Effects of Processing Delay, Formalin Fixation, and Immunohistochemistry on RNA Recovery From Formalin-fixed Paraffin-embedded Tissue Sections : Diagnostic Molecular Pathology

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00019606-200803000-00008ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2008 by Lippincott Williams & Wilkins.17March 2008 p 51-58Effects of Processing Delay, Formalin Fixation, and Immunohistochemistry on RNA Recovery From Formalin-fixed Paraffin-embedded Tissue SectionsOriginal Articlesvan Maldegem, Febe MSc*; de Wit, Mireille BSc*; Morsink, Folkert BSc†; Musler, Alex BSc*; Weegenaar, Jitske BSc*; van Noesel, Carel J. M. MD, PhD**Department of Pathology, Academic Medical Center, Amsterdam†Department of Pathology, University Medical Center, Utrecht, The NetherlandsNo grants or funding have been used to support this study.A supplementary figure is available at http://www.molecularpathology.com.Reprints: Prof Carel J. M. van Noesel, MD, PhD, Department of Pathology, AMC, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands (e-mail: [email protected]).AbstractContemporary pathology involves an emerging role for molecular diagnostics. Current tissue handling procedures [ie, formalin fixation and paraffin embedment (FFPE)] have their origin in the aim to obtain good tissue morphology and optimal results within immunohistochemistry. Unfortunately, FFPE is notorious for its poor RNA conservation capacities. In this study, we have examined the impact of the individual steps in tissue handling processes on the RNA extractability, quality, and usability for reverse-transcription polymerase chain reaction. It was found that a prolonged prefixation time (ie, the time between tissue dissection and fixation) has a measurable impact on RNA integrity when analyzed with the Agilent Bioanalyzer. Surprisingly, however, the deteriorated RNA quality hardly had any consequences for reverse-transcription polymerase chain reaction yields. Furthermore, we assessed the optimal fixation time for RNA preservation, and we found that an RNA heating step, preceding copy DNA synthesis, significantly increases the RNA template length. Finally, we provide a protocol for RNA isolation from immunohistochemically stained FFPE tissue sections. Thus, by applying alterations to tissue handling procedures, archival FFPE tissues become well suitable for RNA-based molecular diagnostics.ArticlePlusClick on the links below to access all the ArticlePlus for this article.Please note that ArticlePlus files may launch a viewer application outside of your web browser.http://links.lww.com/PDM/A4Advances in molecular biology have great impact on our understanding of human disease and have consequently modernized diagnostic and therapeutic medicine. In clinical pathology, the advent of molecular diagnostics is already well established and with it the use of nucleic acid-based assays has increased significantly. Archival formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the most widely available resource for such analyses. The FFPE procedure, historically aimed at tissue conservation and acquisition of optimal morphology, is not necessarily compatible with optimal preservation and extractability of nucleic acids. Genomic DNA can generally be isolated in sufficient quantities out of FFPE tissue. The size of the analyzable DNA fragments obtained, however, does not exceed ±300 to 500 base pairs, posing limitations to the molecular analyses that can be performed. This limited template size is often considered to be the result of strand fragmentation.Similar restraints are true for RNA. In addition, compared with DNA, RNA is much more susceptible to ex vivo tissue handling and to the various processing steps inherent to current pathologic practice. The intrinsic short half-life of RNA molecules, the presumed abundance of RNase enzymes, and the smaller size of RNA strands, makes them more sensitive to degradation and to elution during washing. Despite these limitations, the use of messenger RNA (mRNA) or copy DNA (cDNA), instead of genomic DNA, as molecular template has considerable advantages. mRNAs represent the genes actively transcribed and therefore offer direct information on the actual molecular make-up of cells or