ArticlePDF Available

DNA and RNA Isolated from tissues processed by microwave-accelerated apparatus MFX-800-3 are suitable for subsequent PCR and Q-RT-PCR amplification

Authors:
Csaba Bödör at HCEMM
Csaba Bödör
Otto Schmidt
Otto Schmidt
Otto Schmidt
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Balázs Csernus
Balázs Csernus
Balázs Csernus
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Hajnalka Rajnai at Semmelweis University
Hajnalka Rajnai
Show all 5 authorsHide
Download full-text PDF
Read full-text
Download citation

Abstract and Figures

Over the past decade, methods of molecular biology have appeared in diagnostic pathology and are routinely applied on formalin-fixed, paraffin-embedded histological samples, processed via conventional embedding methods. Due to its reagent- and cost-effectiveness, embedding techniques that utilize microwave acceleration in one or more steps of histoprocessing are increasingly used by numerous laboratories. The demand arises that tissues processed this way should also be suitable for the requirements of molecular pathology. In this study, both conventionally embedded and MFX-800-3 machine-processed tissue samples from the same source were used for isolation of DNA and RNA and for performing PCR and real-time PCR. PCR amplification of the beta-globin gene, as well as the real-time PCR amplification of the ABL mRNA was successful in all cases. Our conclusion is that samples processed by the vacuum assisted automatic microwave histoprocessor MFX-800-3 are perfectly applicable for DNA and RNA isolation and provide appropriate templates for further PCR and realtime PCR studies.
Figures - uploaded by Csaba Bödör
Author content
All figure content in this area was uploaded by Csaba Bödör
Content may be subject to copyright.
Content uploaded by Csaba Bödör
Author content
All content in this area was uploaded by Csaba Bödör
Content may be subject to copyright.
DNA and RNA Isolated from Tissues Processed by Microwave-
Accelerated Apparatus MFX-800-3 are Suitable for Subsequent
PCR and Q-RT-PCR Amplification
Csaba BÖDÖR,1Otto SCHMIDT,2Balázs CSERNUS,1Hajnalka RAJNAI,1Béla SZENDE1
11st Department of Pathology and Experimental Cancer Research, Faculty of Medicine, Semmelweis University;
2Meditest, Budapest, Hungary
METHODS
© 2007 Arányi Lajos Foundation
PATHOLOGY ONCOLOGY RESEARCH Vol 13, No 2, 2007
Article is available online at http://www.webio.hu/por/2007/13/2/0149
Introduction
The methods of molecular pathology became routinely
used in numerous histopathological laboratories over the
past decade.5Recent advances in molecular technologies
allow the utilization of formalin-fixed, paraffin-embedded
histological samples for DNA and/or RNA analysis. Most
of these molecular methods were optimized and validated
on samples processed via the conventional embedding
methods, which proved that conventional methods are in
fact compatible with molecular diagnostics.13
At the same time an increasing number of laboratories
apply embedding techniques that utilize microwave accel-
eration in one or more steps of histoprocessing.3,9 The
Received: Jan 10, 2007; accepted: May 20, 2007
Correspondence: Prof Béla SZENDE, 1st Department of Patholo-
gy and Experimental Cancer Research, Faculty of Medicine,
Semmelweis University, Üllôi út 26, H-1085 Budapest, Hungary,
Tel: +36-1-215-7300 ext: 4433, fax: +36-1-317-1074, e-mail:
bodor@korb1.sote.hu
Over the past decade, methods of molecular biology
have appeared in diagnostic pathology and are rou-
tinely applied on formalin-fixed, paraffin-embed-
ded histological samples, processed via convention-
al embedding methods. Due to its reagent- and
cost-effectiveness, embedding techniques that uti-
lize microwave acceleration in one or more steps of
histoprocessing are increasingly used by numerous
laboratories. The demand arises that tissues
processed this way should also be suitable for the
requirements of molecular pathology. In this study,
both conventionally embedded and MFX-800-3
machine-processed tissue samples from the same
source were used for isolation of DNA and RNA
and for performing PCR and real-time PCR. PCR
amplification of the ββ-globin gene, as well as the
real-time PCR amplification of the ABL mRNA was
successful in all cases. Our conclusion is that sam-
ples processed by the vacuum assisted automatic
microwave histoprocessor MFX-800-3 are perfectly
applicable for DNA and RNA isolation and provide
appropriate templates for further PCR and real-
time PCR studies.
