METHODS 25, 409–418 (2001)

doi:10.1006/meth.2001.1263, available online at http://www.idealibrary.com on

Real-Time PCR Analysis of DNA and RNA Extracted from

Formalin-Fixed and Paraffin-Embedded Biopsies1
Ulrich Lehmann2 and Hans Kreipe

Institute of Pathology, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany

morphology. The feasibility of performing molecular

The archives of departments of pathology represent a unique
source of morphologically defined biopsies derived from normal
and pathologically altered tissues for which extensive clinical data
are available. The exact quantification of nucleic acids in these
biopsies offers a promising extension of current methodology to
study the pathogenesis of many different diseases. The develop-
ment of real-time PCR technology has greatly facilitated the real-
ization of nucleic acid quantification. Now it is feasible to analyze
large series of samples for the exact quantification of nucleic
acids even if the number of target molecules is small and the
amount of material available for analysis is limiting. This review
focuses on our own experiences concerning the extraction of nu-
cleic acids from fixed and embedded biopsies using both conven-
tional approaches and laser-assisted microdissection and the sub-
sequent application of real-time PCR methods for quantification
of mRNA transcripts, gene copy number, and the methylation
status. We provide a number of protocols to assist in the applica-
tion of these techniques. 䉷 2001 Elsevier Science (USA)
Tissue samples collected and processed for pathologi-
cal diagnosis represent a unique source of archived and
morphologically defined disease-specific biological ma-
terial. Retrospective analysis of this archival tissue
would enable the correlation of molecular findings with
the response to treatment and the clinical outcome. In
addition, these findings will guide future prospective
studies analyzing native or freshly frozen biopsies.
Some types of biopsies taken for routine diagnostic
purposes, e.g., bone marrow trephines, exist almost ex-
clusively as fixed and embedded samples because the
processing of fresh biopsies does not yield satisfying
1
This work was supported by DFG Grant Fe 516/1-1.
2 uncertainty. In practical terms, tissue processing can-
To whom correspondence and reprint requests should be ad-
dressed. Fax: ⫹49-(0)511-532-5799. E-mail: Lehmann.Ulrich@MH-
Hannover.de.
genetic analysis of fixed and embedded biopsies will
improve diagnostic procedures and will enable a large
number of research studies on various diseases. The
most widely used fixative in pathology is formalin. It is
comparably cheap, is easy to handle, provides superior
morphological quality, and is compatible with nearly
all relevant antibodies for immunohistochemical stain-
ing. Therefore this article focuses on the extraction
of nucleic acids from formalin-fixed and paraffin-
embedded (FFPE) samples and the subsequent molec-
ular analysis.
In many pathological circumstances quantitative al-
terations in gene copy numbers or mRNA expression
levels are as important as qualitative changes such as
mutations and deletions. The development of real-time
PCR technology (1, 2) has greatly facilitated the realiza-
tion of nucleic acid quantification. The theory and prac-
tice of this new methodology have been extensively dis-
cussed in several recent reviews (3, 4) and are presented
more comprehensively elsewhere in this issue. The pur-
pose of this review is to give an overview of the applica-
tion of real-time PCR technology to the quantitative
analysis of nucleic acids extracted from routinely proc-
essed FFPE pathological specimens performed at our
institution.
GENERAL CONSIDERATIONS
Processing of Tissue Specimens
Tissue processing is a source of great variability and
not be completely standardized in a routine clinical
laboratory or across different laboratories. In Germany