tissues. When expressed, the copy number of a gene transcript in a cell is at least in the order of thousands instead of the 2 DNA copies, implying that less sensitive polymerase chain reaction (PCR) protocols are demanded. For example, clonality determination and B-cell receptor sequencing in lymphoma is less complicated when cDNA is available. Detection of the tumor clone is easier because the immunoglobulin gene is often highly expressed and the noise caused by the presence of silenced nonfunctional rearrangements is reduced.1A qualitative advantage of cDNA-based analysis is that, owing to exclusion of the often sizeable noncoding intron regions in mRNA, consecutive exons can be amplified in 1 piece. Thus, entire coding regions of genes can be covered by a limited number of reverse-transcription PCRs (RT-PCRs). For mutation detection, it is less elaborate to sequence cDNA instead of DNA, as for the latter each exon must often be amplified individually, requiring many primer sets. For example, P53 consists of 11 exons, which can be covered by just 4 sets of primers when sequenced on cDNA.2 cDNA is particularly useful in the search for chromosomal translocations in which the breakpoint regions may span extensive genomic lengths, and which are therefore not suitable for amplification by DNA-PCR. Yet, an RT-PCR on the fusion gene transcripts may provide the means by which the resulting fusion product can be identified.3–6 In addition to somatic mutations or translocations, essential gene products can also become altered in their normal activity or regulation owing to alternative splicing events. An increasing number of reports have linked aberrant splicing with prognosis in cancer, recently reviewed elsewhere.7 Although the identification of underlying mutations in splice sites will still require DNA sequencing, swift screening for the expression of alternative splice variants can be carried out using RT-PCR.In the past years, a number of papers have appeared reporting on specific steps regarding the acquisition of mRNA out of FFPE tissues and its applications.8–20 Here, a systematic analysis was made of the influence of tissue handling and the successive processing steps of daily clinicopathologic practice on the extractability and quality of mRNA. In addition, we provide a protocol for successful RNA isolation from immunohistochemically stained FFPE tissue sections. These findings may aid in optimal RNA extraction from routine archival tissue material, to be used in RT-PCR–based molecular diagnostics.MATERIALS AND METHODSTissue HandlingFresh tissue specimens of 3 livers, obtained directly after hemihepatectomy and ovarium carcinoma tissue, were left unprepared in phosphate-buffered saline (PBS, pH 7.4) for various periods, up to 48 hours, at either 4°C or room temperature, after which the samples were snap-frozen in liquid nitrogen and stored at −80°C until RNA isolation (not more than 5 mo). The same protocol was applied to ovarium carcinoma tissue as a control.Fresh tissue specimens (1.0×1.5 cm) of normal stomach mucosa were fixed in 4% formaldehyde, phosphate-buffered at pH 7.0 (Klinipath, Duiven, The Netherlands) for 2 to 72 hours (standard protocol: 16 to 24 h) at room temperature before further processing in a Tissue-Tek VIP 5 Vacuum Infiltration Processor (Sakura, Zoeterwoude, The Netherlands) according to the following procedure: 5.5 hours, 50% alcohol; 4 hours, 70% alcohol; 6.5 hours, 96% alcohol; 7.5 hours, 100% alcohol; 2 times 1 hour, 100% alcohol; 3 times 1.5 hours, xylene; 3 times 1.5 hours, paraffin; and finally embedded in paraffin.This study was conducted in accordance with the ethical standards in our institutional medical ethical committee on human experimentation, and also in agreement with the Helsinki Declaration of 1975, as revised in 2000.Total RNA Isolation and mRNA IsolationTen to 20 frozen sections of 10-μm thickness were used to isolate total RNA using TRIzol reagent (Invitrogen, Breda, The Netherlands) according to the manufacturer's instructions, and dissolved in RNase-free water.Of the FFPE tissue, 10 to 20 sections (10 μm) were deparaffinized in xylol for 10 minutes. Xylol was replaced by 3 washes with 100% ethanol. After careful removal of the ethanol, the tissue was air-dried for 5 