(Pathology Oncology Research Vol
13, No 2, 149–152)
Key words: paraffin embedding, MFX-800-3 histoprocessor, real-time PCR, molecular pathology
demand arises that tissues processed this way should also
be suitable for the requirements of molecular pathology.
Surprisingly, only limited amount of information is
available in the relevant literature regarding microwave
histoprocessing and molecular pathology.6,8,12 Numerous
laboratories use the vacuum-assisted automatic micro-
wave histoprocessor MFX-800-3; therefore detailed
knowledge would be needed about the possible applica-
tion of tissues processed by this apparatus for methods of
molecular pathology.
In our study, both conventionally embedded and MFX-
800-3 machine-processed tissue samples from the same
source were used for isolation of DNA and RNA and for
performing PCR and real-time PCR.
Materials and Methods
Tissue samples
Four surgically removed tissue samples (two colon car-
cinomas, one gallbladder showing chronic inflammation
and one normal liver tissue) were each divided into two
parts. One part was fixed with 8% neutral formalin and
embedded into paraffin using a TCP 15 Tissue Processor
(Medite, Burgdorf, Germany). The other part was
processed by means of the MFX-800-3 apparatus, as
described earlier.10
DNA isolation and PCR amplification of the β-globin
gene
The paraffin embedded tissue sections were de-waxed
using xylene and absolute ethanol washes (3x5 min).
Genomic DNA isolation from tissue specimens was per-
formed according to the standard salting-out procedure.14
Each PCR reaction contained 100 ng of DNA. Reactions
were carried out using the AmpliTaq Gold™ enzyme system
(Applied Biosystems, Weiterstadt, Germany). Primer
sequences for amplification of the human β-globin gene
were as follows: forward (GH20): 5’-GAAGAGCCAAG-
GACAGGTAC-3’ and reverse (PCO4): 5’-CAACTTCATC-
CACGTTCACC-3’, generating a 267 base pair (bp) long
product.
RNA isolation and real-time PCR amplification
of the ABL gene
Total RNA was extracted using the High Pure RNA
Paraffin kit (Roche Diagnostics, Mannheim, Germany) as
recommended by the manufacturer. One µg of RNA was
reverse transcribed to cDNA using High Capacity cDNA
Archive Kit (Applied Biosystems, Foster City, CA,
USA). The quantitative real-time PCR assay was per-
formed with ABI Prism®7300 Sequence Detection Sys-
tem (Applied Biosystems). For amplification of mRNA of
the Abelson (ABL) gene, the TaqMan®-based technology
was used.
The TaqMan®technology is an absolutely specific and
highly reproducible system for detecting target genes,
since signal is only detected if the target sequence is com-
plementary to the probe used. In this case the primer-
probe set was designed to flank an exon-exon boundary,
which rules out the possibility of genomic DNA amplifi-
cation. The sequences of the probe and the primers were
as follows: TaqMan probe: 5’-FAM-CCATTTTTG-
GTTTGGGCTTCACACCATT-TAMRA-3’, forward primer:
5’-TGGAGATAACACTCTAAGCATAACTAAAGGT-3’
and reverse primer: 5’- GATGTAGTTGCTTGGGACC-
CA-3’.1The estimated product size was 123 bp. All sam-
ples were run in triplicate, in a 20 µl reaction volume con-
taining 100 ng of cDNA. The reaction plate also includ-
ed a non-template control sample, which contained all
reaction components except the cDNA template.
Sequence Detection Software version 1.0 (Applied
Biosystems) was used to analyze the data after ampli-
fication.
Results
DNA isolation and PCR amplification
of the β-globin gene
DNA isolation and the subsequent PCR amplification
were successful from both the conventionally prepared tis-
sue blocks as well as from blocks prepared via the
microwave-accelerated technique. In both cases we were
able to obtain 15-20 µg of genomic DNA with a purity of
1.6-1.7 OD260/OD280 from 5 tissue sections of 30 µm thick-
ness. The results of the β-globin gene amplification are
depicted in Fig. 1. The reaction was carried out success-
fully in all 4 tissue samples, only the gallbladder sample
processed via the conventional method showed a slightly
lighter band.
RNA isolation and real-time PCR amplification
of the ABL gene
Using five 30-µm-thick tissue sections we were able to
obtain 2-5 µg of total RNA (OD260/OD280 = 1.7-1.8) from
tissue blocks prepared with either embedding techniques.
