Introduction

Most tissues from biopsy, surgery, or autopsy origin are usually fixed and paraffin embedded as a routine procedure in hospital departments of pathology. Tissue storage is a fundamental step in the usual clinical procedure for histopathological investigations. The traditional method of tissue preservation is the fixation of tissues, mostly in formalin, followed by paraffin wax embedding. In this way, biochemical, molecular, and structural integrity is ensured also for future retrospective analyses, because there is no further chemical degradation in paraffin. Usually, these tissues have been stored in the archives of pathology departments for decades. Among the archive tissues, postmortem tissues obtained from autopsy represent an important resource for the study of rare diseases, neuropathology, or molecular epidemiology. For these studies in fact, the analysis of both pathological and normal tissues as control is fundamental. The protocols of tissue storage normally suggest the fixation step in buffered formaldehyde solution in the dark for about 24 hr before paraffin wax embedding. This procedure is habitually maintained for biopsy and surgical samples, but autopsy tissues are usually fixed for longer periods of time. Various factors, such as postmortem interval or the type of fixative and the fixation time, could affect the quality and utilization of nucleic acids from archive tissues. Extensive degradation of nucleic acids is often found in archival tissues older than 20 years because nonbuffered formaldehyde solution was frequently used in the past. The most widely used fixative is neutral formalin, but for special purposes other fixatives may be used in histopathology laboratories. Of these, Bouin's solution and acetone can compromise the PCR amplification efficiency.1-1-1 Longer fixation times could also induce unsuccessful PCR amplification due to enhanced degradation of nucleic acids. Of the parameters involved in the degradation of nucleic acids, the postmortem interval is the most important factor affecting the successful extraction of high-molecular weight DNA from frozen autopsy tissues.[2] The delay between death and tissue collection is referred to as the postmortem interval (PMI). Commonly, a cadaver is refrigerated at 2-4°C within a few hours of death; an autopsy may not be performed for legal reasons before 4 to 36 hr or more have passed, depending on different rules and conditions in use.[2]

Generally, the amount of degraded DNA in frozen autopsy tissues is correlated directly with the duration of the postmortem period.[3] Otherwise, in the same type of tissue PMI does not appear to affect significantly the recovery of total RNA or mRNA.[2] Even if formalin fixation compromises the quality and intactness of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA[4] and RNA[5,6] from formalin-fixed and paraffin-embedded postmortem tissues. The use of PCR-based techniques does not require intact nucleic acids for amplification, therefore even an increased degradation may not affect the outcome of the analysis. In our experience, in postmortem tissues, fixed in nonbuffered formalin, DNA fragments longer than 90 bp cannot be amplified. As a consequence of DNA degradation, only very short sequences could be analyzed in postmortem tissues. However, for longer fragment analysis it is possible to perform a partial restoration and reconstruction of DNA length.[4,7] With this procedure it is possible to fill in the DNA breaks and amplify around 300 bp of DNA sequence from autopsy tissues.

As already reported[5,8] it is also possible to extract and analyze RNA from formalin-fixed and paraffin-embedded tissues from autopsy tissues.[5,6] Usually, in routinely treated formalin-fixed material the available fragments of RNA range between 100 and 200 bp. Analysis of RNA obtained from postmortem, formalin-fixed, paraffin-embedded tissues showed higher levels of degradation resulting in an amplification length of not more than 100 bp.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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