minutes. The pellet was dissolved and incubated with 500 μg/mL Proteinase K (Roche Diagnostics, Almere, The Netherlands) in lysis-buffer [10 mM of Tris-HCl/pH 8.0, 0.1 mM of ethylenediaminetetraacetic acid (EDTA)/pH 8.0, 2% sodium dodecyl sulfate/pH 7.3] at 56°C for 2 hours. In case of incomplete lysis, an extra aliquot of Proteinase K was added and the tissue was again incubated for 2 hours at 56°C. Subsequently, RNA was isolated with TRIzol, as described above, with the small alteration that precipitation was carried out in the presence of 1 μL of glycogen to visualize the pellet (20 mg/mL, Roche). Finally, the RNA was dissolved in TE (1 mM of Tris-HCl/0.5 mM of EDTA, pH 8.0).For mRNA isolation, 10 sections of 10 μm of the frozen material were used in the TRIzol RNA isolation procedure. Subsequently, mRNA was isolated using the Micro-FastTrack Kit (Invitrogen) according to the manufacturer's instructions.Quality Control of RNA Using the BioanalyzerThe RNA that was used for quality measurements was subjected to an additional purification step using RNeasy MinElute cleanup columns (QIAGEN, Venlo, The Netherlands). Total RNA and mRNA concentrations were measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). After adjustment of the concentration from 50 to 200 ng/μL using RNase-free water, 1 μL was loaded on an RNA 6000 Nano LabChip and run at a 2100 Bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands). The RNA integrity number (RIN) was calculated using the 2100 Expert software (demo version B.02.02SI238).21,22RT-PCRBefore complementary DNA synthesis, RNA from frozen and FFPE tissues was heated at 65°C for 10 minutes. Alterations in this heating step occurred at 65°C, 70°C, and 80°C for 10 minutes, 2 hours, and 6 hours.For complementary DNA synthesis, the RNA was incubated with 2.5 nmol random hexamer pd(N)6 primers (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) for 10 minutes at 65°C. After cooling on ice, the reaction mixture was added to a final volume of 25 μL. It contained 200 units of Moloney murine leukemia virus RT (Invitrogen), 8 mM of dithiothreitol (DTT, Invitrogen), 1 mM of each dNTP (Amersham), 1× first strand buffer (50 mM of Tris-HCl, pH 8.3; 75 mM of KCl; 3 mM of MgCl2, Invitrogen), and 15 units of RNase inhibitor (Applied Biosystems, Foster City, CA). The reaction was performed for 1 hour at 37°C. Subsequently, the enzyme was inactivated during 10 minutes at 95°C.The resulting cDNA was used in PCRs combined with primer sets specific for 3 different housekeeping genes; hypoxanthine phosphoribosyltransferase (HPRT1), porphobilinogen deaminase, and β-2-microglobin (β2-M). Lengths of the PCR products varied between 157 and 753 bp (Table 1). PCRs were performed in a volume of 25 μL containing 1×PCR-buffer [20 mM of Tris-HCl (pH 8.4), 50 mM of KCl, Invitrogen], 1.25 to 1.5 mM of MgCl2 (Invitrogen), 200 μm of dNTP's (Amersham), 1 unit Platinum Taq polymerase (Invitrogen), and 8 pmol of each primer. The PCR program consisted of a first denaturing step at 95°C for 4 minutes, followed by 40 cycles of successively 30 seconds denaturing at 92°C, 45 seconds annealing at 55°C, 60°C, or 65°C (Table 1) and 45 seconds extension at 72°C, and a final extension step of 5 minutes at 72°C.JOURNAL/dimp/04.03/00019606-200803000-00008/table1-8/v/2021-02-17T195944Z/r/image-tiff Primers Used for the Amplification of the Housekeeping GenesImmunohistochemistryTo test the effects of immunohistochemical staining procedures on RNA integrity, the staining protocol was stopped at each consecutive step, after which the section was scratched off the glass and subjected to RNA isolation and RT-PCR as described before.Tissue sections (4 μm) were mounted on glass slides either by using serum or in a warm water bath. Sections were deparaffinized in xylol and ethanol and blocked for endogenous peroxidase activity by immersion in 0.3% H2O2 in methanol for 20 minutes. Heat-induced epitope retrieval was performed in Tris/EDTA buffer (10 mM/1 mM; pH 9.0) for 10 minutes at 120°C, whereas pepsin pretreatment was performed at a concentration of 0.25% in 0.01 M of HCl/pH 2.0 for 15 minutes at 37°C.Nonspecific binding sites were blocked with 5% normal goat serum or bovine serum albumin (BSA) in PBS, with BSA-c (Aurion, Wageningen, The Netherlands) or with serum-free protein block (DAKO) for 10 minutes. A mouse monoclonal antibody against vimentin (clone V9, DAKO Netherlands B.V., Heverlee, Belgium) was diluted in PBS or in normal antibody diluent (Scytek Laboratories Inc, Logan, UT). Then, the Powervision+poly-HRP detection system (ImmunoVision Technologies, Co, Daly City, CA), or alternatively, Rabbit and Swine-antimouse polyclonal antibodies-biotin labeled (DAKO) in combination with streptABComplex (DAKO) were applied. 