This technical note describes a novel and straightforward experimental strategy for the extraction/capture of DNA using magnetic ionic liquid (MIL) followed by real time-polymerase chain reaction ...(qPCR) analysis. An affordable and low-cost magneto-based multiwell platform was first examined for capturing DNA allowing for simultaneous extractions that increased the analysis throughput of the experimental workflow. This configuration was composed of a series of neodymium rod magnets attached to a multiwell device in which a magneto-active extraction phase (MIL) was suspended for a single drop microextraction (SDME) approach. In this configuration, up to 32 extractions were able to be performed simultaneously, and DNA was successfully extracted from aqueous samples. Furthermore, as a proof-of-concept, this affordable and simple experimental strategy proved to be efficient for the extraction/capture of DNA from challenging samples such as whole blood without any pretreatment. This fact also consists of important feature compared to previous methodologies that required additional steps of sample preparation.
A simple and straightforward methodology using magnetic ionic liquids was proposed for high throughput DNA extraction using a multiwell platform followed by real time polymerase chain reaction.
AbstractChronic use of alcohol and its withdrawal impairs the delicate balance between GABAergic and glutamatergic systems. This unbalance includes changes in GABA receptors – importantly in GABA A ...subtypes – and glutamate receptor – especially in NMDA subtypes. A better comprehension of the different roles of GABA AR and NMDAR subunits could be helpful to define new strategies to counteract the deleterious effects observed during alcohol withdrawal. Taurine, a sulfonated amino acid, has been proposed to attenuate alcohol withdrawal symptoms due to its neuromodulatory properties. In this study, we evaluated the correlations between GABA AR and NMDAR subunits in the hippocampus of rats chronically treated with alcohol or in alcohol withdrawal, and the effects of taurine treatment on these parameters. Male Wistar rats received alcohol (2 g/kg) or water by oral gavage (control), 2x/day, for 28 days. From day 29 to 33, withdrawal rats received water instead of alcohol and all groups were reallocated to receive 100 mg/kg taurine or saline intraperitoneally, once a day. On day 34, rats were euthanized and the hippocampus was dissected for GABA AR α1, α4, δ, and γ2 and NMDAR GluN2A and GluN2B subunits mRNA expression determination by RT qPCR. There were no differences between groups in the studied GABA AR and NMDA subunits. However, we observed a correlation of α1 and γ2 subunits induced by taurine, while in the alcohol group there was a correlation between α4 and GluN2A. In the group treated with alcohol and taurine, we observed an extra correlation, between α1 and GluN2A. After 5 days of withdrawal, a correlation observed in the control group, between δ and GluN2A was reestablished. The correlation found between subunits suggests a neuroadaptation of GABAergic and glutamatergic systems in withdrawal rats. Results from this study contribute to the elucidation of the mechanisms beyond neuroadaptations observed in alcohol use and withdrawal.
Social relations are built and maintained from the interaction among individuals. The oxytocin (OT), vasopressin (VP), estrogen, dopamine, and their receptors are involved in the modulation of sexual ...behavior in females. This study aimed to analyze the impact of OT gene knockout (OTKO) on sexual behavior and the gene expression of oxytocin (OTR), estrogen alpha (ERα), estrogen beta (ERβ), vasopressin (V
1a
R), and dopamine (D
2
R) receptors in the olfactory bulb (OB), prefrontal cortex (PFC), hippocampus (HPC), and hypothalamus (HPT), as well as in the synthesis of VP in the HPT of female mice. Wild-type (WT) littermates were used for comparisons. The
C
DNAs were synthesized by polymerase chain reaction and the gene expression was calculated with the 2
−ΔΔCt
formula. Our results showed that the absence of OT caused an increase in the frequency and duration of non-receptive postures and a decrease in receptive postures in the OTKO. OTKO females showed a significant decrease in the gene expression of OTR in the HPC, V
1a
R in the HPT, and ERα and ERβ in the PFC. There was no significant difference in the gene expression of D
2
R of OTKO. However, OTKO showed an increased gene expression of V
1a
R in the HPC. There is no significant difference in VP mRNA synthesis in the HPT between OTKO and WT. Our findings demonstrate that the absence of OT leads to significant changes in the expression of the studied genes (OTR, ERα, ERβ, V
1a
R), and these changes may contribute to the decreased sexual behavior observed in OTKO females.
