Protecting RNA in fixed tissue: an alternative method for LCM users.

RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection (LCM) followed by RT-PCR. Moreover, RNA isolation kits especially developed for LCM samples are very expensive. Our aim was to determine an easy protocol that ideally must provide an acceptable morphology, allow proper laser capture of selected cells and improve RNA yield and quality. In this study, retinas were dissected, briefly incubated in a RNA preservative and fixed in 2% paraformaldehyde before being cut on a cryostat. LCM was carried out in retinal sections for immediate RNA isolation, by using TRIzol common protocol with minor modifications. Real-time PCR was performed next in order to compare availability of RNA from samples submitted to different protocols. The use of the RNA preservative followed by a fast fixation did not jeopardize tissue morphology, allowing microdissection of selected cells, combined to minor modifications in usual RNA isolation procedures, significantly improved RNA yield and quality. Furthermore, only LCM samples submitted to our protocol provided amplifiable mRNA, as determined by real-time PCR. Taken together, the combination of the described procedures resulted in a reliable alternative for LCM users.

Temporal and topographic ultrastructural alterations of rat heart myofibrils caused by thyroid hormone.

In order to further our understanding regarding the temporal and topographic ultrastructural aspects of the myocardium under thyrotoxicosis, thyroxine (T4; 25 and 100 microg/100 g bw) was administered to young rats 24 hours after birth until 15 days. The animals were then sacrificed, the hearts excised and weighed, and the ventricle tissue samples were then processed for confocal and transmission electron microscopy. At 48/72 hours and 1 week after initiation of T4 treatment with 100 microg/100 g bw, numerous lamellar bodies (probably formed by phospholipids) progressively accumulated in the heart. These bodies were observed in the cytosol, inside mitochondria and in the extracellular matrix. At 2 weeks of T4 treatment with 100 microg/100 g bw, lamellar bodies were virtually absent. Changes in cell shape, disorganization of intercellular junctions, and substantial myofibrillar disarray were observed in many cardiomyocytes. A gradient of myofibrillar disarray, which increased in abundance and intensity from the endocardium to the epicardium, was also observed. Immunocytochemical staining for desmin showed that the arrangement of this protein was disorganized in many cells of T4-treated rats as compared with normal ones, confirming ultrastructural data. The predominant appearance of myofibrillar disarray, associated with disorganization of cytoskeletal proteins in the deep myocardium, may be due to higher mechanical wall stress and consequent higher metabolic demand. Alternatively, differential sensitivity of cardiomyocytes to thyroid hormone in different areas is also a possibility.