3,3-diaminobenzidine, 3-amino-9-ethylcarbazole (AEC), Fuchsine acid, Fast-Red, and Fast-Blue (all obtained from Sigma-Aldrich, Zwijndrecht, The Netherlands) were used as chromogen and hematoxylin was as the counterstain.RESULTSEffects of Delayed Tissue Handling on RNA QualityTo analyze the effects of prefixation time on RNA quality, we chose to use normal liver tissue, assuming that this tissue is relatively sensitive to postponed processing because of the high metabolic activity of the tissue on the one hand and the abundance of proteolytic proteins on the other hand. In concordance with the results published by Godfrey et al,11 no consistent differences in the quantity of isolated total RNA was observed between the samples (data not shown). Figure 1A shows the progressive, time-dependent degradation of total RNA isolated from 1 of the 3 livers, visible as the gradual loss of the 28S ribosomal RNA band and accumulation of degraded RNA below the 18S band. When RNA qualities were depicted in RINs (Fig. 1B), the curves showed that, although the starting qualities (t=0) of RNA from the 3 livers differed significantly, shown in the electropherograms (insets), the course of RNA decay under influence of the different storage conditions was similar among the 3 livers. When the results of the 3 livers were averaged, it became apparent that there was a protective effect on RNA quality when tissues had been kept at 4°C as compared with room temperature; however, this difference was not significant before 12 hours of storage (Fig. 1C). The tissues left at room temperature continued degrading their RNA progressively, whereas decay of the samples kept at 4°C seemed to level off to a near steady-state of RNA quality. Similar experiments on tumorous tissue (ovarium carcinoma) as a control indicated that the velocity of RNA degradation is also determined by the tissue type; the overall RNA quality from the control tissue was much better than the RNA isolated from the livers. More strikingly, there was no significant RNA degradation detectable up to 18 hours, not even when the tissue was stored at room temperature (Fig. 1D). Of importance, the effect of delayed tissue handling on the quality of RNA was also clearly measurable when purified mRNA was analyzed. The mean size of the mRNA fragments decreased with longer prefixation times, visible in the electropherogram as a shift to the left (Fig. 1E).JOURNAL/dimp/04.03/00019606-200803000-00008/figure1-8/v/2021-02-17T195944Z/r/image-jpeg Effects of RNA recovery after delayed tissue processing. RNA was isolated from normal liver and an ovarium carcinoma, which had been left unprepared for 0 to 48 hours, at either 4°C or room temperature, and subsequently snap-frozen. A, A gel-like image of total RNA isolated from liver 1. B, RIN for 3 liver specimens in time is plotted, each curve representing 3 independent RNA isolations from 1 liver specimen. Insets: electropherograms depicting the RNA qualities at the start of the experiments. C, The relative RIN (%RIN), with t=0 set to a 100%, averaged for the 3 livers. D, The RIN of RNAs isolated from ovarium carcinoma in time. E, Electropherogram of purified mRNA, isolated from fresh frozen tissue and from tissue that had been left unprepared for 24 hours at room temperature.Effects of Delayed Tissue Handling on RT-PCRTo study the effect of delayed tissue handling on RNA template length, we performed RT-PCRs for various housekeeping genes on the RNA isolated from the frozen specimens of liver 1, for each time point mentioned above. To our surprise, the maximum PCR product with length of 750 bp could be amplified from all samples, irrespective of storage conditions (Fig. 2). The single exception within these results was the tissue specimen that had been