-2% agarose
or 6-10% polyacrylamide gels and ethidium bromide staining of the DNA
fragments. This procedure will verify that the synthesized DNA fragment is
the expected size. However, to unquestionably verify that the fragment is
indeed the targeted one, it must be subjected to at least one of the following
tests: (A) restriction digestion of the fragment using a known unique enzyme
site, (B) Southern hybridization analysis of the PCR fragments using a specific
probe or, (C) sequencing of the fragment.

Designing PCR Oligonucleotide Primers

The importance of carefully designing primers which meet certain basic

criteria cannot be over-emphasized. There are many free and commercially
available computer programs which can help a researcher to design and/or
test primers (22,41,42,48,33). The following section highlights the basics of
primer design (40).

Generally, the pair of primers are both between 20 and 30 bp in length with
similar A/T and G/C composition. Their calculated annealing temperatures
should be as close to 72°C as possible for optimal stringency. The higher the
stringency, the less likely mis-priming will occur, thereby minimizing non-
specific a m p l i f i c a t i o n . Annealing temperatures can be c a l c u l a t e d using
 REVERSETRANSCRIPTION-PCR 19
empirically derived formulas (49,1,3,17,33,42,54). The formula employed by
the commercial "OLIGO" program is probably the most sophisticated (41,42).
The free-ware program "Oligonucleotide Synthesizing Program" (OSP) has also
met with success (22). After primers have been designed, formamide (44) and
d i m e t h y l s u l f o x i d e c o n c e n t r a t i o n s (16), which r e d u c e hydrogen bond
stabilities, can also be used to adjust annealing stringencies.

Primer-dimers should be avoided. These occur when the 3'-ends of the

primers are complementary to one another and thus, if they anneal, can be
extended by the polymerase. Naturally, this reduces PCR efficiency. It is
thought that as little as a 3bp overlap can result in primer-dimers (42). As
well, the primers should be very specific for a single member of a gene family
to avoid amplifying similar, but undesired fragments (40). Target lengths from
100 bp to 10.8 kb (42) have been amplified, although 300 bp to 2 kb are
generally best. Larger targets require greater care in optimizing the PCR
conditions, while targets less than 300 bp may be confused with primer-
dimers.

RT-PCR Specific Parameters

1) The target sequence should span an intron so that c D N A can be
distinguished from genomic DNA contamination based on size differences.
2) The target sequence defined by the primers should be located as close to
the 3' end of the mRNA as possible (within 2-3 kb) if oligo(dT) is used as
the primer. Thus, even if full-length cDNA is not synthesized, the target can
still be detected (40).
3) Control primers which detect cDNA made against constitutive mRNA (e.g. B-
actin) can be used as a positive control for successful reverse transcription,
especially when the main PCR primers produce a negative result (i.e. the
main target was not present in the sample).
4) When the target mRNA is transcribed from an intron-less gene, it is difficult
to distinguish between amplified cDNA and contaminating genomic DNA.
Perhaps the most direct way of dealing with intron-less genes is the use of
RNA template-specific PCR (RS-PCR) (46). This involves the addition of 30
nucleotides to the 5'-end of the oligo(dT) reverse transcription primer, so
that the resultant cDNA will be 'tagged' with the 30 nucleotides. In the PCR
reaction, primers for the tag and the target sequence will specifically
a m p l i f y the marked cDNA. Untagged DNA will not be a m p l i f i e d .
Furthermore, if problems of carry-over contamination arise (see carry-over
contamination section), one can simply change the tag sequence.
 20 N. W. OHAN and I. J. HEIKKILA

Avoiding Carry.Over Contamination

One of the major problems encountered in the use of PCR is carry-over

contamination from previous reactions. Reagents, primers, and instruments
can become contaminated with previous PCR products and, since the product
of amplification is identical to the source, these 'amplicons' (11) will be
amplified efficiently - subverting all subsequent reactions with the same
target. Many of these problems can be obviated if one routinely follows some
common sense procedures (25, 39). It is also essential to run a negative
control containing all reagents except for the target cDNA, which is usually
prepared as the last sample in a series. Ultraviolet light has been found to be
useful for reducing carry-over contamination (37). Exposure of DNA to
ultraviolet light results in pyrimidine dimers between adjacent pyrimidines,
and thus should prevent the DNA from acting as a template for DNA synthesis.
However, a recent study indicates that its use might be more limited than was
previously thought (45, 4).

