DNA extraction from FFPE tissue samples - a comparison of three procedures.

AIM OF THE STUDY: One of the critical steps in molecular oncology diagnostics is obtaining high quality genomic DNA. Therefore, it is important to evaluate and compare the techniques used to extract DNA from tissue samples. Since formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for both retrospective and prospective studies, we compared three commercially available methods of nucleic acid extraction in terms of quantity and quality of isolated DNA. MATERIAL AND METHODS: Slides prepared from 42 FFPE blocks were macro-dissected. Resulting material was divided and processed simultaneously using three extraction kits: QIAamp DNA FFPE Tissue Kit (QIAGEN), Cobas DNA Sample Preparation Kit (Roche Molecular Systems) and Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega). Subsequently, quantity and quality of obtained DNA samples were analysed spectrophotometrically (NanoDrop 2000, Thermo Scientific). Results of quantitative analysis were confirmed by a fluorometric procedure (Qubit 3.0 Fluorometer, Life Technologies). RESULTS: The results demonstrated that the yields of total DNA extracted using either Maxwell or Cobas methods were significantly higher compared to the QIAamp method (p < 0.001). The Maxwell Extraction Kit delivered DNA samples of the highest quality (p < 0.01). However, the highest total yield of extracted DNA was achieved with the Cobas technique, which may be due to a higher volume of eluate compared to the Maxwell method. CONCLUSIONS: To our knowledge, this is the first paper which directly compares three extraction methods: Cobas, Maxwell and QIAamp. The data herein provide information required for the selection of a protocol that best suits the needs of the overall study design in terms of the quantity and quality of the extracted DNA.

Effects of morphine and methadone treatment on mRNA expression of Gα(i) subunits in rat brains.

Methadone is clinically effective as substitution therapy in patients with opioid dependence. The diversity of methadone and morphine in their intracellular activity is postulated. We compared the effects of repeated daily treatment of Sprague-Dawley rats with morphine (10 mg/kg) and methadone (1 mg/kg) on the expression of the Gα(i1-i3) mRNAs in several rat brain areas using RT-qPCR. We found that both opioid receptor agonists decreased Gα(i3) mRNA in only the nucleus accumbens. Although there was no difference in the influence of morphine and methadone on Gα(i), our results indicate that among Gα(i) subunits, the Gα(i3) is specifically involved in the mechanism of action of both drugs.

Concomitant administration of fluoxetine and amantadine modulates the activity of peritoneal macrophages of rats subjected to a forced swimming test.

Recent studies show that administration of a non-competitive NMDA receptor antagonist, amantadine (AMA), potentiates the action of antidepressant drugs. Since antidepressants may modulate functioning of the immune system and activation of a pro-inflammatory response in depressive disorders is frequently reported, the aim of the present study was to examine whether a combined administration of AMA and the antidepressant, fluoxetine (FLU), to rats subsequently subjected to a forced swimming test (FST) modifies the parameters of macrophage activity, directly related to their immunomodulatory functions, i.e., arginase (ARG) activity and synthesis of nitric oxide (NO). We found that 10 mg/kg AMA and 10 mg/kg FLU, ineffective in FST for antidepressant-like activity when administered alone, increased the ARG/NO ratio in macrophages when administered concomitantly. This effect was accompanied by a decrease of cellular adherence. Concurrently, the basal metabolic activity of the cells measured with reduction of resazurin, and intracellular host defense as assessed by a synthesis of superoxide anion, were not affected by such antidepressive treatment. Our data indicate that co-administration of AMA and FLU decreases the pro-inflammatory properties of macrophages and causes a redirection of immune response toward anti-inflammatory activity, as one can anticipate in the case of an effective antidepressive treatment.

Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain.

Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Galpha(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.

Chronic treatment with electroconvulsive shock may modulate the immune function of macrophages.

OBJECTIVE: To determine the effect of single and chronic electroconvulsive shock (ECS) administration on the immunoregulatory functions of macrophages. METHODS: Male Wistar rats received single or chronic treatment with ECS (150 mA, 50 Hz, 0.5 seconds) delivered through ear clips, once a day for 10 consecutive days, or sham ECS administered likewise. The rats were killed 24 hours after the last treatment, and peritoneal macrophages were cultured in vitro for 3 or 36 hours for a subsequent determination of their metabolic activity. The ability of macrophages to reduce Alamar Blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and nitrotetrazolium blue chloride and pinocytosis, adherence, and vitality, as well as synthesis of nitric oxide and arginase activity, was assessed. RESULTS: We found statistically significant changes in the biological properties of macrophages which occurred after 36 hours of incubation, especially in cultures stimulated with lipopolysaccharide; in contrast, no differences were observed between groups assessed after 3 hours of incubation. Rats receiving chronic 10-fold ECS showed a substantial increase in the metabolic activity of macrophages, reflected as their ability to reduce Alamar Blue and MTT and to increase arginase activity, accompanied with a marked but statistically insignificant decrease in nitric oxide synthesis compared with respective controls. CONCLUSIONS: Our results suggest that chronic treatment with ECS may induce long-lasting changes in the activity of peritoneal macrophages. Attenuation of their proinflammatory properties indicates that ECS can change the primarily immunoregulatory functions of macrophages.

Effect of cocaine on responsiveness of alpha(1)-adrenergic receptors in rat cerebral cortex: modulation by GABA-mimetic drugs.

We investigated the effects of single doses of cocaine (10 mg/kg, ip) and the gamma-aminobutyric acid (GABA)-mimetics tiagabine (10 mg/kg, ip) and vigabatrin (150 mg/kg, ip) injected separately or concomitantly with cocaine, on the responsiveness of cerebral cortical alpha(1)-adrenergic receptors. The accumulation of noradrenaline-stimulated inositol phosphates was estimated in vitro at 2 and 24 h after the drug injection. Cocaine significantly enhanced alpha(1)-adrenergic receptor responsiveness to noradrenaline. Neither tiagabine nor vigabatrin influenced the accumulation of inositol phosphates. Finally, the cocaine-evoked augmentation of alpha(1)-adrenoceptor responsiveness was counteracted by tiagabine but not by vigabatrin. This effect may represent a characteristic feature of tiagabine, not necessarily shared by other GABA-mimetic drugs.