left unprepared at room temperature for 48 hours. Here, the amplifiable template was not above 331 bps.JOURNAL/dimp/04.03/00019606-200803000-00008/figure2-8/v/2021-02-17T195944Z/r/image-jpeg Consequences of delayed tissue handling, at either room temperature or at 4°C, for RNA template length. RT-PCR was performed on commonly used housekeeping genes: HPRT (750, 500, and 225 bp), porphobilinogen deaminase (350 bp), and β2M (150 bp). For location of the primers, see Table 1.Influence of Fixation Time and RNA Preincubation Temperature on cDNA QualityIn sheer contrast to the frozen tissue, a tissue sample that was FFPE did not show the distinctive 28S and 18S ribosomal RNA bands, not even after immediate processing. In addition, the electropherogram showed that the mean length of RNA isolated from FFPE tissue was severely reduced (Fig. 3A). RT-PCR, aimed at housekeeping genes, indicated that an 8 to 16-hour fixation was optimal, in that PCR products of maximal lengths could be acquired (Fig. 3B). After 2 hours of (most likely incomplete) fixation, merely smaller mRNAs were amplifiable. Similar results were obtained for tissue that had been fixed for 72 hours.JOURNAL/dimp/04.03/00019606-200803000-00008/figure3-8/v/2021-02-17T195944Z/r/image-jpeg Effect of FFPE on RNA integrity and determination of an optimal fixation time. A, Electropherogram of RNA isolated from fresh frozen tissue (upper panel) or directly FFPE tissue (lower panel). B, Tissue samples were fixed for 2 to 72 hours, and subsequently paraffin-embedded. RT-PCR for housekeeping genes was performed on RNA isolated from these samples. C, RT-PCR for housekeeping genes, on RNA which was isolated from FFPE tissues, fixed for 2, 8, or 72 hours, and subjected to varying heating steps before the cDNA synthesis.In another series of experiments, the influence of RNA heating preceding the RT-PCR on the length of the amplifiable products was evaluated. FFPE-isolated RNA was subjected to several temperatures for different periods of time before cDNA synthesis. The analyses demonstrated that heating of RNA for 2 hours at 70°C yielded significantly better results than the commonly used 10-minute incubation at 65°C (Fig. 3C, lower panel). Incubation at temperatures above 70°C or for longer periods adversely affected RNA quality (data not shown).These experiments were repeatedly carried out for different tissues. Although the absolute length that could be amplified varied among the samples, the optimum fixation period of 8 to 16 hours was consistently observed within each experiment. Similarly, preincubation of isolated RNA for 2 hours at 70°C before cDNA synthesis was optimal in every experiment.Influence of Individual Steps of Immunohistochemical Staining Procedure on RNA RetrievalWe had experienced that RNA recovery from paraffin sections, stained according to our standard immunohistochemical procedure, was impossible. Therefore, all steps of the staining procedure were tested for their effects on RNA extractability. Figure 4A illustrates the absence of any PCR product right after the blocking step. Further examinations showed that addition of serum, either used for mounting on slides, as a blocking reagent, or as a supplement to the antibody diluents, is a major negative factor. Analysis of RT-PCR, after application of several commonly used staining reagents, showed that the visualization method can also be of great influence, with AEC giving the best results. Thus, RNA is well extractable from immunohistochemically stained tissue sections, provided that (i) mounting of slides is performed using a water bath; (ii) a blocking step is omitted or, when inevitable, performed with BSA-c instead of serum; (iii) antibody dilutions are made in PBS instead of commercially available diluents; and (iv) the eventual staining is carried out using AEC. Figure 4B lists all materials and treatments tested, and their influence on RNA recovery.JOURNAL/dimp/04.03/00019606-200803000-00008/figure4-8/v/2021-02-17T195944Z/r/image-jpeg Optimization of immunohistochemical staining procedure to allow RNA recovery. A, Each step of the immunohistochemical staining protocol (for vimentin) was tested for its influence on the RNA recovery from the tissue section. B, Upper panel, from left to