1046-2023/01 $35.00 409

䉷 2001 Elsevier Science (USA)
All rights reserved.
410 LEHMANN AND KREIPE
for example, reference laboratories receive a consider-
able number of biopsies from hospitals all over the coun-
try. The biopsy may be fixed in a solution provided by
the reference institute or from a different supplier. Also
it may have already been paraffin-embedded. Therefore
the conditions of fixation (time, temperature and fixa-
tive) are quite variable for many biopsies.
For the fixation of all tissue specimens, we use a
phosphate-buffered formaldehyde solution (see Appen-
dix for composition). This solution has a formaldehyde
concentration of approximately 3.7% (w/v) and is some-
times referred to as “10% formalin” in the literature
because the formaldehyde stock solution (approxi-
mately 37%, w/v) is diluted by a factor of 10. This has
and may cause confusion. In an ideal world, the biopsies
should be fixed in the buffered formaldehyde solution
for 24 h at 4⬚C in the dark before the paraffin-embed-
ding procedure starts. In reality most biopsies are fixed
“overnight” but sometimes over the weekend at room
temperature. In our experience this seems to be compat-
ible with subsequent molecular analysis. But prolonged
storage in the formaldehyde solution for more than 1
week destroys nucleic acids. For teaching purposes, sev-
eral institutes store large biopsies in the formaldehyde
solution for years. So far we were unable to retrieve
any nucleic acids from these “wet” archives.
Bone marrow biopsies are fixed in a modified buffered
formaldehyde solution and decalcified for at least 18 h
using EDTA at neutral pH. See the Appendix for the
compositions of the fixative and the “decalcifier.” For
the decalcification, the fixed biopsies are incubated un-
der constant agitation in a large volume of “decalcifier.”
We routinely use approximately 3 liters for 30 bone
marrow trephines. The dehydration and paraffin-em-
bedding steps are performed overnight in an automatic
tissue processing device. For details, the reader is re-
ferred to the excellent textbook of Carson (5). In many
instances we are interested in extracting nucleic acids
from stained tissue sections such as cells isolated by
microdissection (see below). Since nucleic acids are sus-
ceptible to acid- or base-catalyzed hydrolysis, we prefer
to stain tissue sections using a staining solution with
a neutral pH. As with all other protocols developed in
our laboratory for the analysis of archival biopsies, we
try to perform the staining as simply and as fast as
possible to reduce the risk of degradation, cross-contam-
ination, and the hands-on time. In most instances, a
short and simple methylene blue staining as described
in Protocol 1 (Appendix) is sufficient for identification
of the relevant morphological details.
Organization of the Laboratory
The greatest problem with PCR if performed rou-
tinely on a daily basis is cross-contamination of samples
and contamination of samples due to PCR product
“carryover” (6, 7). Because of the very high sensitivity
of the method, a few target molecules are sufficient to
cause false-positive results. If the initial sample to be
analyzed is very small (e.g., microdissected tissue sec-
tions), this kind of contamination may also lead to a
distortion of quantitative measurements.
For these reasons, strictly enforced guidelines con-
cerning the cleaning of instruments and the handling
of samples before and after amplification need to be
followed by all personnel involved. We perform all pre-
amplification steps in a separate laboratory consisting
of two rooms: one for setting up the PCR master mixture
(a “template-free” room) and the other for preparation
of tissue sections, nucleic acid extraction, bisulfite
treatment, and addition of DNA or cDNA to the PCR
mixtures.
For the cutting of paraffin blocks, we use an ordinary
microtome, which is cleaned meticulously after every
sample with a nonhazardous xylol substitute (Xyloler-
satz XEM-200, Vogel, Marburg, Germany). Plastic
labware and the benches are cleaned using a 3% hypo-
chlorite solution. The PCR products are analyzed in a
separate laboratory. In no circumstances should ampli-
fied samples or equipment from this working area be
brought back to the pre-PCR area.
Fragmentation of Nucleic Acids due to Formalin Fixation
The fixation of tissue samples in formaldehyde leads
to extensive crosslinking of all tissue components. As
a consequence of this crosslinking, the nucleic acids
isolated from these specimens are highly fragmented.
The extent of fragmentation (the average fragment size)
depends on the tissue type and the condition of fixation.
In our hands, the average fragment length is around
300–400 bp for DNA and 200 bp for RNA as estimated
from ethidium bromide- or SybrGold-stained gels. This
is in good agreement with other studies reported in the
literature (8).
As a consequence, we modified our extraction proto-
cols and the protocol to treat DNA with bisulfite. For
the bisulfite treatment we developed reaction condi-
tions that are much more gentle but still efficient
enough to avoid complete degradation of the DNA (see
below). It is also necessary to select PCR primers that
produce PCR products that are as short as possible.
This is a general recommendation for real-time PCR
assays to increase amplification efficiency.
Figure 1 demonstrates the effect of different amplicon
 REAL-TIME PCR AND ARCHIVAL BIOPSIES 411

sizes for the analysis of RNA isolated from formalin-
fixed and paraffin-embedded samples. Shown is the de-
tection of the housekeeping gene ␤ -glucuronidase. The
forward primer and the hybridization probe were the
same in all three reactions, but three different reverse
primers were used to generate amplicons of different
sizes. Due to the fragmentation of RNA, a 300-bp frag-
ment was not detectable whereas the 80-bp fragment
gives a strong signal. Using RNA isolated from cultured
cells, all three reverse primers gave comparable signals.
Similar results were reported for the detection of the
p21 mRNA by Specht et al. (9).
Relative Quantification: Theory and Practice
Real-time PCR technology is most often used for the
absolute quantification of nucleic acids (10). However,
the absolute numerical quantification of nucleic acids
has several disadvantages. These include: (i) The prepa-
ration of calibration curve standards with known abso-
lute concentrations is laborious and a serious source of
errors. The OD260nm measurement which is widely used
for determination of nucleic acid concentrations de-
pends on the photometer, the water, buffer substances,
and also the method used for DNA isolation. For these
reasons the variability is quite high. (ii) The stable
storage of quantitative standards is very difficult to
achieve. Highly diluted solutions of nucleic acids are
not stable and preparing dilutions for each use again
introduces an additional source of error. (iii) The ampli-
fication of several standards for generating a calibra-
tion curve for absolute quantification within each run
reduces the amount of samples that can be analyzed in
each run, thereby diminishing the throughput of the
system. (iv) Since the amplification of different tem-
plate preparations (standard versus sample) is com-
pared, the amplification of DNA (or cDNA) from patho-
logical specimens derived from heterogeneous sources
with different degrees of tissue preservation introduces
error. Slightest impurities in the sample and uneven
template fragmentation due to improper fixation se-
verely distort the absolute quantification.
As a consequence we prefer and strongly recommend
a relative quantification algorithm. The CT or thresh-
old value of the target sequence is directly proportional
to the absolute concentration when compared with the
threshold value for reference genes. The factor X by
which the amount of the gene has changed can be calcu-
lated with the formula
X ⫽ 2⫺⌬⌬CT [1]
where ⌬⌬Ct ⫽ (CT,target ⫺ CT,reference)sample ⫺ (CT,target ⫺
CT,reference)normal. The target represents the target gene
of interest and the reference represents a housekeeping
gene. The sample represents the sample of interest (e.g.,
microdissected cancer cells) and the reference repre-
sents the tissue from normal healthy specimens (e.g.,
tissue without any morphological alterations). If the
⌬CT values are the same, no change in the amount of
the target molecule in the sample of interest relative
to the normal tissue has occurred. But if a difference
between the two ⌬CT values is observed, the amount