The real-time PCR amplifications were successful in all
cases (Fig. 2). The average CTvalues obtained for each
sample are summarized in Table 1. The results of the
ABL mRNA amplification clearly reflect that RNA iso-
lated from the same tissue processed by two different
embedding methods provides quantitatively and qualita-
tively similar results. The reaction kinetics was also iden-
tical, therefore, the quantitative results are directly com-
parable.
Discussion
Histoprocessing by use of microwave-based techniques
reduces processing time compared to conventional meth-
ods, and results in approximately ten-fold decrease in the
150 BÖDÖR et al
PATHOLOGY ONCOLOGY RESEARCH
Figure 1. The agarose gel electrophoresis image shows the
results of β-globin gene amplification in different tissues using
conventional and microwave-accelerated histoprocessing. The
amplification was successful in all cases, only the gallbladder
sample processed via the conventional method showed a slight-
ly lighter band. (C: conventional embedding; M: microwave-
accelerated embedding)
Colon carcinoma 1 Colon carcinoma 2
Gall bladder Liver tissue
cost of chemicals, and a perfect preservation of tissue and
cellular structures. Our results show that isolation of
DNA and RNA as well as real-time PCR is possible using
tissue samples processed in a vacuum-assisted
microwave apparatus, and the quality of these reactions
are at least as high as that obtained by the conventional
histoprocessing. The templates after isolation of DNA
and RNA are sufficient for PCR and RT-PCR studies.
These results confirm the data of Hsu et al, who reported
high quality of DNA retrieved for Southern blot
hybridization from microwave-fixed, paraffin-embedded
liver tissues.8Man and Burgar described a novel antigen
unmasking protocol for immunohistochemistry and sub-
sequent PCR amplification, in part also utilizing
microwave oven irradiation.12 Lou et al used microwave
and thermal cycler boiling methods for preparation of cell
samples prior to PCR, for human papillomavirus detec-
tion.11 Ekuni et al found RNA integrity and successful RT-
PCR amplification in dento-alveolar tissues after
microwave-accelerated demineralization.6
Prior to both embedding techniques, formalin, which is
known for its nucleic acid fragmenting effects, was used as
the primary fixative reagent; therefore, we did not expect
any further improvement using the microwave-accelerated
histoprocessing compared to the conventional embedding
regarding DNA or RNA integrity. The literature describes
certain alternative fixatives that cause less nucleic acid
fragmentation;2,16 we are planning to integrate these
reagents into our microwave-assisted histoprocessing pro-
cedure in order to obtain more intact DNA and/or RNA
samples.
The utilization of formalin-fixed, paraffin-embedded tis-
sues for Q-PCR studies is controversial. However, some
studies have successfully used this technique for gene
expression analyses.4,7,15 Since Q-PCR plays a significant
role in modern pathological diagnostics, we aimed to
demonstrate that RNA isolated with microwave technique
is suitable for quantitative expression analysis.
Our results not only confirm previously published data,
but show that the entire fixation and embedding process
performed using a vacuum-assisted microwave apparatus
results in samples perfectly applicable for DNA and
RNA isolation in order to perform PCR and RT-PCR
studies.
References
1. Beillard E, Pallisgaard N, van der Velden VH, et al: Evalua-
tion of candidate control genes for diagnosis and residual dis-
ease detection in leukemic patients using ‘real-time’ quantita-
tive reverse-transcriptase polymerase chain reaction (RQ-
PCR) - a Europe against cancer program. Leukemia 17:2474-
2486, 2003
2. Benchekroun M, DeGraw J, Gao J, et al: Impact of fixative on
recovery of mRNA from paraffin-embedded tissue. Diagn Mol
Pathol 13:116-125, 2004
3. Boon ME, Wals-Paap CH, Visinoni FA, et al: The two-step vac-
uum-microwave method for histoprocessing. Eur J Morphol
33:349-358, 1995
4. Coindre JM, Hostein I, Terrier P, et al: Diagnosis of clear cell
sarcoma by real-time reverse transcriptase-polymerase chain
reaction analysis of paraffin embedded tissues: clinicopatho-
logic and molecular analysis of 44 patients from the French sar-
coma group. Cancer 107:1055-1064, 2006
5. Crocker J: Demystified... Molecular pathology in oncology.
Mol Pathol 55:337-347, 2002.