right, the RT-PCRs that were obtained with either the original protocol, without immunohistochemistry, and after the altered staining procedure, referring to the schematic representation of the influence of immunohistochemistry on the RNA quality in the lower panel: the treatments that impede successful RNA extraction are depicted on the left side, whereas the (alternative) steps on the right side include a protocol that allows for proper RNA isolation. ABC includes streptavidin-biotin complex; DAB, diaminobenzidine tetrahydrochloride; HIER, heat-induced epitope retrieval; NGS, normal goat serum; RamBIO, Rabbit-antimouse polyclonal antibody-biotin labeled; SarBIO, Swine-antirabbit polyclonal antibody-biotin labeled.DISCUSSIONFFPE results in chemical cross-linking of proteins and nucleic acids, thereby preventing RNA degradation mediated by endonucleases present in the tissue. Still, retrieval of good quality RNA from FFPE tissues is often difficult to achieve. Despite the rising efforts to set up frozen tissue archives in many clinical centers, it is common practice that FFPE tissue is the only material available for (molecular) diagnostics. We evaluated the influence of the individual steps of tissue handling procedures on RNA extractability and integrity and searched for improvements in the RNA isolation protocol. When discussing the topic of RNA isolation, 2 dogmas generally emerge. First, the tissue material should be fixed or frozen as soon as possible, or it should be kept cold to prevent RNA degradation by RNases. Second, RNA isolated from FFPE material is largely fragmented and is therefore not very useful for molecular assays that require certain template lengths.To study the effects of delayed tissue handling, we chose liver material assuming that such active tissue, full of enzymes, is one of the most difficult tissues in which to preserve the RNA. This assumption appeared more true than expected, as 2 out of 3 livers used in the delayed tissue handling experiment did not yield good quality RNA, not even from the samples that had been frozen directly after surgical dissection. This result contrasted with the constant quality of the RNA in the ovarium carcinoma samples. RNA decay in these livers presumably started before they were included in our experiment. Liver tissue is metabolically active, which renders the tissue sensitive to hypoxia, inducing tissue damage and the subsequent release of various degradation enzymes. Extensive ischemia is common during a (hemihepatectomy) hepatectomy, with surgery taking 3 to 5 hours. Consequently, during surgery, the tissue is exposed to hypoxia and subsequently proteases and nucleases act for several hours at body temperature, which is optimal for their activity. Indeed, further study of the surgical reports elucidated that liver 1, qualitatively the best of 3, had undergone the shortest surgical operation (still 3.5 h) as compared with the other 2 livers, which were dissected during operations lasting at least 5.5 hours. This finding indicates that warm-ischemia can have dramatic consequences for RNA integrity in the tissue. Nevertheless, with RINs of 8.6, 5.5, and 3.2, representing good, intermediate, and poor RNA quality at time point zero, respectively, we were able to measure RNA degradation in time, independent of the degradation status at start. Remarkably all 3 samples showed a similar speed of decay in time. As expected, it was found that prolonged storage of the material before freezing gradually reduced the RNA integrity when this was measured with the Bioanalyzer. Cold storage protected the RNA in the tissue from degradation to some degree. However, this protective effect was not found to be significant before the prefixation time exceeded 8 to 12 hours; a delay of tissue handling that normally does not occur in daily practice.To relate the Bioanalyzer results to the functional consequences of the RNA decay, we chose to use conventional RT-PCR as measurement since most of our current molecular diagnostic assays depend on the presence or absence of a PCR product for readout. We found that the RNA which was isolated from all time points of liver 1 could still serve as template for a successful RT-PCR. Supporting our findings, similar results were obtained