FIG. 1. Effect of the amplicon size on the RT-PCR analysis of RNA isolated from formalin-fixed biopsies. The amplification plots for
different real-time PCR assays using the same cDNA as a template are shown. The forward primer and the fluorescence-labeled hybridization
probe are the same for all three reactions; only the reverse primer is different, thereby producing the three different PCR products.
412 LEHMANN AND KREIPE
of the target molecule has increased or decreased, de-
pending on the ⌬⌬CT value. The theory and applications
of the 2⫺⌬⌬CT method are discussed elsewhere in this
issue (29).
Principally, it would be sufficient to measure one sam-
ple of normal tissue as a source for the ⌬Ct,reference.
To determine the variability of this value due to fixa-
tion artifacts or sample impurities we measured the
⌬Ct,reference for all genes of interest in more than 60
microdissected samples of normal breast epithelium
and calculated the mean ⌬Ct,reference (11). The standard
deviation of these values was in the range 0.5 to 1 cycle.
To apply relative quantification, the following as-
sumptions have to be correct: (i) The efficiencies of the
PCR for the target and chosen references must be equal.
The efficiency is measured as a constant ⌬CT value over
a broad range of template concentrations. To mimic the
assay conditions in the samples of interest as closely
as possible, this test should be performed with nucleic
acids isolated from fixed and embedded specimens.
(ii) Expression of the housekeeping genes should be
constant in the tissue and the pathological situation
of interest.
In conclusion, relative quantification is not only a
more simple and economical method than absolute
quantification, but also a more reliable and exact
method for the analysis of nucleic acids extracted from
FFPE specimens. In performing relative calculation, all
distortions of the amplification efficiency due to fixation
artifacts or sample impurities are eliminated.
As a caveat, we and others have observed that the
CT values generated by different lots of the same probe
are not identical. No German suppliers of hybridization
probes guarantee absolute uniformity of different lots
of the same sequence. Therefore, one has to compare
the amplification plots of a newly purchased lot of a
hybridization probe with amplification plots generated
with aliquots from the former lot. In some cases we
have observed differences of nearly two cycles.
METHODOLOGY
Isolation of DNA
Our protocol for isolating genomic DNA from archival
FFPE specimens was developed for the analysis of tis-
sue sections isolated by laser-assisted microdissection
(12). Only several hundred cell profiles or fewer were
lysed in the lysis buffer. To minimize the loss of mate-
rial, our aim was to lyse the cells in a buffer that is
compatible with PCR without any additional purifica-
tion. Therefore our lysis buffer contains Tween 20 in-
stead of SDS, because the latter compound is an effec-
tive inhibitor of DNA polymerases. We also use a low
concentration of EDTA (see Protocol 2 Appendix).
Heat inactivation of proteinase K leads to a complete
denaturation of the double-stranded genomic DNA,
which facilitates the subsequent amplification by PCR.
Following proteinase K treatment, the samples can be
stored safely for months at ⫺20⬚C. We have used this
simple and crude preparation of genomic DNA from
small tissue samples for the reproducible and successful
analysis of several hundred biopsies.
In situations where several thousand cells are iso-
lated by microdissection (e.g., large intraductal breast
cancer lesions or large suspicious lymphoid infiltrates
in the bone marrow or lymph node) the sample is lysed
in a larger volume of 50–200 ␮l. To concentrate the
DNA and to remove the stain and other impurities, we
precipitate these samples after the heat inactivation
step by adding 0.1 vol of sodium acetate, pH 7, con-
taining 100 ␮g/ml Dextran (MW 500,000) as a carrier
(Sigma Chemicals, Deisenhofen, Germany) and 2.5 vol
of absolute ethanol. After incubation at ⫺20⬚C for at
least 24 h, the samples are centrifuged (20 min, 14,000g,
4⬚C), washed once with 70% ethanol, air dried, and
dissolved in 20–50 ␮l TE buffer (10 mM Tris–HCl pH
8, 1 mM EDTA).
If the DNA has to be manipulated enzymatically after
isolation, e.g., with restriction endonucleases, further
purification steps are required to obtain reproducible
results. For the clonality analysis of hepatic angiomyo-
lipoma employing the HUMARA assay (13) it was nec-
essary to exhaustively extract the DNA isolated from
manually microdissected sections of FFPE biopsies
with phenol/chloroform followed by precipitation of
the DNA.
Isolation of RNA
The isolation of RNA from archival biopsies requires
more steps than DNA extraction. It is necessary to use
a buffer that not only lyses the tissue but also rapidly
denatures the proteins that may degrade the RNA. We
developed a simple and rapid one-step extraction
protocol (see Protocol 3, Appendix) based on the dena-
turation solution originally described by Chomczynski
and Sacchi (14).
The concentration of the RNA is measured from the
absorbance at 260 nm. Yields of 3 to 30 ␮g of total RNA
can be isolated depending on the size of the biospy. The
RNA may be used directly for cDNA synthesis (0.5 to
3 ␮g is recommended). In the vast majority of samples
 REAL-TIME PCR AND ARCHIVAL BIOPSIES
prepared in our institute, the average size of the frag-
mented RNA was found to be about 200 nucleotides as
estimated from a formaldehyde–agarose gel.
In comparison to published procedures ((8, 15) and
references therein) the method described here has sev-
eral advantages: (i) Omission of deparaffinization and