6. Ekuni D, Firth JD, Putnins EE, et al: RNA integrity and in situ
RT-PCR in dento-alveolar tissues after microwave accelerated
demineralisation. Arch Oral Biol 51:164-169, 2006
7. Gjerdrum LM, Sorensen BS, Kjeldsen E, et al: Real-time quan-
titative PCR of microdissected paraffin-embedded breast carci-
noma: an alternative method for HER-2/neu analysis. J Mol
Diagn 6:42-51, 2004
8. Hsu HC, Peng SY, Shun CT, et al: High quality of DNA
retrieved for Southern blot hybridization from microwave-
fixed, paraffin-embedded liver tissues. J Virol Methods
31:251-261, 1991
9. Kok LP, Visser PE, Boon ME, et al: Histoprocessing with the
microwave oven: an update. Histochem J 20:323-328, 1988
151PCR and Q-RT-PCR from MFX-800-3 Processed Samples
Vol 13, No 2, 2007
Table 1. The average CT values obtained for each sample
Sample Embedding Average CT
Colon carcinoma C 34.10
M 33.74
Colon carcinoma 2 C 33.40
M 33.63
Gall Bladder C 32.21
M 32.70
Liver tissue C 34.52
M 33.92
C: conventional embedding; M: microwave embedding
Figure 2. The amplification plots show successful amplifications
of the ABL mRNA from different tissues using conventional
and microwave-accelerated histoprocessing. Similar CTvalues
were obtained from samples processed by either embedding
methods (conventional vs. microwave-accelerated).
Delta Rn vs. Cycle
10. Kovacs L, Szende B, Elek G, et al: Working experience with a
new vacuum-accelerated microwave histoprocessor. J Pathol
180:106-110, 1996
11. Lou YK, Qin H, Molodysky E, et al: Simple microwave and
thermal cycler boiling methods for preparation of cervicovagi-
nal lavage cell samples prior to PCR for human papillomavirus
detection. J Virol Methods 44:77-81, 1993
12. Man YG, Burgar A: An antigen unmasking protocol that satis-
fies both immunohistochemistry and subsequent PCR amplifi-
cation. Pathol Res Pract 199:815-825, 2003
13. Mies C: Molecular biological analysis of paraffin-embedded
tissues. Hum Pathol 25:555-560, 1994
14. Miller SA, Dykes DD, Polesky HF, et al: A simple salting out
procedure for extracting DNA from human nucleated cells.
Nucleic Acids Res 16:1215, 1988
15. Ryan JL, Fan H, Glaser SL, et al: Epstein-Barr virus quantita-
tion by real-time PCR targeting multiple gene segments: a
novel approach to screen for the virus in paraffin-embedded tis-
sue and plasma. J Mol Diagn 6:378-385, 2004
16.
Vincek V, Nassiri M, Nadji M, et al: A tissue fixative that pro-
tects macromolecules (DNA, RNA, and protein) and histo-
morphology in clinical samples. Lab Invest 83:1427-1435,
2003
152 BÖDÖR et al
PATHOLOGY ONCOLOGY RESEARCH
... It was found that 50% of the microwave processed tissues contained pure DNA compared to only 40% of conventionally processed tissues showing uncontaminated DNA. These results are in accordance with findings of Bodor et al who were able to obtain the genomic DNA with a purity of 1.6-1.7 OD260/OD280 from both of the processing techniques evaluated in this study (23). Also, Hsu et al were able to retrieve excellent quality DNA from in situ hybridization when microwave fixed tissue were used, further supporting the outcome of this study (24). ...
... Banarjee SK et al and Aydin et al who had described a microwave based DNA extraction procedure from FFPE tissues arrived at a similar conclusion i.e. the derived DNA was of sufficient quantity (25,26). This parameter was also studied by Bodor et al who were successfully able to extract good quality and quantity of DNA after having the tissue processed by microwave apparatus (23). ...
... On the basis of the results of the GAPDH gene expression post PCR, it can be stated that DNA retrieved from microwave method can be as effectively amplified as those processed by the conventional overnight technique; hence, equally good quality from both the technique. Likewise, conclusions were drawn by Banarjee et al and Bodor et al who carried out positive amplification of ki-ras and β-globin gene in human tissues via PCR (23,26). These results were further in congruence with the findings of Aydin et al, who reported successful PCR amplification of the DNA from microwave processed tissues (25). ...
