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
genetic analysis of fixed and embedded biopsies will
source of morphologically defined biopsies derived from normal
and pathologically altered tissues for which extensive clinical data
improve diagnostic procedures and will enable a large
are available. The exact quantification of nucleic acids in these number of research studies on various diseases. The
biopsies offers a promising extension of current methodology to most widely used fixative in pathology is formalin. It is
study the pathogenesis of many different diseases. The develop- comparably cheap, is easy to handle, provides superior
ment of real-time PCR technology has greatly facilitated the real- morphological quality, and is compatible with nearly
ization of nucleic acid quantification. Now it is feasible to analyze all relevant antibodies for immunohistochemical stain-
large series of samples for the exact quantification of nucleic ing. Therefore this article focuses on the extraction
acids even if the number of target molecules is small and the of nucleic acids from formalin-fixed and paraffin-
amount of material available for analysis is limiting. This review embedded (FFPE) samples and the subsequent molec-
focuses on our own experiences concerning the extraction of nu-
ular analysis.
cleic acids from fixed and embedded biopsies using both conven-
tional approaches and laser-assisted microdissection and the sub-
In many pathological circumstances quantitative al-
sequent application of real-time PCR methods for quantification terations in gene copy numbers or mRNA expression
of mRNA transcripts, gene copy number, and the methylation levels are as important as qualitative changes such as
status. We provide a number of protocols to assist in the applica- mutations and deletions. The development of real-time
tion of these techniques. 䉷 2001 Elsevier Science (USA) 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
Tissue samples collected and processed for pathologi- more comprehensively elsewhere in this issue. The pur-
cal diagnosis represent a unique source of archived and pose of this review is to give an overview of the applica-
morphologically defined disease-specific biological ma- tion of real-time PCR technology to the quantitative
terial. Retrospective analysis of this archival tissue analysis of nucleic acids extracted from routinely proc-
would enable the correlation of molecular findings with essed FFPE pathological specimens performed at our
the response to treatment and the clinical outcome. In institution.
addition, these findings will guide future prospective
studies analyzing native or freshly frozen biopsies.
Some types of biopsies taken for routine diagnostic GENERAL CONSIDERATIONS
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 Processing of Tissue Specimens
Tissue processing is a source of great variability and
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- not be completely standardized in a routine clinical
Hannover.de. 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- Organization of the Laboratory

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

sizes for the analysis of RNA isolated from formalin- reduces the amount of samples that can be analyzed in
fixed and paraffin-embedded samples. Shown is the de- each run, thereby diminishing the throughput of the
tection of the housekeeping gene ␤ -glucuronidase. The system. (iv) Since the amplification of different tem-
forward primer and the hybridization probe were the plate preparations (standard versus sample) is com-
same in all three reactions, but three different reverse pared, the amplification of DNA (or cDNA) from patho-
primers were used to generate amplicons of different logical specimens derived from heterogeneous sources
sizes. Due to the fragmentation of RNA, a 300-bp frag- with different degrees of tissue preservation introduces
ment was not detectable whereas the 80-bp fragment error. Slightest impurities in the sample and uneven
gives a strong signal. Using RNA isolated from cultured template fragmentation due to improper fixation se-
cells, all three reverse primers gave comparable signals. verely distort the absolute quantification.