rehydration saves time, prevents the risk of RNA degra-
dation during rehydration, and reduces sample cross-
contamination from repeated pipetting. In our hands,
deparaffinization of samples does not significantly in-
crease the yield of RNA but increases the risk of losing
tissue, especially if very small biopsies are processed.
(ii) The incubation time for extraction of the tissue spec-
imens is much shorter than in most protocols (overnight
compared with several days). (iii) Expensive prefabri-
cated reagents (e.g. TRIzol or RNAzol) are not neces-
sary. (iv) Most importantly, the hands-on time is dra-
matically reduced because only one extraction step and
one precipitation are necessary, reducing the contact to
hazardous chemicals and minimizing the risk of sample
contamination and loss of material during repeated
pipetting.
To date, we have extracted RNA from several hun-
dred specimens. Following our protocol for RNA extrac-
tion, we have an overall efficiency of greater than 98%
for the recovery of amplifiable RNA if the specimen has
been processed in our institution. The efficiency is much
lower when paraffin-embedded biopsies from other in-
stitutes are analyzed, indicating that the tissue proc-
essing protocols are not compatible with molecular
studies.
FIG. 2. Relative quantification of kappa and lambda immunoglobulin light chain transcripts in bone marrow trephines displaying reactive
lymphoid hyperplasia. Shown is the CT value difference for the kappa and lambda transcripts in 37 archival bone marrow trephines.
413
Quantitative Detection of RNA: An Example:
To explore the efficiency, reliability, and reproducibil-
ity of quantitative mRNA expression studies in bone
marrow biopsies, we determined the ratio of kappa and
lambda immunoglobulin light chain transcripts using
a newly developed real-time consensus PCR.3 By align-
ment of all known light chain sequences, we have identi-
fied a highly conserved region long enough to design a
hybridization probe and appropriate primers to amplify
all light chain mRNAs with one set of probe and prim-
ers. This is the first protocol for the exact quantification
of light chain expression in FFPE samples and comple-
ments and improves existing methods which are at best
semiquantitative (immunohistochemistry or in situ hy-
bridization). In a series of samples from different pa-
tients with reactive lymphoid hyperplasia, the ratio of
these two mRNA species should be nearly constant as
demonstrated in Fig. 2. These results clearly show
that it is possible to obtain meaningful quantitative
mRNA expression data from FFPE and even decalci-
fied biopsies.
We extended these studies to the analysis of bone
marrow biopsies with suspicious lymphoid infiltrates
to measure light chain restriction (i.e., nonphysiological
preferential expression of one light chain) quantita-
tively on the level of the mRNA transcripts.3 Since light
3
Lehmann, U., Bock, O., Länger, F. and Kreipe H. Demonstration
of light chain restricted clonal B-lymphoid infiltrates in archival bone
marrow trephines by quantitative real-time PCR. Submitted for pub-
lication.
414 LEHMANN AND KREIPE
chain restriction is an indicator of a monoclonal B-cell
proliferation, this new quantitative assay could serve
as a complementation of existing clonality assays.
Quantitative Detection of DNA Methylation in Archival
Biopsies
The methylation of cytosine residues in the promoter
region of tumor-suppressor genes is now widely recog-
nized as an alternative mechanism of gene inactivation
in cancer (16). To address the question of whether pro-
moter methylation is an early event in the process of
malignant transformation, small and precancerous le-
sions need to be analyzed. However, these lesions are
nearly exclusively available as archival FFPE patholog-
ical specimens. Therefore, CpG methylation has to be
analyzed in FFPE biopsies to answer important biologi-
cal and pathophysiological questions. In several situa-
tions it will be necessary to microdissect tissue sections
for the analysis of homogenous cell populations (see
(17)).
Most studies analyzing DNA methylation are based
on the treatment of the DNA with bisulfite (18). This
bisulfite conversion translates the epigenetic modifica-
tion of the original DNA into a difference of the primary
sequence by converting unmethylated cytosine to uracil
(which is amplified as thymine in subsequent PCR
assays) whereas methylated cytosine remains un-
changed (see Fig. 3).
Our method of treating DNA with bisulfite is de-
scribed in detail in Protocol 4 (Appendix). The major
modification introduced by us is the much shortened
incubation time for the bisulfite treatment. Tradition-
ally samples were incubated overnight (approximately
16 h) to achieve complete conversion. It has been our
experience that near-complete destruction of DNA oc-
curs after 16 h incubation in the highly concentrated
FIG. 3. Schematic representation of the bisulfite treatment of geno-
mic DNA. Methylated cytosine remains as cytosine, whereas unmeth-
ylated cytosine is converted to uracil, which is amplified as thymine
in a subsequent PCR.
bisulfite solution at an acidic pH (pH 5.0). This bisulfite-
mediated degradation of DNA is especially a problem
when analyzing DNA isolated from archival specimens
because the DNA is already highly fragmented. Real-
time PCR was used to measure the DNA before and
after bisulfite treatment. We demonstrate that after 3
h unconverted DNA is not detectable and the specific
signals for the converted DNA have already reached
their maximum (U. Lehmann, unpublished). These ob-
servations have recently been confirmed by another