•Variations of maternal care are linked with differentiated gene expression in OB.•The OB of HL mothers expressed more Drd1a, Prlr, Htr1a, Htr1b and Esr1 than LL.•No difference of gene expression was ...found in the HP, PFC or ST of mothers LL or HL.•There were differences of gene expression in the HP between lactating and virgins.•LL showed a decrease in the frequency of nursing and remained longer off the pups.
For most mammalian species, maternal behavior has an essential role in the development of the offspring. The frequency of licking/grooming (LG) the pups has been used as a parameter to evaluate maternal care, having mothers with high (HL) or low (LL) frequencies of LG. This study aimed to analyze the gene expression of the receptors for dopamine (Drd1a), prolactin (Prlr), serotonin (Htr1a, Htr1b), estrogen (Esr1, Esr2), and of Bdnf in the olfactory bulb (OB), hippocampus (HP), prefrontal cortex (PFC), and striatum (ST) of Wistar rats from three groups: LL (n=8); HL (n=8); virgin females in diestrus (D; n=6). Maternal behavior was studied between the 1st and 7th postpartum days. Brain parts were analyzed by qRT-PCR. LL showed a decrease in the frequency of nursing, and an increase of remaining off the pups. There was an increase in gene expression of Drd1a, Prlr, Htr1a, Htr1b and Esr1 in the OB of HL, compared to LL. In the HP, Drd1a, Prlr and Htr1a were differently expressed when comparing HL, or LL, with D. The main finding is that HL had higher gene expression levels in the OB, which is a crucial structure to promote behavioral differences.
Taste perception plays a key role in determining individual food preferences and dietary habits and may influence nutritional status. This study aimed to investigate the association of TAS1R2 ...(Ile191Val - rs35874116) and TAS1R3 (-1266 C/T - rs35744813) variants with food intake and nutritional status in children followed from birth until 7.7 years old. The nutritional status and food intake data of 312 children were collected at three developmental stages (1, 3.9 and 7.7 years old). DNA was extracted from blood samples and the polymorphisms were analyzed by real-time polymerase chain reactions (qPCR) using hydrolysis probes as the detection method. Food intake and nutritional status were compared among individuals with different single nucleotide polymorphism (SNP) genotypes. At 3.9 years old, children homozygous (Val/Val) for the TAS1R2 Ile191Val polymorphism ingested less sugar and sugar-dense foods than children who were *Ile carriers. This finding demonstrated that a genetic variant of the T1R2 taste receptor is associated with the intake of different amounts of high sugar-content foods in childhood. This association may provide new perspectives for studying dietary patterns and nutritional status in childhood.
Purpose
To investigate the prevalence of human papillomavirus (HPV) in cervical samples of pregnant and non-pregnant women in South-Brazil.
Methods
A prospective study of 91 pregnant and 92 ...non-pregnant women with no previous history of cervical dysplasia or cancer was carried out. Cervical samples for HPV testing and cytology were collected in each trimester of pregnancy and in the puerperium for pregnant women and at matched intervals for the non-pregnant women. All samples were analyzed through PCR with consensus primers GP5+/GP6+. Genotyping was performed using specific primers. To control for confounding factors, the analysis of multivariate logistic regression was applied. The measure of odds ratio (OR) and the 95 % confidence interval (95 % CI) were used. The level of statistical significance was set at 5 % (
P
≤ 0.05).
Results
HPV DNA was detected in 23/91 (25.3 %) cervical samples from the pregnant women and in 12/92 (13 %) cervical samples from non-pregnant women (
P
= 0.035). There was a significant association among cervical HPV infection and young age, number of lifetime sexual partners, and the presence of abnormal cervical cytology. HPV16 and HPV18 were the viral types more frequently detected. Out of the 23 HPV-positive pregnant women, 17 (73.9 %) had normal cervical cytology.
Conclusion
Our results suggest a higher prevalence of HPV infection in pregnant vs. non-pregnant women. This finding may be related to the relative immunosuppression observed in pregnant women, outlining the importance of the appropriate monitoring of the viral infection in this specific population.
Byline: Adriana V Roehe (1), Ana Paula G Frazzon (1), Grasiela Agnes (1), Andrea P Damin (1), Antonio A Hartman (1), Marcia S Graudenz (1) Author Affiliation: (1) Avenida Ijui, 635/402 Petropolis, ...Porte Alegre, Rio Grande do Sul, CEP, 90460-200, Brazil Article History: Registration Date: 10/05/2006 Received Date: 08/05/2006 Accepted Date: 09/05/2006 Online Date: 27/01/2007
A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by ...reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex).
Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder.
Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively.
Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene.
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