Effect of Conventional and Microwave Tissue Processing Technique on DNA Integrity: A Comparative Molecular Analysis
Article
Full-text available
  • Sep 2018
Background Methods of diagnostic molecular biology are routinely applied on formalin-fixed, paraffin-embedded tissues processed via conventional method. Recently, there has been a growing interest to use microwave technology in histopathology laboratories to overcome the deficiencies of the conventional processing method. Thefore, this study was aimed to compare and analyze the quality and quantity of DNA obtained from tissues processed by conventional and microwave tissue processing techniques and to further ascertain the applicability of the latter for PCR (polymerase chain reaction based research). Methods Thirty fresh tissues of oral squamous cell carcinoma (OSCC) were included, and each sample was cut into two equivalent halves. One tissue half was processed by conventional manual method whereas the other half was processed using a domestic microwave oven. DNA was obtained from all the tissues which were then subjected to Polymerase chain reaction (PCR) to evaluate GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene expression. Results The results revealed better DNA yield from microwave processed tissue while the quality of the DNA was alike from both the techniques. Conclusion On the basis of the results obtained, it can be concluded that DNA produced by microwave processed tissues was similar to that obtained by conventional processing technique in terms of quantity and quality. Thus, microwave processed tissue samples can be successfully used for further molecular studies and researches.
View
... Hence the urgent need to develop a suitable fixation procedure to overcome the problems inherent in the use of aldehydes. Bödör et al. [42] set out to demonstrate that RNA isolated with the help of MW energy is suitable for quantitative expression analysis. Starting from the reported description of some alternative fixatives causing reduced nucleic acid fragmentation [43,44], they planned to integrate these reagents into their MW-assisted histoprocessing procedure in order to obtain more intact DNA and/or RNA samples. ...
... Their results not only confirmed previously reported data, but also showed that the entire fixation and embedding process as performed in a vacuum-assisted MW apparatus provided samples amenable to DNA and RNA isolation for PCR and RT-PCR tests [42]. In addition, histoprocessing by use of MW-based sample preparation reduced processing times relative to conventional methods; also, it decreased chemical costs by a factor of ten and allowed perfect preservation of tissue and cellular structures. ...
View
... Our spectrophotometric analysis findings were appropriate for qualitative PCR-based screening techniques. Likewise, the studies were successfully able to extract good quality and quantity of DNA after having the tissue processed by microwave irradiation, making it detectable by PCR [14,32]. As a result of the qualitative screening analysis, we revealed that microwave irradiation did not have a noticeable effect on GMO screening or plant-specific elements. ...
Screening of P-35S, P-FMV, and T-NOS genetic elements in microwave-treated genetically modified cereal flours
Article
Full-text available
  • Apr 2023
  • MOL BIOL REP
Background Reliable and efficient methods for detecting genetically modified organisms (GMOs) in unprocessed and processed food will be essential for establishing an effective system for traceability all along the supply chain. It is important to understand the detection of GMOs following microwave treatment, which is a common processing method used in various food products such as flours. Therefore, this study aimed to detect the presence of Cauliflower mosaic virus (CaMV) 35S promoter (P-35S), Figwort mosaic virus (FMV) promoter (P-FMV), and T-NOS (nopaline synthase terminator) genetic elements in DNA samples from untreated and microwave-treated genetically modified (GM) cereal flour samples using the qualitative polymerase chain reaction (PCR) based screening method. Methods and results DNA was extracted from all samples, and the efficiency of the qualitative PCR screening technique was tested by the verification studies. We performed an inhibition study with plant-specific actin (ACT) gene to the effectiveness of confirming the DNA extraction method. Then, we made the confirming of the qualitative PCR system by method performance testing criteria. The high quality and quantity of the DNA extracts from untreated and microwave-treated flour samples indicated the applicability of qualitative PCR screening assays. The results showed that microwave radiation does not significantly impact the genetic element screening in flour materials. Conclusion Untreated and microwave-treated flour samples had amplifiable DNA for the simultaneous screening of three genetic elements. The qualitative screening tests conducted in this study produced dependable outcomes, thus, can be successfully used for monitoring in control laboratories.
View
... Compared to immersion fixation, fixation acceleration methods, including both microwave-accelerated and ultrasound-accelerated fixation have been reported to improve the yield of high-molecular-weight DNA and PCR success from FFPE tissue [64][65][66][67][68]. RNA analyses appear to be more acceleration method specific. ...