Similar results were reported for the detection of the As a consequence we prefer and strongly recommend
p21 mRNA by Specht et al. (9). 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
Relative Quantification: Theory and Practice threshold value for reference genes. The factor X by
Real-time PCR technology is most often used for the which the amount of the gene has changed can be calcu-
absolute quantification of nucleic acids (10). However, lated with the formula
the absolute numerical quantification of nucleic acids
has several disadvantages. These include: (i) The prepa- X ⫽ 2⫺⌬⌬CT [1]
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 where ⌬⌬Ct ⫽ (CT,target ⫺ CT,reference)sample ⫺ (CT,target ⫺
for determination of nucleic acid concentrations de- CT,reference)normal. The target represents the target gene
pends on the photometer, the water, buffer substances, of interest and the reference represents a housekeeping
and also the method used for DNA isolation. For these gene. The sample represents the sample of interest (e.g.,
reasons the variability is quite high. (ii) The stable microdissected cancer cells) and the reference repre-
storage of quantitative standards is very difficult to sents the tissue from normal healthy specimens (e.g.,
achieve. Highly diluted solutions of nucleic acids are tissue without any morphological alterations). If the
not stable and preparing dilutions for each use again ⌬CT values are the same, no change in the amount of
introduces an additional source of error. (iii) The ampli- the target molecule in the sample of interest relative
fication of several standards for generating a calibra- to the normal tissue has occurred. But if a difference
tion curve for absolute quantification within each run 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- compatible with PCR without any additional purifica-
pending on the ⌬⌬CT value. The theory and applications tion. Therefore our lysis buffer contains Tween 20 in-
of the 2⫺⌬⌬CT method are discussed elsewhere in this stead of SDS, because the latter compound is an effec-
issue (29). tive inhibitor of DNA polymerases. We also use a low
Principally, it would be sufficient to measure one sam- concentration of EDTA (see Protocol 2 Appendix).
ple of normal tissue as a source for the ⌬Ct,reference. Heat inactivation of proteinase K leads to a complete
To determine the variability of this value due to fixa- denaturation of the double-stranded genomic DNA,
tion artifacts or sample impurities we measured the which facilitates the subsequent amplification by PCR.
⌬Ct,reference for all genes of interest in more than 60 Following proteinase K treatment, the samples can be
microdissected samples of normal breast epithelium stored safely for months at ⫺20⬚C. We have used this
simple and crude preparation of genomic DNA from
and calculated the mean ⌬Ct,reference (11). The standard
small tissue samples for the reproducible and successful
deviation of these values was in the range 0.5 to 1 cycle.
analysis of several hundred biopsies.
To apply relative quantification, the following as-
In situations where several thousand cells are iso-
sumptions have to be correct: (i) The efficiencies of the
lated by microdissection (e.g., large intraductal breast
PCR for the target and chosen references must be equal. cancer lesions or large suspicious lymphoid infiltrates
The efficiency is measured as a constant ⌬CT value over in the bone marrow or lymph node) the sample is lysed
a broad range of template concentrations. To mimic the in a larger volume of 50–200 ␮l. To concentrate the
assay conditions in the samples of interest as closely DNA and to remove the stain and other impurities, we
as possible, this test should be performed with nucleic precipitate these samples after the heat inactivation
acids isolated from fixed and embedded specimens. step by adding 0.1 vol of sodium acetate, pH 7, con-
(ii) Expression of the housekeeping genes should be taining 100 ␮g/ml Dextran (MW 500,000) as a carrier
constant in the tissue and the pathological situation (Sigma Chemicals, Deisenhofen, Germany) and 2.5 vol
of interest. of absolute ethanol. After incubation at ⫺20⬚C for at
In conclusion, relative quantification is not only a least 24 h, the samples are centrifuged (20 min, 14,000g,
more simple and economical method than absolute 4⬚C), washed once with 70% ethanol, air dried, and
quantification, but also a more reliable and exact dissolved in 20–50 ␮l TE buffer (10 mM Tris–HCl pH
method for the analysis of nucleic acids extracted from 8, 1 mM EDTA).
FFPE specimens. In performing relative calculation, all If the DNA has to be manipulated enzymatically after
distortions of the amplification efficiency due to fixation isolation, e.g., with restriction endonucleases, further
artifacts or sample impurities are eliminated. purification steps are required to obtain reproducible
results. For the clonality analysis of hepatic angiomyo-
As a caveat, we and others have observed that the
lipoma employing the HUMARA assay (13) it was nec-
CT values generated by different lots of the same probe
essary to exhaustively extract the DNA isolated from
are not identical. No German suppliers of hybridization
manually microdissected sections of FFPE biopsies
probes guarantee absolute uniformity of different lots
with phenol/chloroform followed by precipitation of
of the same sequence. Therefore, one has to compare the DNA.
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. Isolation of RNA
The isolation of RNA from archival biopsies requires
more steps than DNA extraction. It is necessary to use
METHODOLOGY 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
Isolation of DNA protocol (see Protocol 3, Appendix) based on the dena-
turation solution originally described by Chomczynski
Our protocol for isolating genomic DNA from archival and Sacchi (14).