in a study by Godfrey et al11 that measured the influence of prefixation times on relative gene expression with quantitative RT-PCR. Altogether, the benefit of keeping tissues at 4°C between dissection and further handling seems to be limited.Concerning the second dogma, our results unequivocally showed that RNA extracted from FFPE tissues indeed was of inferior quality compared with frozen tissue samples. However, FFPE material is often the only source of RNA for molecular diagnostics. In our attempt to improve RNA recovery from FFPE material, we determined the optimal fixation time. We suspected that insufficient fixation would render the tissue insufficiently protected against the continuing enzymatic degradation, whereas prolonged fixation could result in irreversible fragmentation. Indeed, an optimum was found for fixation time; the best PCR results were obtained when the samples had endured formalin fixation between 8 and 16 hours. As the optimal fixation time depends on the tissue type and specimen size, a standardized fixation protocol should involve a formalin incubation of at least 4 hours, up to a maximum of 24 hours for regular size fragments. For core or needle biopsies, the optimal fixation time is probably shorter. These findings may have implications for current tissue handling procedures; for example, in case of dissections carried out on Fridays, it is not desirable to leave the tissue in formalin throughout the weekend.The finding that RNA yield can be improved by introducing a heating step of 2 hours at 70 degrees before cDNA synthesis is noteworthy. This finding is in agreement with the study of Masuda et al,23 who showed that formalin treatment of RNA oligos causes monomethylol addition to the amino bases. In case of extensive fixation, also methylene bridging between 2 amino groups occurs. Most of the methylol additions can be removed by heat incubation. Hence, the improvement of the RT-PCR after a heating step contradicts the supposed fragmentation of RNA due to formalin fixation. On the other hand, the methylene bridging is more difficult to break and can therefore cause irreversible restriction of the RNA template length, which may account for the poorer quality of RNA isolated from FFPE as compared with frozen tissue.In some cases, it may be desirable not to use entire tissue sections for RNA isolation, but instead to focus on a certain population of cells that can be identified immunohistochemically. For example, when there are low numbers of tumor cells in the sample, enrichment by laser microdissection may improve analysis. Of the various studies conducted on RNA isolation after immunohistochemical staining, however, most were performed on frozen tissue sections and mainly focussed on quantitative RT-PCR for which only short RNA templates are required.14,24–28 In our experience, RNA isolation is not possible after standard immunohistochemistry on FFPE tissue sections. Analysis of the impact of individual steps of the immunohistochemistry protocol on RNA retrieval revealed that the blocking step and the use of commercially available diluents and amplification kits form the main culprits. Serum turned out to be the constituent that is responsible for this interference, because replacing the serum with BSA-c or omitting it by using PBS-diluted antibodies greatly improved RNA recovery. Furthermore, best results were obtained when AEC was used as a substrate/chromogen. Using this modified protocol, mRNAs of at least 225 bp can be recovered from immunohistochemically stained FFPE tissue sections.Obviously, fresh frozen tissue is preferable over FFPE material for its use in RNA-based assays.29 Consequently, increasing numbers of Biobanks of nonfixed frozen tissue are being established. Furthermore, alternative alcohol-based fixatives are under development, which will enable improved RNA preservation and retrieval. Still, the main source of material for molecular diagnostics in daily practice includes FFPE material. In our study, we have shown that, with small alterations to current tissue handling and isolation procedures, RNA can be successfully recovered from archival FFPE tissue that is with an acceptable quality for standard RT-PCR–based molecular diagnostic assays.REFERENCES1. Aarts WM, Bende RJ, Steenbergen EJ, et al. 