group (19). For a comprehensive discussion of quantita-
tive real-time PCR for measuring DNA methylation see
the accompanying article in this issue (30).
Quantification of Gene Copy Number in Laser-Assisted
Microdissected Archival Breast Cancer Specimens
Nearly all studies employing real-time PCR technol-
ogy for the quantification of gene copy number describe
the analysis from fresh frozen biopsies (20). However,
small and precancerous lesions are nearly exclusively
available as FFPE biopsies. These lesions in general
are quite small and may show lower levels of gene am-
plification than invasive tumor cells. Therefore, it is
important to analyze samples obtained by microdissec-
tion of stained tissue sections that are free of contami-
nating bystander cells. For these reasons we combined
the laser-assisted microdissection (see below) to obtain
pure samples of suspicious cells with real-time PCR
technology to accurately quantify low levels of amplifi-
cation (12). In comparison to our method, studies using
alternative in situ methods such as fluorescence in situ
hybridization face major limitations (21). These include
a lack of preservation of histological detail and difficul-
ties in the reproducible detection and objective scoring
of low levels of amplification. The simple lysis protocol
described in Protocol 2 (Appendix) provided DNA of
sufficient quality for the reproducible quantification of
gene copy number. This protocol contains as few steps
as possible to reduce the risk of sample cross-contami-
nation and loss of material. If too much dye is present
in the lysate or if several samples have to be pooled to
increase the yield of DNA, a simple precipitation using
ethanol and sodium acetate containing dextran as a
carrier may be performed (for details see above).
Employing this technology, we separated intraductal
breast cancer cells from surrounding invasive carci-
noma cells and nonmalignant bystander cells by laser-
assisted microdissection. Figure 4 demonstrates the
contamination-free isolation of intraductal and inva-
sive breast cancer cells from the very same tissue sec-
tion. Exact gene copy number was quantified by real-
time PCR. For the copy number determination of the
 REAL-TIME PCR AND ARCHIVAL BIOPSIES
growth regulatory genes c-erb-B2 topoisomeraseII␣, c-
myc, and cyclinD1, we applied the relative quantifica-
tion approach described above. We have chosen two
chromosomal regions for which no numerical aberration
has been described for the copy number determination
of the growth regulatory genes c-erb-B2, topoisomer-
aseII␣, c-myc, and cyclinD1. To increase the adherence
of the sections to the slides, the membranes on which
the sections are mounted following microdissection are
FIG. 4. Laser-microdissection and isolation of pure intraductal and
pure invasive tumor cells using laser-assisted microdissection. (A)
Section of a ductal-invasive carcinoma of the breast. The reduced
optical quality is due to the fact that the slides are dried and not
coverslipped for microdissection. (B) Section after removal of the
intraductal and the invasive component, respectively. (C) Isolated
intraductal (left) and invasive (right) tumor cells in the lid of a reac-
tion tube. (A–C) Methylene blue-stained; original magnification:
⫻100.
415
coated with a 0.1% solution of poly (L-lysine) (Sigma
Chemicals, Deisenhofen, Germany). After sectioning,
the slides are incubated for 15 min at 55⬚C. After they
are mounted, the sections are deparaffinized and
stained as described in Protocol 1 (Appendix). It is
necessary to dry the stained sections at least over-
night at 40⬚C. Otherwise residual liquid between the
membrane and the slide will prevent the microdissec-
tion since the laser cannot cut through a layer of liquid
and one will create only air bubbles. After microdissec-
tion, the DNA was isolated as described in protocol 2
(Appendix). In an extension of this initial study, we
systematically compared the gene copy number of sev-
eral important growth regulatory genes in intraductal
breast cancer cells with the histological classification
of the samples (11).
CONCLUDING REMARKS
As mentioned above, the protocol for isolating DNA
from archival specimens was developed for the analy-
sis of small microdissected samples of fewer than 1000
cell profiles. The procedure was kept as simple as
possible to reduce the risk of contamination and loss
of material during preparation. We have compiled four
protocols from the literature to isolate DNA from non-
microdissected fixed biopsies (see Table 1).
The quantitative analysis of RNA extracted from
archival biopsies is a quite new application of real-
time PCR technology. Therefore it might be interest-
ing to compare our protocols and experiences with the
few other publications that are available. To facilitate
this we have compiled five references with a short
description of the methodology in Table 2.
Following the protocols described above, DNA and
RNA isolated from FFPE biopsies can be analyzed
by all variations of conventional and real-time PCR
assays. One must keep in mind, however, that the
nucleic acids are fragmented and that fragmentation
will occur to a different extent. This means that the
amplicon size has to be minimized and that relative
quantification approaches with internal controls are
much more reliable and easier to perform than an
absolute quantification with external calibration
curves.
Keeping these caveats in mind, reliable quantita-
tive data can be obtained as convincingly and indepen-
dently demonstrated by several groups. A vast area
of research has been opened up which will lead to a
wealth of new insights into not only the pathogenesis
416 LEHMANN AND KREIPE