Molecular Pathology and Pre-Analytic Variables: Impact on Clinical Practice From a Breast Pathology Perspective
Article
Full-text available
  • Jun 2018
Purpose of Review Cancer therapy is increasingly becoming dependent on the ability to target specific molecular pathways that drive disease progression. Consequently, the identification of specific molecular pathways requires the use of companion diagnostic assays that are accurate and reproducible. This review will discuss the data and concerns surrounding pre-analytic variables in the evaluation of breast cancer tissue. Recent Findings Although ASCO/CAP guidelines offer recommendations for the collection and preservation of diagnostic clinical tissues, standard clinical practice has paid little attention to the suitability of these tissues for further molecular analysis. Current research suggests that alterations in the molecular integrity of tissue during the pre-analytic stage may result in inaccurate results and potentially sub-standard patient care. Summary Threshold recommendations for optimal molecular analyses associated with pre-analytic variables are limited. Future recommendations regarding pre-analytic variables and molecular diagnostics of breast cancer tissue will require additional research on the effects of pre-fixation, fixation, processing, and storage on nucleic acid integrity, comparing fixed material with fresh and/or frozen controls.
View
... Nevertheless, microwave-assisted reactions 53 in molecular biology largely remained unexplored. Microwave 54 energy has been used successfully for protein assay [6], enzyme 55 immobilization [7], isolation of nucleic acids [8,9], gene and oligo-56 nucleotide delivery [10], parallel DNA amplification [11], DNA sen-57 sing [12], immunohistochemistry [13,14], and enzyme-linked 58 immunosorbent assay [15]. Hydrolysis of adenosine triphosphate 59 by microwave irradiation has also been studied [16]. ...
View
Pre-analytics, Current Testing Technologies, and Limitations of Testing
Chapter
  • Jan 2020
Molecular evaluation of genetic abnormalities requires basic knowledge of genetics and molecular pathology. It also requires basic knowledge of pre-analytic variables that affect these tests and the limitations for each test used to detect these abnormalities. Many limitations of testing start before the tests are even performed. In this chapter, we will review pre-analytical variables affecting the molecular evaluation, the landscape of current testing technologies, and the limitations of testing of some of the most commonly used tests. The aim is to cover the most commonly used tests in clinical practice, and tests not commonly used or mainly used for research are not covered in this chapter.
View
Application of Microwave Irradiation to Bone Decalcification and Its Effect on DNA Quality
Chapter
  • Mar 2017
Microwave (MW) irradiation has been widely used in the field of histopathology to reduce the total time to prepare tissue sections for microscopy. The processes before tissue sectioning include fixation, dehydration, and embedding in paraffin, and MW irradiation accelerates these reactions. In histological studies of bone, a process to soften compact bone, “decalcification”, is necessary. Many studies have applied MW irradiation to this time-consuming process, and positive results of MW irradiation for bone decalcification are accumulating. In recent years, genetic analysis has become popular in this field through the techniques of in situ hybridization and reverse transcription-polymerase chain reaction as well as genotyping of DNA extracted from decalcified specimens. The effect of MW irradiation on the quality of nucleic acids is now a major consideration. This chapter presents the MW effects on bone decalcification in terms of bone histology, the progress of decalcification techniques based on MW, and trends concerning the applicability of specimens decalcified with MWs for genetic analysis.
View
Food Engineering Series
Chapter
  • Oct 2012
Microwave irradiation can be of assistance to accelerate some steps in genomics, proteomics, metabolomics, and their derived subdisciplines. The particular operations that can be expedited by using microwaves (MWs) differ among omics, and so do the specific MW devices used to this end, which include focused or multimode MWs, single continuous or high-throughput formats, and laboratory-adapted, commercial, or dedicated equipment. The specific operations most efficiently assisted by MW in genomic applications are cell fixation, DNA extraction, deparaffination, digestion, PCR hybridization, rolling circle amplification, and metal-enhanced fluorescence. Proteomics can benefit from the effects of MWs for operations such as enzyme quenching and proteolysis (whether enzymatic or chemical), identification and characterization of post-translational modifications or metal-catalyzed reaction sites on proteins and lipase selectivities, dissociation of protein complexes and protein quantitation with commercially available procedures such as ICATR and iTRAQR, or traditional procedures based on sensitive phenomena such as fluorescence or chemiluminescence. In any case, it is metabolomics that has profited most greatly from MW assistance, especially for purposes such as drying, digestion, solid–liquid extraction (or, more accurately, “leaching”), steam distillation, liquid–liquid extraction, and derivatization of a large variety of metabolites from widely different matrices.