FFPE specimens was developed for the analysis of tis- The concentration of the RNA is measured from the
sue sections isolated by laser-assisted microdissection absorbance at 260 nm. Yields of 3 to 30 ␮g of total RNA
(12). Only several hundred cell profiles or fewer were can be isolated depending on the size of the biospy. The
lysed in the lysis buffer. To minimize the loss of mate- RNA may be used directly for cDNA synthesis (0.5 to
rial, our aim was to lyse the cells in a buffer that is 3 ␮g is recommended). In the vast majority of samples
 REAL-TIME PCR AND ARCHIVAL BIOPSIES 413

prepared in our institute, the average size of the frag- Quantitative Detection of RNA: An Example:
mented RNA was found to be about 200 nucleotides as To explore the efficiency, reliability, and reproducibil-
estimated from a formaldehyde–agarose gel. ity of quantitative mRNA expression studies in bone
In comparison to published procedures ((8, 15) and marrow biopsies, we determined the ratio of kappa and
references therein) the method described here has sev- lambda immunoglobulin light chain transcripts using
eral advantages: (i) Omission of deparaffinization and a newly developed real-time consensus PCR.3 By align-
rehydration saves time, prevents the risk of RNA degra- ment of all known light chain sequences, we have identi-
dation during rehydration, and reduces sample cross- fied a highly conserved region long enough to design a
contamination from repeated pipetting. In our hands, hybridization probe and appropriate primers to amplify
deparaffinization of samples does not significantly in- all light chain mRNAs with one set of probe and prim-
crease the yield of RNA but increases the risk of losing ers. This is the first protocol for the exact quantification
tissue, especially if very small biopsies are processed. of light chain expression in FFPE samples and comple-
(ii) The incubation time for extraction of the tissue spec- ments and improves existing methods which are at best
imens is much shorter than in most protocols (overnight semiquantitative (immunohistochemistry or in situ hy-
compared with several days). (iii) Expensive prefabri- bridization). In a series of samples from different pa-
cated reagents (e.g. TRIzol or RNAzol) are not neces- tients with reactive lymphoid hyperplasia, the ratio of
sary. (iv) Most importantly, the hands-on time is dra- these two mRNA species should be nearly constant as
matically reduced because only one extraction step and demonstrated in Fig. 2. These results clearly show
one precipitation are necessary, reducing the contact to that it is possible to obtain meaningful quantitative
hazardous chemicals and minimizing the risk of sample mRNA expression data from FFPE and even decalci-
contamination and loss of material during repeated fied biopsies.
pipetting. We extended these studies to the analysis of bone
To date, we have extracted RNA from several hun- marrow biopsies with suspicious lymphoid infiltrates
dred specimens. Following our protocol for RNA extrac- to measure light chain restriction (i.e., nonphysiological
tion, we have an overall efficiency of greater than 98% preferential expression of one light chain) quantita-
for the recovery of amplifiable RNA if the specimen has tively on the level of the mRNA transcripts.3 Since light
been processed in our institution. The efficiency is much
lower when paraffin-embedded biopsies from other in- 3
Lehmann, U., Bock, O., Länger, F. and Kreipe H. Demonstration
stitutes are analyzed, indicating that the tissue proc-
of light chain restricted clonal B-lymphoid infiltrates in archival bone
essing protocols are not compatible with molecular marrow trephines by quantitative real-time PCR. Submitted for pub-
studies. lication.

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.
414 LEHMANN AND KREIPE

chain restriction is an indicator of a monoclonal B-cell bisulfite solution at an acidic pH (pH 5.0). This bisulfite-
proliferation, this new quantitative assay could serve mediated degradation of DNA is especially a problem
as a complementation of existing clonality assays. 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
Quantitative Detection of DNA Methylation in Archival after bisulfite treatment. We demonstrate that after 3
Biopsies h unconverted DNA is not detectable and the specific
signals for the converted DNA have already reached
The methylation of cytosine residues in the promoter
their maximum (U. Lehmann, unpublished). These ob-
region of tumor-suppressor genes is now widely recog-
servations have recently been confirmed by another
nized as an alternative mechanism of gene inactivation
group (19). For a comprehensive discussion of quantita-
in cancer (16). To address the question of whether pro-
tive real-time PCR for measuring DNA methylation see
moter methylation is an early event in the process of
the accompanying article in this issue (30).
