ABSTRACT
Aim: To evaluate the baseline expression of the immune genes in PBMCs of responder and non-responder patients with chronic Hepatitis C
Background: Although the contribution of peripheral blood mononuclear cell [PBMC] gene expression in treatment outcome of hepatitis C virus [HCV] infection is supposed, it has remained to be distinctly delineated. The baseline expression of the immune genes inside PBMCs may reflect the responsiveness status following IFN treatment
Methods: Totally, 22 chronic HCV encompasses 10 responders and 12 non-responsive cases enrolled randomly regarding medical records. The PBMCs from the peripheral blood samples were isolated and then incubated for 6 hours in the culture media. The baseline expression of TLR7, SOCS1 and ISG15 was measured by Real time PCR
Results: The gene expression pattern in PBMCs of both groups showed a similar trend. The expression of SOCS1 and TLR7 genes showed higher levels in non-responder group [P>0.05]. The result of ISG15 showed a higher but non-significant expression in the responder group [P>0.05]
Conclusion: The similar pattern of TLR7, SOCS1 and ISG15 expression in the responder and non-responder patients indicated their poor discriminating and predictive value in PBMCs sample
ABSTRACT
Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA [cfDNA] is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control [EDC] is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time
Methods: A DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/ 500 microl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples
Results: Comparison of real time PCR threshold cycle [Ct] for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them
Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis
ABSTRACT
Background: asthma is very common in children and its diagnosis is based on clinical manifestations, which can be misdiagnosed as other respiratory diseases with similar signs and symptoms
Objective: to analyze the expression of ST2L and CD203c in the diagnosis of pediatric asthma
Methods: basophils were purified from whole blood samples of patients and healthy controls using Ficol-Paque gradient and Basophil Isolation Kit. RNA extraction was done by RNX-Plus solution and after synthesis of cDNA, the gene expression was analyzed by means of real time PCR
Results: patients expressed significantly higher levels of CD203c than healthy controls [p=0.01]. Although there was an increase in the transcription level of ST2L gene in patients, the results were not statistically significant compared to those obtained from the healthy controls [p>0.05]. A Specificity of 60% and a sensitivity of 73% were foundusing ROC curve for CD203c expression. Patients with positive family history of asthma exhibited more CD203c and ST2L expression [p<0.05]
Conclusion: it is proposed that determining CD203c expression by real time PCR may be an effective technique for diagnosis of pediatric asthma
ABSTRACT
Background: Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions
Materials and Methods: In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA [cffD-NA] was extracted from maternal plasma. Real-time quantitative polymerase chain reaction [qPCR] for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 [RASSF1A] gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively
Results: Out of 48 fetuses between 8 and 32 weeks [wks] of gestational age [GA], we correctly diagnosed 45 cases [93.75%] of RHD positive fetuses and 2 cases [4.16%] of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative
Conclusion: Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration
Subject(s)
Humans , Female , Genotype , Prospective Studies , Cohort Studies , Real-Time Polymerase Chain Reaction , Cell-Free System , DNA , Pregnancy , Rh-Hr Blood-Group System , Genotyping TechniquesABSTRACT
Kisspeptin and RFamide-related peptide-3 [RFRP-3] are known to affect GnRH/luteinizing hormone [LH] in several species, including the rat. It has been hypothesized that GnRH/LH changes during the rat estrous cycle may result from changes in the expression of KiSS1 and RFRP-3 genes. Therefore, the present study investigates KiSS1 and RFRP-3 gene expression at the transcriptional level in the rat hypothalamus during the estrous cycle. In the present experimental study, 36 adult female Sprague-Dawley rats [3-4 months old] were used to study the expression of KiSS1 and RFRP-3 mRNA in the hypothalamus during the estrous cycle. Four rats were ovariectomized, whereas the remainder were allotted to four different phases of the estrous cycle [n=8 per estrus phase]. Rats were decapitated, and the hypothalami were immediately dissected and frozen in liquid nitrogen. Expressions of KiSS1 and RFRP-3 mRNAs were analyzed by real-time PCR. The expression of KiSS1 mRNA during estrus was lower than other phases of the cycle [p<0.01]. Expression of KiSS1 mRNA during the metestrus phase was lower than the proestrus phase [p<0.01]. The expression of RFRP-3 mRNA during proestrus was lower than the diestrus phase [p<0.01]. Results of the present study showed the role of coordinated expression of KiSS1 and RFRP-3 mRNA in the hypothalamus in the control of the rat estrous cycle