Background: Formalin fixed and paraffin wax embedded tissues of necropsy origin are an important source for molecular analysis especially in rare diseases, neuropathology, or molecular epidemiology studies. Because of DNA degradation, only short sequences can be amplified from this type of tissue, very often less than 100 bases. This poses problems because studies on polymorphism and mutations occurring in large genes often require the analysis of long sequences. Methods: The development of a simple treatment to obtain longer fragments of DNA for the analysis of archival postmortem paraffin wax embedded tissues. Results: It was possible to amplify longer sequences ranging up to 300 bases from postmortem tissues, with no modification to the usual DNA extraction procedures. To obtain longer stretches of DNA, a pre-PCR restoration treatment was required, by filling single strand breaks, followed by a vigorous denaturation step. Conclusions: The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp–250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy.

All tissues from biopsy or surgery origin are fixed and paraffin embedded as a routine procedure in the hospital departments of pathology. The traditional method of tissue preservation is the fixation in formalin, followed by paraffin embedding. In this way tissue's integrity is ensured also for future analyses, because there is no further chemical degradation of nucleic acids and proteins in tissues embedded in paraffin. After few sections for the histopathological examination the tissues are stored for decades in the hospital archives. Even if formalin fixation compromises the quality and integrity of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA and RNA from these archive's tissues, even of autopsy origin. Protein analysis is on the contrary completely blocked, due to the fact that formalin fixation creates covalent links between proteins and the only way to study protein expression is immunohistochemistry. In this study we present our results concerning the use of a new formalin free fixative, the FineFIX®. After extraction of nucleic acids, PCR and RT-PCR analyses were performed in DNA and RNA respectively. For DNA analysis it was possible to obtain amplicons of 2400 bps, while in formalin-fixed samples the maximum length achieved was less than 400 bps. RT-PCR analysis show that it was possible to study RNA fragments of 600 bps from FineFIX® fixed tissues, against a maximum length of about 150 bps achieved by formalin-fixed tissues. These tissues were analyzed also by Western Blot analysis, showing that the proteins obtained from FineFIX® treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues. Protein extracts from FineFix® treated tissues were also compared with fresh tissues'ones by two dimensional electrophoresis, demonstrating that the protein pattern were well comparable for number and distribution of the spots.

Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young’s modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines’ elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM.

Formalin-fixed tissues represent the most abundant clinical material for retrospective studies. However, formalin highly affects macromolecules, impairing their extraction and analysis. In this study, the suitability of some potential substitutes of formalin for RNA-based applications has been considered. Conventional formalin was compared with methacarn and the commercial FineFIX. Their impact on overall RNA preservation was investigated in a cell line-based model fixed during a time course treatment and in a series of fixed human tissues. RNA yield was detected by Nanodrop; ribosomal RNA (rRNA) integrity by electrophoresis and the Agilent Bioanalyzer; messenger RNA (mRNA) integrity by Northern blot and endpoint reverse transcription-polymerase chain reaction; and mRNA amount by real-time polymerase chain reaction. In the cell line model, formalin fixation showed time-dependent detrimental effects on overall RNA preservation. Methacarn and FineFIX were more conservative on both rRNA and mRNA preservation and their impact was time-independent. In tissues, high rRNA degradation levels were found in all fixed specimens, contrasting with the results found in the cells. Conversely, the effects of the fixatives on mRNA integrity reflected the observations shown in the cell line model. In methacarn-fixed samples mRNA amount was also preserved, whereas in formalin and FineFIX-fixed samples it was notably lower when compared with the fresh frozen control. Alcohol-based fixatives are a good solution for long-term fixation of both cytologic and tissue samples by virtue of their time-independent effects on mRNA preservation. In fixed tissue samples, however, the potential effects of preanalytical tissue-related factors should be considered when performing mRNA quantitative analysis.