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Link]1469531600019606-200803000-0000800125140_2003_6_43_fritsch_characteristics_|00019606-200803000-00008#xpointer(id(citation_FROM_JRF_ID_d2938e789_citationRF_FLOATING))|11065404||ovftdb|SL00125140200364311065404citation_FROM_JRF_ID_d2938e789_citationRF_FLOATING[Full Text]00125140-200306010-0000600019606-200803000-0000800125140_2003_6_43_fritsch_characteristics_|00019606-200803000-00008#xpointer(id(citation_FROM_JRF_ID_d2938e789_citationRF_FLOATING))|11065213||ovftdb|SL00125140200364311065213citation_FROM_JRF_ID_d2938e789_citationRF_FLOATING[CrossRef]10.1007%2Fs10024-002-0013-100019606-200803000-0000800125140_2003_6_43_fritsch_characteristics_|00019606-200803000-00008#xpointer(id(citation_FROM_JRF_ID_d2938e789_citationRF_FLOATING))|11065405||ovftdb|SL00125140200364311065405citation_FROM_JRF_ID_d2938e789_citationRF_FLOATING[Medline Link]12375129 Primers Used for the Amplification of the Housekeeping Genes Effects of RNA recovery after delayed tissue processing. RNA was isolated from normal liver and an ovarium carcinoma, which had been left unprepared for 0 to 48 hours, at either 4°C or room temperature, and subsequently snap-frozen. A, A gel-like image of total RNA isolated from liver 1. B, RIN for 3 liver specimens in time is plotted, each curve representing 3 independent RNA isolations from 1 liver specimen. Insets: electropherograms depicting the RNA qualities at the start of the experiments. C, The relative RIN (%RIN), with t=0 set to a 100%, averaged for the 3 livers. D, The RIN of RNAs isolated from ovarium carcinoma in time. E, Electropherogram of purified mRNA, isolated from fresh frozen tissue and from tissue that had been left unprepared for 24 hours at room temperature. Consequences of delayed tissue handling, at either room temperature or at 4°C, for RNA template length. RT-PCR was performed on commonly used housekeeping genes: HPRT (750, 500, and 225 bp), porphobilinogen deaminase (350 bp), and β2M (150 bp). For location of the primers, see Table 1. Effect of FFPE on RNA integrity and determination of an optimal fixation time. A, Electropherogram of RNA isolated from fresh frozen tissue (upper panel) or directly FFPE tissue (lower panel). B, Tissue samples were fixed for 2 to 72 hours, and subsequently paraffin-embedded. RT-PCR for housekeeping genes was performed on RNA isolated from these samples. C, RT-PCR for housekeeping genes, on RNA which was isolated from FFPE tissues, fixed for 2, 8, or 72 hours, and subjected to varying heating steps before the cDNA synthesis. Optimization of immunohistochemical staining procedure to allow RNA recovery. A, Each step of the immunohistochemical staining protocol (for vimentin) was tested for its influence on the RNA recovery from the tissue section. B, Upper panel, from left to right, the RT-PCRs that were obtained with either the original protocol, without immunohistochemistry, and after the altered staining procedure, referring to the schematic representation of the influence of immunohistochemistry on the RNA quality in the lower panel: the treatments that impede successful RNA extraction are depicted on the left side, whereas the (alternative) steps on the right side include a protocol that allows for proper RNA isolation. ABC includes streptavidin-biotin complex; DAB, diaminobenzidine tetrahydrochloride; HIER, heat-induced epitope retrieval; NGS, normal goat serum; RamBIO, Rabbit-antimouse polyclonal antibody-biotin labeled; SarBIO, Swine-antirabbit polyclonal antibody-biotin labeled.Electropherograms of tissue samples from liver 1, stored in varying conditions before freezing: tissues were kept for 2, 4, 8, 12, 24 or 48 hrs, at either room temperature (RT) or 4°C, and subsequently snap-frozen in liquid nitrogen and stored at-80°C. After RNA isolation, samples were analyzed with the bioanalyzer.Effects of Processing Delay, Formalin Fixation, and Immunohistochemistry on RNA Recovery From Formalin-fixed Paraffin-embedded Tissue Sectionsvan Maldegem Febe MSc; de Wit, Mireille BSc; Morsink, Folkert BSc; Musler, Alex BSc; Weegenaar, Jitske BSc; van Noesel, Carel J. M. MD, PhDOriginal ArticlesOriginal Articles117p 51-58

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