A direct means of controlling carry-over contamination is to alter the PCR

products such that they may be discriminated against in subsequent PCR
reactions. Longo et al. (30) have done this by replacing dTTP with dUTP in the
PCR assay. Thus, all PCR products will incorporate dUTP. When the next
reaction is performed, the reaction tube containing all reagents and DNA are
treated with uracil DNA g l y c o s y l a s e which will cleave any c a r r y - o v e r
products, without significantly affecting the DNA sample. The glycosylase is
then denatured in the first PCR cycle. When tested with RT-PCR, it resulted in
a 10 million-fold reduction in the synthesis of amplicons (38). Even then, some
trace contamination was still detected.

RT-PCR Applications

The high sensitivity of RT-PCR, coupled with the extremely low RNA quantities
required, has made it the most powerful method available for the detection of
rare transcripts. Some of its recent applications are listed in Table 1. A
possible problem with RT-PCR is that it is t o o sensitive. Chelly et aL (6) have
reported the detection of the expression of tissue-specific genes in non-
specific cells a phenomenon they term 'illegitimate transcription'. The
potential e x i s t e n c e of this basal expression suggests that a method of
quantitation using RT-PCR would be useful to distinguish different levels of
 REVERSE TRANSCRIPTION-PCR m

Table 1: Examples of Uses of RT-PCR

Purpose Target RNA Reference

Source
HUMAN
Rapid clinical Respiratory Nasopharyn- Paton et al. (1992) J.
Virology
detection Syncytial gel Aspirates Clin. Micro. 30: 901-904.
Virus
Virology Detection Hepatitus C Serum Castillo et al. (1992) J,
methods Virus Vir. Meth. 38:71-80.

Oncology Detection of Wilms tumor Kidney Brenner et al. (1992)

alternatively susceptibility Oncogene 7: 1431-1433.
spliced gene mRNA (WT1)
Oncology Detection for PML-RAR- Acute Pro- Chen et al. (1992)
diagnostic alpha fusion myelocytic Oncogene 7:1223-1232.
purposes gene isoforms leukemia cells
PIG
St. Louis Brain (and Howe et al. (1992) J. Vir.
Virology Detection Encephalitis mosquito Meth. 36: 101-110.
viral RNA abdomen)
MOUSE
Distinguishing Interleukin-3 Bone Marrow, Fung et al. (1992) J.
Growth Expression and IL-3 Cell lines Immun. Meth. 149: 97-
Factors Levels receptor-like 103.
mRNA
Development Detection of Alkaline Hahnel et al. (1990)
stages of Phosphatase Embryo Development 110:555-
expression 564.
RAT
Detection and Calcium/
Kinase relative Calmodulin Brain Beamen-Hall et al. (1992)
subunit quantitation Dependent J. Neurochem. 58: 1259-
mRNAs (non-corn- ProteinKinase 1267
petitive assay) II Isoenzymes
Localization, Endothelin
Nephrology Quantitation Receptor Kidney Terada et al. (1992) J.
Clin. Invest. 90: 10%112.
via Southerns mRNA
Xenopus
laevis
Differential Various Shuldiner et al. (1991)
Development developmental Insulin genes e m b r y o n i c Proc. Natl. Acad. Sci.
expression sta~es 88:7679-7683.
22 N.W. OHANand J. I. HEIKK/LA
gene expression. Also, this would allow the comparison of gene expression
under various environmental conditions.

Quantitative RT-PCR

The PCR reaction proceeds in an exponential manner until reagent limits are
met (12). If one can determine the final quantity of products (X), and one
knows the amplification efficiency (E, max. value = 1), and the number of
cycles (n), the initial amount of starting template (I) can be calculated using
the following equation (19):

X = I(I +E) n

The final yield, (X), can be determined from densitometric scans of ethidium
bromide stained gels (19), or the radioactive labelling of a primer or dNTP in
the PCR reaction mix which can then be quantitated via scintillation counting
of excised bands or d e n s i t o m e t r i c s c a n n i n g of its a u t o r a d i o g r a m
(35,7,12,19,40). Finally, the yield of PCR product can also be obtained by
Southern blot analysis, followed by the densitometric scanning of the resultant
autoradiogram (7,5).

