A pressure cooking-based DNA extraction from archival formalin-fixed, paraffin-embedded tissue

As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin-fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high-quality DNA extraction from archival FFPE tissue specimens remains complex and time-consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit and OD 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with methylation-specific polymerase chain reaction can be used for epigenetic studies with the advantages of rapidity and high quality and may contribute to the development of biomarkers in clinical studies.     

DNA extraction from archival formalin-fixed,paraffin-embedded tissues: heat-induced retrieval in alkaline solution

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5–12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.     

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies

Formaldehyde fixation of biopsy specimens for routine purposes has often been held responsible for the poor reproducibility of immunohistochemical studies. Recently, antigen retrieval (AGR) using microwave irradiation was described as a potential tool to enhance immunostaining. A comparison of conventional staining and staining after microwave heating was performed for 52 markers, using tissues fixed in formaldehyde for 24 h, 1 to 6 weeks and 3 years respectively, as well as consultant case material. After adequate duration of fixation (24 h), only a few markers (17%) showed better results after AGR, but this percentage was increased to 50% when tissues were fixed for longer periods. Maximal enhancement was obtained in the group of consultant cases (58% of tested markers demonstrated better staining results), in which the period of fixation and tissue processing was unknown. To achieve reliable enhancement with AGR, continuous heating (100° C) should not be shorter than 20 min. In conclusion, AGR may become the most important tool to simplify and equalize immunohistochemical techniques, if critically evaluated.     

Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis.

From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. "The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue". This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of integral(Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as integral (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = integral (Pffpe)/ integral (Pf) x 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf x R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe= Pf. Further studies are designed to test the limitations of the proposed hypothesis.     

Mining the archival formalin-fixed paraffin-embedded tissue proteome: opportunities and challenges

The significant potential of tissue-based proteomic biomarker studies can be restricted by difficulties in accessing samples in optimal fresh-frozen form. While archival formalin-fixed tissue collections with attached clinical and outcome data represent a valuable alternate resource, the use of formalin as a fixative which induces protein cross-linking, has generally been assumed to render them unsuitable for proteomic studies. However, this view has been challenged recently with the publication of several papers accomplishing variable degrees of heat-induced reversal of cross-links. Although still in its infancy and requiring the quantitative optimisation of several critical parameters, formalin-fixed tissue proteomics holds promise as a powerful tool for biomarker-driven translational research. Here, we critically review the current status of research in the field, highlighting challenges which need to be addressed for robust quantitative application of protocols to ensure confident high impact inferences can be made.     

An efficient method for the extraction of DNA from formalin-fixed, paraffin-embedded tissue by sonication

A method was developed for fast and efficient isolation of DNA from formalin-fixed, paraffin-embedded tissue sections for subsequent use in PCRs and DNA hybridization assays. The method relies on the use of a sonicating water bath to disrupt tissue samples to which a small amount of micro-sized glass beads have been added. The sonicating glass beads provide fast and efficient physical shearing of fixed tissue sections, allowing for quick release and solubilization of the DNA. The extraction process from paraffin section to amplifiable target DNA takes 30 minutes. The method eliminates the need for repetitive solvent extractions and exhaustive proteinase K digestion. PCR amplification of human genomic and viral target sequences was successfully carried out on DNA isolated from a number of different types of normal and infected tissues.     

Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.     

Novel protein extraction approach using micro-sized chamber for evaluation of proteins eluted from formalin-fixed paraffin-embedded tissue sections

We describe a novel antigen-retrieval method using a micro-sized chamber for mass spectrometry (MS) analysis to identify proteins that are preferentially eluted from formalin-fixed paraffin-embedded (FFPE) samples. This approach revealed that heat-induced antigen retrieval (HIAR) from an FFPE sample fixed on a glass slide not only improves protein identification, but also facilitates preferential elution of protein subsets corresponding to the properties of antigen-retrieval buffers. Our approach may contribute to an understanding of the mechanism of HIAR.     

Protein-embedding technique: a potential approach to standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections.

A serial study was performed to develop a protein-embedding technique for standardization of immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue sections. A protein carrier matrix must have two phases, a liquid phase to allow uniform mixing of a protein and a solid phase forming a 'block' that can be fixed and processed in the same manner as human tissue. This standardized protein block would serve as a source of thin sections for control of IHC and therefore must also withstand the boiling conditions of antigen retrieval (AR). After multiple experiments, a method was developed utilizing polymer microsphere (beads) as a support medium for protein. The method showed particular promise for quantitative IHC.     

Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates

Background

Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.

Principal Findings

In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.

Conclusions

These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.     

Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues

A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA.     

Southern and dot blot analysis of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas

Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffin-embedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA. Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.     

Southern and dot blot analysis of DNA from formalin-fixed,paraffin-embedded tissue samples from colonic carcinomas

Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffinembedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA. Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.     

Extraction of cells from paraffin-embedded tissue sections for single-cell DNA cytophotometry

A simple technique is presented for the isolation of cells from paraffin-embedded tissues for Feulgen DNA cytophotometric investigations. Tissue fragments from paraffin blocks were deparaffinized in xylene, rehydrated and refixed in a formalin solution and incubated in a solution of 0.5 pepsin in 0.25% hydrochloric acid. After filtration through a 70 micron mesh and centrifugation, the cells were smeared upon a glass slide. Comparison between the results obtained with freshly prepared imprints and with pepsin-extracted cells of the same tumor showed that the extraction technique does not influence the Feulgen reaction or the DNA distribution pattern. Investigations carried out on bladder and embryonal carcinomas have demonstrated that the method permits an analysis of histologically or histochemically identified tumor cells within individual tissue areas.