Global gene expression profiling of formalin-fixed paraffin-embedded tumor samples: a comparison to snap-frozen material using oligonucleotide microarrays

Oligonucleotide microarrays are widely used to investigate gene expression in a large-scale approach. A major limitation is the dependency on frozen material to obtain high-quality ribonucleic acid because most clinical specimens are formalin-fixed and paraffin-embedded (FFPE). The ability to analyze these samples using microarrays would enlarge the investigable sample stocks manifold. We conducted a comparison of snap-frozen and FFPE tissues investigating two malignomas. Gene expression profiles were obtained from both materials of the tumors. Independently processed triplicates of snap-frozen and FFPE specimen, respectively, were two-round-amplified and hybridized on Affymetrix GeneChips (Palo Alto, CA, USA). Differentially expressed genes were identified in both FFPE and frozen material. All replicates had a correlation coefficient (R) of greater than 0.95 after normalization. Only direct comparison of FFPE to frozen replicates resulted in a mean R of 0.86, rendering a "mixed" investigation unfeasible. More than 50% (419 genes) of the more than fivefold differentially expressed genes (800 in FFPE, 685 in frozen material) were detected concomitantly regardless of the material used, which is similar to other comparisons of different gene expression analysis platforms. Thus, global gene expression analyses using solely FFPE material seem to be feasible with nearly comparable results to frozen tissue studies.     

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: An analysis of resected gastric carcinoma

Although several factors affecting the sensitivity of potymer-ase chain reaction (PCR) amplification from formalin-fixed tissues have been Investigated mostly by experiments, the feasibility of archival formalln-flxed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2–3 days and 14% (4/28) of the samples fixed for 4–6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified pS3 products from archival tissues using a non-isotopic method, temperature gradient gel elec-trophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding Just after 1 day or less fixation for subsequent use in PCR amplification.     

Comparison of different tissue sampling methods for protein extraction from formalin-fixed and paraffin-embedded tissue specimens

Aims

Protein extracts from formalin-fixed and paraffin-embedded (FFPE) tissue for proteomic analysis has recently gained attention. In this study, we explored the possibility to standardize tissue sampling from paraffin blocks and compared the protein extracts with those obtained from fresh frozen material.

Materials and methods

Fresh frozen and FFPE material was obtained from five patients with pancreatic ductal adenocarcinoma either by cutting sections with a microtome or by stamping a cylinder with tissue micro-array technology. All samples were weighed, forwarded to protein extraction and analyzed by polyacrylamide gel electrophoresis and Western blotting. Immunohistochemistry allocated proteins in tissue sections.

Results

Sampling of tissue was highly reproducible, as assessed by sample weight. While protein concentrations were significantly higher in fresh frozen material compared to FFPE material, equal amounts of protein were extracted from FFPE using either paraffin sections or core cylinders in SDS-PAGE, all three procedures showed comparable protein patterns. In Western blotting, annexin I had the same molecular weight independent of the sample source and sampling procedure.

Conclusions

The sampling of FFPE specimens for protein extraction and analysis can be standardized, uncovering equal amounts of tissue and protein. In addition, the proteins extracted from FFPE tissue seem to be the same compared with those extracted from fresh frozen tissue.     

Quantitative DNA methylation analysis of laser capture microdissected formalin-fixed and paraffin-embedded tissues

We developed an assay to quantify DNA methylation in breast cancer cells isolated by laser capture microdissection (LCM). The assay uses methylation sensitive restriction enzyme (MSRE) digestion and quantitative polymerase chain reaction (qPCR). To assess the validity and precision of the assay, we prepared standard samples with expected methylation percentage (MP) for two gene promoters (PLAU (plasminogen inhibitor, urokinase) and TIMP3 (TIMP metallopeptidase inhibitor 3)) that we compared with measured MPs. We found good linearity of MSRE digestion and qPCR procedures for both promoters (β = 0.90-1.19 ± 0.05-0.10 and r = 0.95-0.98; all P < 0.0001). Moreover, results remained similar after addition of a purification step between MSRE digestion and qPCR procedures. The validity of this technique was also confirmed by successfully replicating previously published MPs of four cell lines for PLAU and TIMP3 promoters. We assessed the consistency of our approach by comparing MPs of PLAU and TIMP3 promoters from nine breast cancer patients and two cell lines using LCM frozen tissues and their corresponding formalin-fixed paraffin-embedded tissues. We found good consistency (intraclass correlation coefficient = 0.93) of MPs between frozen tissues and formalin-fixed paraffin-embedded tissues. Our data demonstrate that this assay based on digestion with MSRE and qPCR procedures is a good technique to quantify MP on limited amounts of DNA and may find clinical applications.     

Setting out the frame conditions for feasible use of FFPE derived RNA

Introduction

The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.

Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy₀ quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy₀ method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~ 1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples.     