Determining the efficiency of the reaction is difficult because the amplification

is exponential. Thus, the effects of unavoidable small differences in the
preparation of each reaction will also be amplified. Therefore, one must be
able to account for tube-to-tube variations. Several methods have been
devised (5,7,18,19,26,31,35). To date, the most successful and accurate one
seems to be competitive RT-PCR (18,19,47). Competitive RT-PCR involves the
co-amplification of known quantities of a competitive template which uses the
same primers as the target, in the same reaction tube. Earlier attempts at
using two primer sets, one for the target and one for a control target,
encountered problems due to different annealing efficencies of the primers.
The competitor and target sequences can be distinguished by a small (100 bp)
intron in the competitor, or a single mutational difference resulting in the gain
or loss of a restriction site (these can be generated using PCR-based site-
specific mutagenesis (21)). When one runs samples with constant amounts of
target cDNA, and a dilution series of known competitor concentrations, it is
possible to determine the initial amount of target template. This is determined
from a graph of the ratio of the final yield of competitor: target product yield
vs. the known concentration of initially added competitor template. The
quantity at which the ratio is l : l is most representative of the initial amount
 REVERSETRANSCRWrION-PCR
of target template. Gilliland et al. (18,19) have used this technique to
quantitate less than 1 pg of target c D N A from 1 ng of total m R N A , and could
d i s t i n g u i s h t w o - f o l d d i f f e r e n c e s in m R N A c o n c e n t r a t i o n s . T o a c c o u n t for
differences in reverse transcription efficiencies, an R N A competitor, using the
same primers as the target can be used in a co-reverse transcription and co-
a m p l i f i c a t i o n reaction (50). Refer to Table 2 for some current examples of
quantitative R T - P C R .

Table 2: Recent Applications of Quantitative RT-PCR

Target/(Source) Method of Reference

Quantitation
Cytokines: Interleukin-2 Competitive RT-PCR Carding et al. (1992) J.
IL-4, interferon-gamma (DNA competitor) Immuno. Meth. 151:277-287.
(Human)
HIV-1 viremia Competitive RT-PCR Menzo et al. (1992) J. Clin.
(Human) (RNA competitor) Micro. 30: 1752-1757.
Human c - e r b B - 2 proto- g-Actin internal control Ninomiya et al. (1992)Br. J.
oncogene (gastric to standardize quantities Cancer 66:84-87.
cancer cells)
Cytotoxic cell proteinase Competitive RT-PCR Prendergast et al. (1992) J.
mRNA (tumor cells) (RNA competitor) Biol. Chem. 67: 5090-5095.

Multi-drug resistance-1 Normalized by in- Murphy et al. (1990)

P-glycoporotein dependent (i.e. 2 primer Biochemistry 29: 10351-10356.
(Human colon sets) amplification of a
carcinoma cell lines) control gene
Cytokines: granulocyte- The O r i g i n a l
macrophage colony- Competitive RT-PCR Gilliland et al. (1990) Proc.
stimulating factor, IL-3 (DNA competitor, Natl. Acad. Sci. USA. 87: 2725-
(Human) identical primers) 2729.
Aldose reductase (Calf Normalization with l]- Ohtaka et al. (1992)
pulmonary artery actin quantities in a Diabetologia 35: 730-734.
endothelial cells) separate reaction tube
Dystrophin mRNA Co-RT and co-amplified Chelly et al. (1990) Eur. J.
(cultured mouse brain exogenous m R N A Biochem. 187: 691-698.
ceils) control (2 primer sets)
Laminin A, B1, B2 chains Competitive RT-PCR Horikoshi et al. (1992) Kidney
(mouse kidney mRNAs) (RNA competitor) Intl. 42:764-769.

Renin gene expression Competitive RT-PCR lwaiet al. (1992) J.

(rat CNS) IRNA competitor) Hypertension 10:717-724

Differential Display of Eukaryotic mRNA

A recent R T - P C R innovation has been developed to c o m p a r e the differential

