Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of β-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues

The effects of individually ventilated cages on the respiratory systems of male and female Wistar rats from birth until adulthood

OBJECTIVE: To evaluate the respiratory systems of male and female rats maintained in individually ventilated cages (IVCs) from birth until adulthood. METHODS: Female Wistar rats were housed in individually ventilated cages or conventional cages (CCs) and mated with male Wistar rats. After birth and weaning, the male offspring were separated from the females and kept in cages of the same type until 12 weeks of age. RESULTS: The level of food consumption was lower in male offspring (IVC=171.7±9; CC=193.1±20) than in female offspring (IVC=100.6±7; CC=123.4±0.4), whereas the water intake was higher in female offspring (IVC=149.8±11; CC=99.2±0) than in male offspring (IVC=302.5±25; CC=249.7±22) at 11 weeks of age when housed in IVCs. The cage temperature was higher in individually ventilated cages than in conventional cages for both male (IVCs=25.9±0.5; CCs=22.95±0.3) and female (IVCs=26.2±0.3; CCs=23.1±0.3) offspring. The respiratory resistance (IVC=68.8±2.8; CC=50.6±3.0) and elastance (IVC=42.0±3.9; CC=32.4±2.0) at 300 µm/kg were higher in the female offspring housed in ventilated cages. The ciliary beat values were lower in both the male (IVCs=13.4±0.2; CC=15±0.4) and female (IVC=13.5±0.4; CC=15.9±0.6) offspring housed in individually ventilated cages than in those housed in conventional cages. The total cell (IVC=117.5±9.7; CC=285.0±22.8), neutrophil (IVC=13.1±4.8; CC=75.6±4.1) and macrophage (IVC=95.2±11.8; CC=170.0±18.8) counts in the bronchoalveolar lavage fluid were lower in the female offspring housed in individually ventilated cages than in those housed in conventional cages. CONCLUSIONS: The environmental conditions that exist in individually ventilated cages should be considered when interpreting the results of studies involving laboratory animals. In this study, we observed gender dimorphism in both the water consumption and respiratory mechanics of rats kept in ventilated cages

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of β-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues

The effects of individually ventilated cages on the respiratory systems of male and female Wistar rats from birth until adulthood

OBJECTIVE: To evaluate the respiratory systems of male and female rats maintained in individually ventilated cages (IVCs) from birth until adulthood. METHODS: Female Wistar rats were housed in individually ventilated cages or conventional cages (CCs) and mated with male Wistar rats. After birth and weaning, the male offspring were separated from the females and kept in cages of the same type until 12 weeks of age. RESULTS: The level of food consumption was lower in male offspring (IVC=171.7±9; CC=193.1±20) than in female offspring (IVC=100.6±7; CC=123.4±0.4), whereas the water intake was higher in female offspring (IVC=149.8±11; CC=99.2±0) than in male offspring (IVC=302.5±25; CC=249.7±22) at 11 weeks of age when housed in IVCs. The cage temperature was higher in individually ventilated cages than in conventional cages for both male (IVCs=25.9±0.5; CCs=22.95±0.3) and female (IVCs=26.2±0.3; CCs=23.1±0.3) offspring. The respiratory resistance (IVC=68.8±2.8; CC=50.6±3.0) and elastance (IVC=42.0±3.9; CC=32.4±2.0) at 300 µm/kg were higher in the female offspring housed in ventilated cages. The ciliary beat values were lower in both the male (IVCs=13.4±0.2; CC=15±0.4) and female (IVC=13.5±0.4; CC=15.9±0.6) offspring housed in individually ventilated cages than in those housed in conventional cages. The total cell (IVC=117.5±9.7; CC=285.0±22.8), neutrophil (IVC=13.1±4.8; CC=75.6±4.1) and macrophage (IVC=95.2±11.8; CC=170.0±18.8) counts in the bronchoalveolar lavage fluid were lower in the female offspring housed in individually ventilated cages than in those housed in conventional cages. CONCLUSIONS: The environmental conditions that exist in individually ventilated cages should be considered when interpreting the results of studies involving laboratory animals. In this study, we observed gender dimorphism in both the water consumption and respiratory mechanics of rats kept in ventilated cages

Exposure to fine particulate matter in the air alters placental structure and the renin-angiotensin system

<div><p>Transforming growth factor beta 1 (TGFβ1), the uteroplacental renin-angiotensin system (RAS) and vascular endothelial growth factor A (VEGF-A) participate in the placentation process. The aim of this study was to investigate the effects of exposure to pollutants on the placenta.</p><p>Methods</p><p>Female Wistar rats were exposed to filtered air (F) or to concentrated fine particulate matter (P) for 15 days. After mating, the rats were divided into four groups and again exposed to F or P (FF, FP, PF, PP) beginning on day 6 of pregnancy. At embryonic day 19, the placenta was collected. The placental structure, the protein and gene expression of TGFβ1, VEGF-A, and its receptor Flk-1 and RAS were evaluated by indirect ELISA and quantitative real-time PCR.</p><p>Results</p><p>Exposure to P decreased the placental mass, size, and surface area as well as the TGFβ1, VEGF-A and Flk-1 content. In the maternal portion of the placenta, angiotensin II (AngII) and its receptors AT₁ (AT₁R) and AT₂ (AT₂R) were decreased in the PF and PP groups. In the fetal portion of the placenta, AngII in the FP, PF and PP groups and AT₂R in the PF and PP groups were decreased, but AT₁R was increased in the FP group. VEGF-A gene expression was lower in the PP group than in the FF group.</p><p>Conclusions</p><p>Exposure to pollutants before and/or during pregnancy alters some characteristics of the placenta, indicating a possible impairment of trophoblast invasion and placental angiogenesis with possible consequences for the maternal-fetal interaction, such as a limitation of fetal nutrition and growth.</p></div

Surface area of the maternal placenta.

<p>FF = filtrated air before and during pregnancy, FP = filtrated air before and polluted air during pregnancy, PF = polluted air before and filtrated air during pregnancy, PP = polluted air before and during pregnancy. *p<0.05 vs. FF. #p<0.05vs. PF.</p

Primers used for qPCR gene expression protocol.

<p>Primers used for qPCR gene expression protocol.</p

Mass, thickness, longitudinal and transversal diameters and surface area of the placenta.

<p>Mass, thickness, longitudinal and transversal diameters and surface area of the placenta.</p

Antibodies used for ELISA protein expression protocol.

<p>Antibodies used for ELISA protein expression protocol.</p