of all kinds of diseases and but also the mechanisms 3. Rehydrate with a drop of water.
of normal development and differentiation. 4. Stain 3 min with methylene blue (Löfflers Methy-
lenblau, Merck, Germany, diluted 1:5 with water).
5. Wash three times with water.
APPENDIX 6. Dry overnight at 40⬚C.
B. Paraffin Sections
Solutions
1. Select to deparaffinization (2 ⫻ 10 min xylol sub-
Buffered formalin: 3.7% formaldehyde, 29 mM stitute; 2 ⫻ 5 min 100% ethanol, 1 ⫻ 5 min 96%, 1 ⫻
NaH2PO4, 45.8 mM Na2HPO4. 5 min 70%; 1 ⫻ 5 min water).
Decalcifier: 270 mM EDTA, 210 mM Tris–HCl, 2. Stain 3 min with methylene blue (Löfflers Methy-
pH 7–7.2. lenblau, Merck, Germany, diluted 1:5 with water).
Fixative for bone marrow trephines: 0.1 M potassium 3. Wash three times with water.
acetate, 0.5% glutaraldehyde, 1.1% formaldehyde. 4. Dry overnight at 40⬚C.

Protocol 1: Methylene Blue Staining of Histological Protocol 2: Isolation of DNA from Microdissected
Sections Samples
A. Cryo Sections The P.A.L.M. Laser-MicroBeam System (P.A.L.M.,
Bernried, Germany) is currently used at our institute.
1. Fix immediately with a drop of 100% ethanol.
2. Air dry. 1. The microdissected samples are catapulted into