View
A Review of Preanalytical Factors Affecting Molecular, Protein, and Morphological Analysis of Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue: How Well Do You Know Your FFPE Specimen?
Article
  • Nov 2014
Context: Formalin fixation and paraffin embedding is a timeless, cost-efficient, and widely adopted method of preserving human tissue biospecimens that has resulted in a substantial reservoir of formalin-fixed, paraffin-embedded blocks that represent both the pathology and preanalytical handling of the biospecimen. This reservoir of specimens is increasingly being used for DNA, RNA, and proteomic analyses. Objective: To evaluate the impact of preanalytical factors associated with the formalin fixation and paraffin embedding process on downstream morphological and molecular endpoints. Data sources: We surveyed the existing literature using the National Cancer Institute's Biospecimen Research Database for published reports investigating the potential influence of preanalytical factors associated with the formalin fixation and paraffin embedding process on DNA, RNA, protein, and morphological endpoints. Conclusions: Based on the literature evidence, the molecular, proteomic, and morphological endpoints can be altered in formalin-fixed, paraffin-embedded specimens by suboptimal processing conditions. While the direction and magnitude of effects associated with a given preanalytical factor were dependent on the analyte (DNA, RNA, protein, and morphology) and analytical platform, acceptable conditions are highlighted, and a summary of conditions that could preclude analysis is provided.
View
Fast track biopsy method: A rapid approach to preoperative diagnoses
Article
  • Apr 2010
It is well known that the use of a microwave oven greatly reduces the time for histoprocessing. Objective: Apply the microwave-based histoprocessing method "Fast Track Biopsy" (FTB), previously described for breast needle-core biopsies, to samples of other organs. From 01/09/2008 to 31/12/2008, 125 normal and neoplastic tissue samples (thickness from 0.2 and 1.2 cm) were collected, processed according to the FTB technique, stained with haematoxylin/eosin (H/E) and analysed with tissue-specific immunohistochemical (IHC) markers. The quality of both H/E and IHC stained sections was comparable to that obtained with standard methods. Sample thickness less than 0.5 cm gave better results. the FTB method is suitable for most tissue types, and is thus useful for preoperatory diagnoses.
View
View
View
A Simple salting out procedure for extracting DNA from Human Nucleated Cells
Article
  • Jan 1988
  • NUCLEIC ACIDS RES
We investigate the contribution of the Iberian bat fauna to the cryptic diversity in Europe using mitochondrial (cytb and ND1) and nuclear (RAG2) DNA sequences. For each of the 28 bat species known for Iberia, samples covering a wide geographic range within Spain were compared to samples from the rest of Europe. In this general screening, almost 20% of the Iberian species showed important mitochondrial discontinuities (K2P distance values > 5%) either within the Iberian or between Iberian and other European samples. Within Eptesicus serotinus and Myotis nattereri, levels of genetic divergence between lineages exceeded 16%, indicating that these taxa represent a complex of several biological species. Other well-differentiated lineages (K2P distances between 5–10%) appeared within Hypsugo savii, Pipistrellus kuhlii and Plecotus auritus, suggesting the existence of further cryptic diversity. Most unsuspected lineages seem restricted to Iberia, although two have crossed the Pyrenees to reach, at leas...
View
Epstein-Barr Virus Quantitation by Real-Time PCR Targeting Multiple Gene Segments
Article
  • Nov 2004
Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamH1W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.