Evaluation of the new VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of human immunodeficiency virus type 1 RNA

BackgroundThe VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of HIV-1 RNA has recently been introduced.ObjectivesIn this study, the performance of the VERSANT HIV-1 RNA 1.0 Assay (kPCR) was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 2.0.Study designAccuracy, linearity, interassay and intra-assay variations were determined, and a total of 196 routine clinical samples including a high number of HIV-1 subtype non-B samples were investigated.ResultsWhen accuracy of the new kit was tested, all of the quantifiable results were found to be within ?0.5 log₁₀ unit of the expected panel results. Determination of linearity resulted in a quasilinear curve up to the initial concentration of 3.4 × 10⁵ copies/mL. The interassay variation ranged from 12 to 20%, and the intra-assay variation ranged from 8 to 16%. When clinical samples were tested by the VERSANT HIV-1 RNA 1.0 Assay (kPCR) and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, the results for 95% of all samples with positive results by both tests were found to be within ±1.0 log₁₀ unit. The viral loads for all samples measured by the Siemens and Roche assays showed a high correlation (R² = 0.94); quantitative results obtained by the Siemens assay were usually found to be lower than those obtained by the Roche assay.ConclusionsThe new VERSANT HIV-1 RNA 1.0 Assay (kPCR) proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.     

Allele-specific associated polymorphism analysis: Novel modification of SSCP for mutation detection in heterozygous alleles using the paradigm of resistance to thyroid hormone

Allele-specific polymorphism (ASAP) analysis is a modification of single-strand conformation polymorphism (SSCP) mutation screening under optimized temperature conditions in a minigel format withethidium bromide detection. ASAP analysis was used to screen for and identify mutations within thehuman thryoid hormone receptor-ß (hTR-ß) gene. These mutations are the underlying cause ofresistance to thyroid hormone (RTH). Eleven dissimilar known hTR-ß mutations and six previouslyuncharacterized mutations were accurately identified. ASAP screening extends to unique ASAP-DNAfingerprinting as an identifying signature for each novel hTR-ß mutation detected thus far. Gel-plugsfrom the SSCP gels containing polymorphic single-stranded DNA alleles were used without elution toprepare solid-phase sequencing templates for mutant allele PCR and sequencing (MAPS). The couplingof ASAP analysis with MAPS has eliminated many of the interpretative and technical problemsassociated with the sequencing of heterozygous alleles. Together, this convenient screening and sequencing methodology offers accuracy, reproducibility, speed, and the potential elimination of allradioactivity, providing a general strategy for future automated detection and characterization of genetic mutations. © 1995 Wiley-Liss, Inc.     

A PCR-based integrated protocol for the structural analysis of the 13th exon of the human beta-myosin heavy chain gene (MYH7): development of a diagnostic tool for HCM disease

Familial Hypertrophic Cardiomyopathy (FHC) constitutes a genetic disease of the sarcomere characterized by a Mendelian pattern of inheritance. A variety of different mutations affecting the at least eight sarcomeric gene products has been identified and the majority of them appear to function through a dominant negative mechanism. Family history analysis and genetic counseling have been widely adopted as integral tools for the evaluation and management of individuals with Hypertrophic Cardiomyopathy (HCM). Genetic testing of the disease has been progressively released into the clinical mainstream, thus rendering the development of novel and potent molecular diagnostic protocols an inevitable task. To this direction, we have evolved an integrated PCR-based molecular protocol, which through the utilization of novel “exonic” primers allows, among others, the structural analysis of the 13th exon of the human β-myosin heavy chain gene locus (MYH7) mainly characterized by the critical for HCM Arginine residue 403 (R⁴⁰³). Interestingly, through a DNA sequencing approach, a single nucleotide substitution from “G” to “T” was detected in the adjacent 13th intron, thus divulging the versatile potential of the present molecular protocol to clinical practice.     

Defining the anatomical localisation of subsets of the murine mononuclear phagocyte system using integrin alpha X (Itgax, CD11c) and colony stimulating factor 1 receptor (Csf1r, CD115) expression fails to discriminate dendritic cells from macrophages

The murine mononuclear phagocyte (MNP) system comprises a diverse population of cells, including monocytes, dendritic cells (DC) and macrophages. Derived from the myeloid haematopoietic lineage, this group of cells express a variety of well characterized surface markers. Expression of the integrin alpha X (Itgax, CD11c) is commonly used to identify classical DC, and similarly expression of colony stimulating factor 1 receptor (Csf1r, CD115) to identify macrophages. We have characterized the expression of these markers using a variety of transgenic mouse models. We confirmed previous observations of Itgax expression in anatomically defined subsets of MNPs in secondary lymphoid organs, including all MNPs identified within the germinal centres. The majority of MNPs in the intestinal lamina propria and lung express Itgax. All mucosal Itgax expressing cells also express Csf1r suggesting Csf1-dependent haematopoietic derivation. This double-positive population included germinal centre MNPs. These data reveal that Itgax expression alone does not specifically define classical DC. These results suggest more cautious interpretation of Itgax-dependent experimentation and direct equation with uniquely DC-mediated activities, particularly in the functioning of non-lymphoid MNPs within the intestinal lamina propria.