TABLE 1
Comparison of Protocols for DNA Extraction from Nonmicrodissected FFPE Biopsies for Real-Time PCR

Purpose of the study DNA extraction method Reference

Quantitative detection of
mycobacteria in sarcoidosis
Quantitative detection of HHV-8
Quantitative detection of
Toxoplasma gondii
Quantitative microsatellite analysis
Dexpat kit (Takara, Tokyo, Japan), organic extraction and precipitation (22)
Deparaffinization and proteinase K digestion (0.1 mg/ml) for 3–5 days in 100 mM (23)
NaCl/10 mM Tris–HCl pH 8.4/25 mM EDTA/0.5% SDS followed by organic
extraction and precipitation
Genomic Prep Kit (Amersham Pharmacia), involves proteinase K digestion, depro- (24)
teination, and precipitation
Deparaffinization and proteinase K digestion (0.5 mg/ml) for 3–5 days in 100 (25)
mM NaCl/10 mM Tris–HCl pH 8/25 mM EDTA/0.5% SDS followed by organic
extraction and precipitation

TABLE 2
Comparison of Protocols for RNA Extraction from FFPE Biopsies for Real-Time PCR

Reference transcript(s) RNA extraction method Reference

␤2-Microglobulin Deparaffinization and proteinase K digestion (0.5 mg/ml) for 16 h in 20 mM Tris–HCl, pH 7.6/ (26)
20 mM EDTA/1% SDS at 55⬚C; RNA purified further from this lysate using TRIzol (Gibco/
Life Technologies)
␤ -Glucuronidase Deparaffinization and proteinase K digestion (6 mg/ml) for 3 days in 1 M GTC/25 mM 2-ME/0.5% (8)
Sarcosyl 20/20 mM Tris–HCl, pH 7.5; after the first and second days, proteinase K again added
(same concentration), extraction with acidic phenol/chloroform; following precipitation, RNA
purified twice using TRIzol (Gibco/Life Technologies)
GAPDH Deparaffinization and use of Micro RNA isolation Kit (Stratagene) (27)
GAPDH Deparaffinization, then homogenization of tissue in lysis buffer using a tube pestle; Purification (28)
using PUREscipt RNA isolation kit (Gentra Systems) within 3 h 55⬚C
HPRTa PGK Deparaffinization and proteinase K digestion (0.5 mg/ml) for 16 h in 10 mM Tris–HCl pH 8/0.1 (9)
mM EDTA/2% SDS at 60⬚C followed by organic extraction and precipitation
a
HPRT, human hypoxanthine phosphoribosyltransferase; PGK, phosphoglycerate kinase.
 REAL-TIME PCR AND ARCHIVAL BIOPSIES
the lid of a 0.5-ml reaction tube. Only the tubes offered
by P.A.L.M. can be used for this, because for this appli-
cation, the distance between the section and the inner
side of the lid is too great in the tubes made by other
manufacturers.
2. Apply 30–50 ␮l proteinase K digestion buffer (50
mM Tris–HCl, pH 8.1, 1 mM EDTA, 0.5% Tween 20,
0.1 mg/ml proteinase K) to the lid and close the tubes
in this inverted position. Leave the tubes standing on
their lids.
3. Incubate samples overnight at 40⬚C in this in-
verted position in a small hybridization oven.
4. Centrifuge the tubes for 3 min at 14,000g, and
incubate the samples at 95⬚C for 10 min in a ther-
moblock with a heated lid to inactivate the protein-
ase K.
5. Centrifuge the samples briefly and store at ⫺20⬚C.
Protocol 3: Extraction of RNA from Formalin-Fixed and
Paraffin-Embedded Biopsies
1. Cut one to six 10-␮m sections from the paraffin
block and place them directly in a 1.5-ml screw-cap tube
without deparaffinization or rehydration.
2. Add 850 ␮l denaturation solution (4 M guanidi-
nium isothiocyanate/0.25 M sodium citrate/0.5%
sarcosyl/0.1 M 2-mercaptoethanol) and 250 ␮l protein-
ase K solution (20 mg/ml in H2O) and incubate the tubes
overnight at 55⬚C in a vigorously agitating ther-
moshaker at 1400 rpm.
3. The next day, centrifuge the samples at 4⬚C for
5 min at 14,000g. After centrifugation a white cap of
solidified paraffin forms on the surface of the solution.
4. Transfer the digested sample to a clean 2-ml safe-
lock tube, carefully avoiding any transfer of undigested
material and solidified paraffin.
5. Add to this solution 100 ␮l 3 M sodium acetate,
pH 5.2, 630 ␮l water-saturated phenol, and 270 ␮l chlo-
roform. Mix thoroughly and leave on ice for 15 min.
6. After centrifugation at 4⬚C for 20 min at 14,000g,
remove approximately 1 ml of the upper aqueous phase,
carefully avoiding the transfer of any material collected
at the interface.
7. Add to the RNA-containing supernatant 1 ␮l gly-
cogen (20 mg/ml, Roche Molecular Diagnostics) and 1
ml isopropanol. After mixing, store samples overnight
at ⫺20⬚C.
8. Centrifuge the samples at 4⬚C for 20 min at
14,000g. Wash the pellet once with 70% ethanol, and
dissolve the air-dried pellet in 20 to 100 ␮l diethyl pyro-
carbonate (DEPC)-treated H2O. Avoid overdrying the
pellet.
Note. This solution may be stored safely for a few
417
weeks without degradation at ⫺20⬚C. For longer stor-
age, we precipitate the RNA again by adding 0.1 vol