expression of eukaryotic mRNA in different ceils, or under different
24 N.W. OHAN and J. J. HEIKKILA
environmental conditions. Liang and Pardee (28) employed RT-PCR to amplify
a sub-set of the mRNA population. This was accomplished using a primer
which is anchored to the poly(A) tail of eukaryotic mRNA, plus two additional
3'-bases, e.g. 5 ' - T l l C A . There is a 1/3 chance of the first 5'-base prior to the
poly(A) tail of the mRNA being a G, excluding A as a possibility. There is a 1/4
chance of the second 5'-base being a T. Thus, statistically speaking, this primer
should select one twelfth of the mRNA during reverse transcription. This same
primer is then used in the PCR reaction in conjunction with a very short (a
random 10mer) second primer. Due to the short sequence of the second
primer, it is likely to anneal with several different cDNAs, at varying positions
relative to the first primer. This results in the amplification of many different
cDNAs which can then be separated and visualized on a sequencing gel. The
resultant banding pattern is characteristic of the mRNAs present in a cell or
under different environmental conditions, and the pattern can be reproduced
in duplicate experiments. Side-by-side comparisons are possible for different
samples run on the gel. Unique fragments can be isolated directly from the gel
and cloned for further study. Liang and Pardee applied their technique to
normal and tumorigenic mouse cells. From the 50-100 cDNAs amplified, they
were able to detect and clone a differentially expressed mRNA in each cell
type. Using different primer pairs, other sub-populations of mRNA could also
be analyzed. A similar method using low stringency annealing of an arbitrary
primer for cDNA synthesis and the first PCR cycle followed by 30 high
stringency cycles has also been described (51). This method amplifies a very
small subset of cDNA molecules (10-20) and comparisons can be made as with
Liang and Pardee's procedure.

Concluding Remarks

In this overview of the reverse transcription-polymerase chain reaction, we

have attempted to present some of the important criteria for successfully
performing RT-PCR in the laboratory. Aspects of RNA isolation, reverse
t r a n s c r i p t i o n , PCR v a r i a b l e s , p r i m e r design, and the p r e v e n t i o n of
contamination have been presented. Knowledge of their details should enable
more efficient application of the technique as a means of sensitively detecting
and quantitating mRNA, and now, identifying and cloning d i f f e r e n t i a l l y
expressed eukaryotic mRNAs. As more information becomes available, the
power of highly sensitive detection will become more of an advantage than a
disadvantage in RT-PCR - facilitating its accurate and controlled use as one of
the most powerful methods of RNA analysis.
 REVERSETRANSCRIFI'ION-PCR 25

References

1. Baldino Jr., F., Chesselet, M.-F, Lewis, M. (1989) High-resolution in situ

hybridization histochemistry. Meth. Enzymol. 168, 761-777.

2. Bloch, W. (1991) A biochemical perspective of the polymerase chain

reaction. Biochemistry 30, 2735-2747.

3. Borer, P., Dengler, B., Tinoco Jr., I., Uhlenbeck, O. (1974) Stability of
ribonucleic acid double-stranded helices. J. Mol. Biol. 86, 843-853.

4. Briscoe, P., Jorgensen, T. (1991) Parameters affecting susceptibility of PCR

contamination to UV inactivation. BioTechniques 10, 591-595.

5. Buck, K., Harris, R., and Sikela, J. (1991) A general method for quantitative
PCR analysis of mRNA levels for members of gene families: application to
G A B A A receptor subunits. BioTechniques 11, 636-641.

6. Chelly, J., J.-P. Concordet, J.-C. Kaplan, and A. Kahn (1989) Illegitimate
transcription: transcription of any gene in any cell type. Proc. Natl. Acad.
Sci. USA. 86, 2617-2621.

7. Chelly, J., Montarras, D., Pinset, C., Berwald-Netter, Y., Kaplan, J.-C., Kahn, A.
(1990) Quantitative estimation of minor mRNAs by cDNA-polymerase
chain reaction. Eur. J. Biochem. 187, 691-698.

8. Chirgwin, J., Przbyla, A., MacDonald, R., and Rutter, W. (1979) Isolation of
biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry 18, 5294-5299.

9. Chomczynski, P., and Sa~chi, N. (1987) Single-step method of RNA isolation

by acid guanidinium 'thiocyanate-phenol-chloroform extraction. Anal.
Biochem. 162, 156-159.

10. Chou, Q., M. Russell, D. Birch, J. Raymond and W. Bloch (1992) Prevention
of pre-PCR mis-priming and primer dimerization improves low-copy-
number amplifications. Nucl. Acids Res. 20, 1717-1723.

11. Cimino, G., Metchette, K., Tessman, J., Hearst, J., and Isaacs, S. (1991) Post-
PCR sterilization: a method to control carryover contamination for the
polymerase chain reaction. Nucl. Acids Res. 19, 99-107.