malignant transformation, small and precancerous le-
sions need to be analyzed. However, these lesions are
nearly exclusively available as archival FFPE patholog- Quantification of Gene Copy Number in Laser-Assisted
ical specimens. Therefore, CpG methylation has to be Microdissected Archival Breast Cancer Specimens
analyzed in FFPE biopsies to answer important biologi-
Nearly all studies employing real-time PCR technol-
cal and pathophysiological questions. In several situa-
ogy for the quantification of gene copy number describe
tions it will be necessary to microdissect tissue sections
the analysis from fresh frozen biopsies (20). However,
for the analysis of homogenous cell populations (see
small and precancerous lesions are nearly exclusively
(17)).
available as FFPE biopsies. These lesions in general
Most studies analyzing DNA methylation are based
are quite small and may show lower levels of gene am-
on the treatment of the DNA with bisulfite (18). This
plification than invasive tumor cells. Therefore, it is
bisulfite conversion translates the epigenetic modifica-
important to analyze samples obtained by microdissec-
tion of the original DNA into a difference of the primary
tion of stained tissue sections that are free of contami-
sequence by converting unmethylated cytosine to uracil
nating bystander cells. For these reasons we combined
(which is amplified as thymine in subsequent PCR
the laser-assisted microdissection (see below) to obtain
assays) whereas methylated cytosine remains un-
pure samples of suspicious cells with real-time PCR
changed (see Fig. 3).
technology to accurately quantify low levels of amplifi-
Our method of treating DNA with bisulfite is de-
cation (12). In comparison to our method, studies using
scribed in detail in Protocol 4 (Appendix). The major
alternative in situ methods such as fluorescence in situ
modification introduced by us is the much shortened
hybridization face major limitations (21). These include
incubation time for the bisulfite treatment. Tradition-
a lack of preservation of histological detail and difficul-
ally samples were incubated overnight (approximately
ties in the reproducible detection and objective scoring
16 h) to achieve complete conversion. It has been our
of low levels of amplification. The simple lysis protocol
experience that near-complete destruction of DNA oc-
described in Protocol 2 (Appendix) provided DNA of
curs after 16 h incubation in the highly concentrated
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-
FIG. 3. Schematic representation of the bisulfite treatment of geno-
sive breast cancer cells from the very same tissue sec-
mic DNA. Methylated cytosine remains as cytosine, whereas unmeth-
ylated cytosine is converted to uracil, which is amplified as thymine tion. Exact gene copy number was quantified by real-
in a subsequent PCR. time PCR. For the copy number determination of the
 REAL-TIME PCR AND ARCHIVAL BIOPSIES 415

growth regulatory genes c-erb-B2 topoisomeraseII␣, c- coated with a 0.1% solution of poly (L-lysine) (Sigma
myc, and cyclinD1, we applied the relative quantifica- Chemicals, Deisenhofen, Germany). After sectioning,
tion approach described above. We have chosen two the slides are incubated for 15 min at 55⬚C. After they
chromosomal regions for which no numerical aberration are mounted, the sections are deparaffinized and
has been described for the copy number determination stained as described in Protocol 1 (Appendix). It is
of the growth regulatory genes c-erb-B2, topoisomer- necessary to dry the stained sections at least over-
aseII␣, c-myc, and cyclinD1. To increase the adherence night at 40⬚C. Otherwise residual liquid between the
of the sections to the slides, the membranes on which membrane and the slide will prevent the microdissec-
the sections are mounted following microdissection are 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
FIG. 4. Laser-microdissection and isolation of pure intraductal and absolute quantification with external calibration
pure invasive tumor cells using laser-assisted microdissection. (A) curves.