sodium acetate, pH 5.2, and 2.5 vol absolute ethanol.
To prevent cross-contamination, all items that come
into contact with the samples including microtome
blade, forceps, and block holder are cleaned with xylol
or 2% hypochlorite solution before the next block is cut.
Protocol 4: Bisulfite Treatment of DNA Isolated from
Archival Specimens
1. Incubate the DNA in a total volume of 50 ␮l of 200
mM NaOH at 42⬚C for 30 min for complete separation of
both strands.
2. Add 275 ␮l 3.6 M bisulfite containing 1 mM hydro-
quinone (freshly prepared) and incubate the samples
for 3 h at 55⬚C in the dark.
3. Add 375 ␮l 6 M sodium iodine containing 1 mM
2-mercaptoethanol and 5 ␮l glass milk suspension (Qia-
gen, Hilden, Germany) and mix thoroughly.
4. After 10 min at room temperature, centrifuge the
samples at 14,000g for 3 min at 4⬚C and discard the su-
pernatant.
5. Wash the pellet three times with 70% ethanol.
6. Resuspend the pellet in 20 mM NaOH/90% etha-
nol and incubate for 5 min at room temperature.
7. Wash the pellet twice with 90% ethanol.
8. Elute the air-dried pellet with 25 ␮l TE buffer for
15 min at 55⬚C.
9. Centrifuge again (14,000g, 3 min, 4⬚C) and trans-
fer the supernatant to a fresh tube; 3 to 6 ␮l of this
solution is used for methylation-specific PCR.
ACKNOWLEDGMENTS
We thank Oliver Bock, Martina Mühlenhoff, Nils von Neuhoff, and
Thomas Schmittgen for many stimulating discussions and critical
remarks, which have improved this article.
REFERENCES
1. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H.
(1991) Proc. Natl. Acad. Sci. USA 88, 7276–7280.
2. Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W., and Deetz, K.
(1995) PCR Methods Appl. 4, 357–362.
3. Wittwer, C. T., Ririe, K. M., Andrew, R. V., David, D. A., Gundry,
R. A., and Balis, U. J. (1997) Biotechniques 22, 176–181.
4. Lie, Y. S., and Petropoulos, C. J. (1998) Curr. Opin. Biotechnol.
9, 43–48.
5. Carson, F. L. (1997) Histotechnology: A Self-Instructional Text,
ASC Press.
6. Kwok, S., and Higuchi, R. (1989) Nature 339, 237–238.
7. Rys, P. N., and Persing, D. H. (1993) J. Clin. Microbiol. 31,
2356–2360.
418 LEHMANN AND KREIPE
8. Godfrey, T. I., Kim, S.-H., Chavira, M., Ruff, D. W., Warren, R. S.,
Gray, J. W., and Jensen, R. H. (2000) J. Mol. Diagn. 2, 84–91.
9. Specht, K., Richter, T., Muller, U., Walch, A., Werner, M., and
Hofler, H. (2001) Am. J. Pathol. 158, 419–429.
10. Freeman, W. M., Walker, S. J., and Vrana, K. E. (1999) Biotech-
niques 26, 112–122, 124–125.
11. Glöckner, S., Lehmann, U., Wilke, N., Kleeberger, W., Länger, F.,
and Kreipe, H. (2001) Lab. Invest. 81, 565–571.
12. Lehmann, U., Glöckner, S., Kleeberger, W., von Wasielewski,
H. F., and Kreipe, H. (2000) Am. J. Pathol. 156, 1855–1864.
13. Flemming, P., Lehmann, U., Becker, T., Klempnauer, J., and
Kreipe, H. (2000) Hepatology 32, 213–217.
14. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,
156–159.
15. Krafft, A. E., Duncan, B. W., Bijwaard, K. E., Taubenberger, J. K.,
and Lichy, J. H. (1997) Mol. Diagn. 2, 217–230.
16. Jones, P. A., and Laird, P. W. (1999) Nat. Genet. 21, 163–167.
17. Lehmann, U., Hasemeier, B., Lilischkis, R., and Kreipe, H. (2001)
Lab. Invest. 81, 635–638.
18. Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt,
F., Grigg, G. W., Molloy, P. L., and Paul, C. L. (1992) Proc. Natl.
Acad. Sci. USA 89, 1827–1831.
19. Grunau, C., Clark, S. J., and Rosenthal, A. (2001) Nucleic Acids
Res. 29, E65.
20. Bieche, I., Olivi, M., Champeme, M. H., Vidaud, D., Lidereau,
R., and Vidaud, M. (1998) Int. J. Cancer 78, 661–666.
21. Wisecarver, J. L. (1999) Am. J. Clin. Pathol. 111, 299–301.
22. Ishige, I., Usui, Y., Takemura, T., and Eishi, Y. (1999) Lancet
354, 120–123.
23. Kennedy, M. M., Biddolph, S., Lucas, S. B., Howells, D. D., Picton,
S., McGee, J. O., Silva, I., Uhlmann, V., Luttich, K., and O’Leary,
J. J. (1999) J. Clin. Pathol. 52, 569–573.
24. Lin, M. H., Chen, T. C., Kuo, T. T., Tseng, C. C., and Tseng, C. P.
(2000) J. Clin. Microbiol. 38, 4121–4125.
25. Nigro, J. M., Takahashi, M. A., Ginzinger, D. G., Law, M.,
Passe, S., Jenkins, R. B., and Aldape, K. (2001) Am. J. Pathol.
158, 1253–1262.
26. Bijwaard, K. E., Aguilera, N. S., Monczak, Y., Trudel, M.,
Taubenberger, J. K., and Lichy, J. H. (2001) Clin. Chem. 47,
195–201.
27. Goldsworthy, S. M., Stockton, P. S., Trempus, C. S., Foley, J. F.,
and Maronpot, R. R. (1999) Mol. Carcinogen. 25, 86–91.
28. Sheils, O. M., O’Leary, J. J., and Sweeney, E. C. (2000) J. Pathol.
192, 32–36.
29. Livak, K. J., and Schmittgen, T. D. (2001) Methods 25.
30. Trinh, B. N., Long, T. I., and Laird, P. W. (2001) Methods 25.