12. Delidow, B., Peluso, J. and White, B. (1989) Quantitative measurement of

mRNAs by polymerase chain reaction. Gene Anal. Techn. 6, 120-124.
26 N.W. OHANand I. J. HEIKKILA
13. Eckert, A. and Kunkel, T. (1990) High fidelity DNA synthesis by the
Thermus aquaticus DNA polymerase. Nucl. Acids Res. 18, 3739-3744.

14. Erlich, H., Gelfand, D. and Saiki, R. (1988) Specific DNA amplification.
Nature 331, 461-462.

15. Erlich, H., Gelfand, D., Sninsky, J. (1991) Recent advances in the
polymerase chain reaction. Science 252, 1643-1651.

16. Filichkin, S. and S. Gelvin (1992) Effect of dimethyl sulfoxide concentration

on specificity of primer matching in PCR. BioTechniques 12, 828-830.

17. Frier, S., Kierzek, R., Jaeger, J., Sugimoto, N., Caruthers, M., Neilson, T., and
Turner, D. (1986) Improved free-energy parameters for predictions of
RNA duplex stability. Proc. Natl. Acad. Sci. USA. 83, 9373-9377.

18. Gilliland, G., Perrin, S., and Bunn, H. (1990) Competitive PCR for
Quantitation of mRNA. In PCR Protocols: A Guide to Methods And
Applications, Innis, M., Gelfand, D., Sninsky, J. and White, T. (eds.).
Academic Press, Orlando. p60-69.

19. Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H. (1990) Analysis of
cytokine mRNA and DNA: detection and quantitation by competitive
polymerase chain reaction. Proc. Natl. Acad. Sci. USA. 87, 2725-2729.

20. Haft, L., Atwood, J., DiCesare, J., Katz, E., Picozza, E., Williams, J., and
Woudenberg, T. (1991). A high-performance system for automation of the
polymerase chain reaction. BioTechniques 10, 102-112.

21. Higuchi, R., Krummel, B., and Saiki, R. (1988) A general method of in vitro
preparation and specific mutagenesis of DNA fragments: study of protein
and DNA interactions. Nucl. Acids Res. 16, 7351-7367.

22. Hillier, L. and P. Green (1991) OSP: A computer program for choosingPCR
and DNA sequencing primers. PCR Methods and Amplifications. 1, 124-
128.

23. Ivinson, A. (1991) Optimizing PCR conditions for HLA class II SSO typing.
Eur. J. Immunogen. 18, 23-32.

24. Kawasaki,, E., and Wang, A. (1989) Detection of gene expression. In PCR
Technology, H.A. Erlich, ed. Stockton Press, New York. 89-96.

25. Kwok, S., and Higuchi, R. (1989) Avoiding false positives with PCR. Nature
339, 237-238.
 REVERSETRANSCRIPTION-PCR 27
26. Landgraf, A., Reckmann, B., Pingoug, A. (1991) Quantitative analysis of
polymerase chain reaction (PCR) products using primers labelled with
biotin and a fluorescent dye. Anal. Biochem. 193, 231-235.

27. Larrick, J.W. (1992) Message amplification phenotyping (MAPPing) -

principles, practice and potential. Trends Biotechnol. 10, 146-151.

28. Liang, P. and A.B. Pardee (1992) Differential display of eukaryotic

messenger RNA by means of the polymerase chain reaction. Science 257,
967-971.

29. Lin, P. and D. M. Brown (1992) Synthesis of oligodeoxyribonucleotides

containing degenerate bases and their use as primers in the polymerase
chain reaction. Nucl. Acids Res. 20, 5149-5152.

30. Longo, M., Berninger, M., and Hartley, J. (1990) Use of uracil DNA
glycosylase to control carry-over contamination in polymerase chain
reactions. Gene 93, 125-128.

31. Lundeberg, J., Wahlberg, J., and Uhl6n, M. (1991) Rapid colorimetric
quantification of PCR-amplified DNA. BioTechniques 10, 68-72.

32. Maniatis, T., Fritsch, E., and Sambrook, J. eds. (1989) Molecular Cloning: A
Laboratory Manual, 2nd ed.. Cold Spring Harbour Laboratory, Cold Spring
Harbour NY.

33. McGraw, R.,Steffe, E., Baxter, S. (1990) Sequence-dependent

oligonucleotide-target duplex stabilities: rules from empirical studies with
a set of twenty-mers. BioTechniques 8, 674-677.

34. Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction.
Scientific American. April, 56-65.

35. Murphy, L., Herzog, C., Rudick, J., Fojo, A., and Bates, S. (1990) Use of the
polymerase chain reaction in the quantitation of rndr-1 gene expression.
Biochemistry 29, 10351-10356.

36. Myers, T. and Gelfand, D. (1991) Thermostable reverse transcriptase/DNA

polymerase. Amplifications 7, 5-8.

37. Ou, C.-Y, Moore, J., Schochetman, G. (1991) Use of UV irradiation to reduce
false positivity in polymerase chain reaction. BioTechniques 10, 442-445.

38. Pang, J., J. Modlin and R. Yolken (1992) Use of modified nucleotides and
uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-
based amplification of RNA. Mol. Cell. Probes 6, 251-256.
28 N.W. OHANand J. J. HEIKKILA
39. Prince, A.M. and L. Andrus (1992) PCR: How to kill unwanted DNA.
BioTechniques 12, 358-360.

40. Rappolee, D.A., Wang, A., Mark, D., Werb, Z. (1989) Novel method for
studying mRNA phenotypes in single or small numbers of cells. J. Cell.
Biochem. 39, 1-11.

41. Rychlik, W. and Rhoads, R. (1989) A computer program for choosing

optimal oligonucleotides for filter hybridization, sequencing and in vitro
amplification of DNA. Nucl. Acids Res. 17, 8543-8551.

42. Rychlik, W., Spencer, W.J., Rhoads, R.E. (1990) Optimization of the
annealing temperature for DNA amplification in vitro. Nucl. Acids Res. 18,
6409- 6412.

43. Saiki, R., Gelfand, D., Stoffel, S. Scharf, S., Higuchi, R., Horn, G., Mullis,
Erlich, H. (1988) Primer-directed enzymatic amplification of DNA with
thermostable DNA polymerase. Science 239, 487-491.

44. Sarkar, G., Kapelner, S., Sommer, S. (1990) Formamide can dramatically
improve the specificity of PCR. Nucl. Acids Res. 18, 7465.

45. Sarkar, G. and Sommer, S. (1991) Parameters affecting susceptibility of

PCR contamination to UV inactivation. BioTechniques 10, 591-593.

46. Shuldiner, A., Tanner, K., Moore, C., Roth, J. (191) RNA template-specific
PCR: an improved method that dramatically reduces false positives in RT-
PCR. BioTechniques 11, 760-763.

47. Siebert, P. D. and J.W. Larrick (1992) Competitve PCR. Nature 359, 557-
558.

48. Siebert, P. (1991) RT-PCR: Methods and applications, Book 1, Clontech

Laboratories, Inc., San Diego.

49. Suggs, S., Hirose, T., Miyake, E., Kawashima, M., Johnson, K., Wallace, R.
(1981) Use of synthetic deoxyribonucleotides for the isolation of specific
cloned DNA sequences. In D.D. Brown (ed.), ICN-UCLA Symp. Dev. Biol.
Using Purified Genes. Academic Press Inc., New York, Vol. 23, pp 683-693.

50. Wang, A. and Mark, D. (1990) Quantitative PCR. In PCR Protocols: A Guide
to Methods and Applications. Innis, M., Gelfand, D., Sninsky, J. and White,
T. (eds.). Academic Press, Orlando. p70-75.

51. Welsh, J., K. Chada, S. Dalai, R. Cheng, D. Ralph and M. McClelland (1992)
Arbitrarily primed PCR fingerprinting of RNA. Nucl Acids Res. 20, 4965-
4970.
 REVERSETRANSCRIPTION-PCR 29

52. Wittwer, C., Fillmore, G., Garling, D. (1990) Minimizing the time required
for DNA amplification by efficient heat transfer to small samples. Anal.
Biochem. 186, 328-331.

53. Wright, P. and Wynford-Thomas, D. (1990) The polymerase chain reaction:

miracle or mirage? A critical review of its uses and limitations in
diagnosis and research. J. Pathol. 162, 99-117.

54. Wu, D., Ugozzoli, L., Pal, B., Qian, J., Wallace, R. (1991) Laboratory Methods:
The effect of temperature and oligonucleotide primer length on the
specificity and efficiency of amplification by the polymerase chain
reaction. DNA Cell Biol. 10, 233-238.