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 Keeping these caveats in mind, reliable quantita-
coverslipped for microdissection. (B) Section after removal of the tive data can be obtained as convincingly and indepen-
intraductal and the invasive component, respectively. (C) Isolated dently demonstrated by several groups. A vast area
intraductal (left) and invasive (right) tumor cells in the lid of a reac-
tion tube. (A–C) Methylene blue-stained; original magnification: of research has been opened up which will lead to a
⫻100. 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 Dexpat kit (Takara, Tokyo, Japan), organic extraction and precipitation (22)
mycobacteria in sarcoidosis
Quantitative detection of HHV-8 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
Quantitative detection of Genomic Prep Kit (Amersham Pharmacia), involves proteinase K digestion, depro- (24)
Toxoplasma gondii teination, and precipitation
Quantitative microsatellite analysis 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 417

the lid of a 0.5-ml reaction tube. Only the tubes offered weeks without degradation at ⫺20⬚C. For longer stor-
by P.A.L.M. can be used for this, because for this appli- age, we precipitate the RNA again by adding 0.1 vol
cation, the distance between the section and the inner sodium acetate, pH 5.2, and 2.5 vol absolute ethanol.
side of the lid is too great in the tubes made by other To prevent cross-contamination, all items that come
manufacturers. into contact with the samples including microtome
2. Apply 30–50 ␮l proteinase K digestion buffer (50 blade, forceps, and block holder are cleaned with xylol
mM Tris–HCl, pH 8.1, 1 mM EDTA, 0.5% Tween 20, or 2% hypochlorite solution before the next block is cut.
0.1 mg/ml proteinase K) to the lid and close the tubes
in this inverted position. Leave the tubes standing on
their lids. Protocol 4: Bisulfite Treatment of DNA Isolated from
3. Incubate samples overnight at 40⬚C in this in- Archival Specimens
verted position in a small hybridization oven. 1. Incubate the DNA in a total volume of 50 ␮l of 200
4. Centrifuge the tubes for 3 min at 14,000g, and mM NaOH at 42⬚C for 30 min for complete separation of
incubate the samples at 95⬚C for 10 min in a ther- both strands.
moblock with a heated lid to inactivate the protein- 2. Add 275 ␮l 3.6 M bisulfite containing 1 mM hydro-
ase K. quinone (freshly prepared) and incubate the samples
5. Centrifuge the samples briefly and store at ⫺20⬚C. 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-
Protocol 3: Extraction of RNA from Formalin-Fixed and gen, Hilden, Germany) and mix thoroughly.
Paraffin-Embedded Biopsies 4. After 10 min at room temperature, centrifuge the
1. Cut one to six 10-␮m sections from the paraffin samples at 14,000g for 3 min at 4⬚C and discard the su-
block and place them directly in a 1.5-ml screw-cap tube pernatant.
without deparaffinization or rehydration. 5. Wash the pellet three times with 70% ethanol.
2. Add 850 ␮l denaturation solution (4 M guanidi- 6. Resuspend the pellet in 20 mM NaOH/90% etha-
nium isothiocyanate/0.25 M sodium citrate/0.5% nol and incubate for 5 min at room temperature.
sarcosyl/0.1 M 2-mercaptoethanol) and 250 ␮l protein- 7. Wash the pellet twice with 90% ethanol.
ase K solution (20 mg/ml in H2O) and incubate the tubes 8. Elute the air-dried pellet with 25 ␮l TE buffer for
overnight at 55⬚C in a vigorously agitating ther- 15 min at 55⬚C.
moshaker at 1400 rpm. 9. Centrifuge again (14,000g, 3 min, 4⬚C) and trans-
3. The next day, centrifuge the samples at 4⬚C for fer the supernatant to a fresh tube; 3 to 6 ␮l of this
5 min at 14,000g. After centrifugation a white cap of solution is used for methylation-specific PCR.
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 ACKNOWLEDGMENTS
material and solidified paraffin.
5. Add to this solution 100 ␮l 3 M sodium acetate, We thank Oliver Bock, Martina Mühlenhoff, Nils von Neuhoff, and
Thomas Schmittgen for many stimulating discussions and critical
pH 5.2, 630 ␮l water-saturated phenol, and 270 ␮l chlo- remarks, which have improved this article.
roform. Mix thoroughly and leave on ice for 15 min.
6. After centrifugation at 4⬚C for 20 min at 14,000g,
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