View
High quality of DNA retrieved for Southern blot hybridization from microwave-fixed, paraffin-embedded liver tissues
Article
  • Feb 1991
  • J VIROL METHODS
To overcome the degradation problem encountered in DNA extracted from formalin-fixed, paraffin-embedded tissue blocks, several methods of tissue fixation were examined in order to improve the quality of the DNA recovered for use in nucleic acid analysis. The fixation methods included formalin fixation alone, alcohol fixation alone, and microwave fixation with tissues immersed in phosphate-buffered saline (PBS), alcohol, or formalin. Unfixed fresh frozen tissue served as the control. Using hepatitis B virus (HBV) DNA sequences and the type I human procollagen gene as markers and liver tissue as a target, microwave fixation, with formalin omitted, not only preserved the DNA very well, but also the labile viral antigen. Both high molecular weight-integrated and free-form HBV DNAs were well preserved, and suitable for polymerase chain reaction and Southern blot analysis. The restriction enzyme fragment pattern of DNA recovered from these paraffin blocks was identical to that of unfixed fresh frozen tissue. Microwave fixation also preserved the labile preS2 epitope of the hepatitis B surface antigen (HBsAg) considerably better than formalin. These results suggest that microwave fixation is superior to routine formalin fixation for the preservation of excellent quality of genomic and viral DNAs for nucleic acid hybridization analysis. Alcohol, often used for nucleic acid purification, was also a good fixative for preserving DNA and the antigenicity of the labile antigen, especially when carried out in combination with microwave fixation.
View
Histoprocessing with the microwave oven: An update
Article
  • Jun 1988
This paper evaluates and extends the novel method of preparing tissue blocks for paraffin sections within 30 to 60 min, that was proposed in early 1985 in a paper by Boon et al. (1986). More than 2 years' additional experience and testing various microwave ovens has led to new protocols reported in this paper. Results are given of testing (i) an especially designed microwave oven for histoprocessing, (ii) microwavable reagents, (iii) processing larger numbers of specimens simultaneously, (iv) handling different types and sizes of tissue. It is concluded that effective temperature control offers substantial advantages. In addition, the possibilities of performing routine diagnostic pathology omitting formalin altogether are sketched.
View
Miller SA, Dykes DD, Polesky HF. A simple salting-out procedure for extracting DNA from nucleated cells. Nucleic Acids Res 16: 1215
Article
  • Mar 1988
Full textFull text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (113K), or click on a page image below to browse page by page. 1215
View
Molecular biological analysis of paraffin-embedded tissues
Article
  • Jul 1994
  • Hum Pathol
Molecular biology techniques have been adapted to the analysis of paraffin-embedded tissues (PETs), expanding their clinical utility. In vitro amplification with the polymerase chain reaction (PCR) promises to be the most useful means of retrospective analysis because it can be performed successfully on nucleic acids that have been partially degraded during fixation, paraffin embedding, and the extraction process. Five clinical situations in which DNA analysis of PETs can be helpful are: (1) confirmatory molecular diagnosis of lymphoma in which fresh tissue has not been obtained at the time of surgery, (2) identification of infectious agents, (3) genetic characterization of a putative inherited disease in which the affected individual has died, (4) confirmation of donor cell malignancy in transplant recipients, and (5) specimen identification. The role of the pathologist in molecular diagnosis will grow because of the feasibility of using PETs, a venue unique to our profession.
View
Simple microwave and thermal cycler boiling methods for preparation of cervicovaginal lavage cell samples prior to PCR for human papillomavirus detection
Article
  • Oct 1993
  • J VIROL METHODS
Sample preparation is an important step in the detection of viral DNA by the polymerase chain reaction (PCR) technique. The method used should achieve release of cellular DNA with the minimum of manipulation steps so as to reduce the possibility of contamination. The present report demonstrates that either microwaving or 20 min of boiling in the heating block of a thermal cycler lead to satisfactory results in the detection by PCR of human papillomavirus in cervicovaginal epithelial cell specimens obtained by lavage. Since each of these methods uses only one step the possibility of contamination is greatly reduced compared to the widely used proteins K/nonionic detergent extraction procedure.
View
The two-step vacuum-microwave method for histoprocessing
Article
  • Dec 1995
When microwaving and vacuum is combined, decrease of boiling temperature can be exploited in the histoprocessing procedures allowing a completely novel approach for impregnating tissue with paraffin. We found that, if the pressure is sufficiently low in the paraffin step, no ethyl-alcohol step is necessary for the dehydration. In that case, the fixed tissue blocks can be directly placed into the isopropanol and, after that, in the hot paraffin, in which the final dehydration of the tissue takes place. The full histoprocessing procedure can thus be shortened into two steps only, with a processing time (for 72 biopsies) of a mere 40 min. Working completely without ethyl alcohol might be of interest for countries in which ethyl alcohol is difficult or expensive to obtain. We conclude that vacuum-microwave histoprocessing allows us to omit ethyl-alcohol dehydration and to replace xylene, resulting in a two-step 'green' (ecological) method.
View