Real-time qPCR Techniques Research Articles

MicroRNA-155 and 194 alter expression of Th17 and T regulatory-related transcription factors in the patients with severe coronavirus disease 2019 (COVID-19)

IntroductionIt has been demonstrated that the patients with severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) suffer from severe inflammation. Due to the ethnics, the immune responses may be different. Additionally, microRNAs may alter immune responses in the patients. The current study was aimed to evaluate the expression of T helper subsets-related transcription factors, some T helper 17 (Th17) products, and two microRNAs, including miR-155 and miR-194, in the Iranian hospitalized patients. MethodsIn this study, T-box expressed in T cells (T-bet), GATA binding protein 3, The retinoid orphan receptor gamma t (RORγt), forkhead box P3 (FOXP3), interleukin (IL)-17A, IL-8, and CC ligand 20 (CCL20) mRNA levels and, miR-155 and miR-194 levels were evaluated in 70 patients suffered from severe coronavirus disease 2019 (COVID-19) and 70 healthy subjects using Real-Time qPCR technique. ResultsThe findings showed that RORγt, and FOXP3 mRNA levels were significantly increased, while IL-17A, IL-8, and CCL20 mRNA levels were significantly decreased in the hospitalized SARS-CoV-2 infected patients. Although the levels of miR-155 and miR-194 were not different between groups, miR-194 has negative and positive correlations with RORγt and IL-17A in the Iranian healthy controls. ConclusionThis study reports although RORγt was up-regulated, IL-17A, IL-8, and CCL20 mRNA levels were significantly decreased in the hospitalized SARS-CoV-2 infected patients. It may be concluded that up-regulation of FOXP3, via development of T regulatory lymphocytes suppresses Th17 functions and neutralizes Th17 activities. MiR-194 may play crucial roles in regulation of RORγt and IL-17A expression in healthy people, the phenomenon that is disrupted in the severe SARS-CoV-2 infected patients.

Effect of PRECEDE-PROCEED-Model-Based Health Education on the Life Quality and 5-HTT Gene Expression of Patients with Gastric Cancer after Surgery.

This study aimed to investigate the effect of PRECEDE-PROCEED-Model-based health education on the life quality of patients with gastric cancer after surgery. Also, the effect of this model was evaluated on 5-HTT gene expression as a gene related to depression. For this purpose, a total of 32 gastric cancer patients who were hospitalized in this hospital between March 2019 and September 2020 were enrolled in this study after surgery and, according to the time of admission, were divided into the control group and observation group, with 16 patients in each group. Patients in the control group were nursed regularly, while those in the observation group, in addition to the regular nursing, would receive the PRECEDE-PROCEED-Model-based health education. Post-surgery life quality of patients in two groups was compared from the following aspects: Rehabilitation process, pain assessment, rate of complications and The Short-Form (SF-36) Health Survey. The expression of the 5-HTT gene was performed by the Real-time qPCR technique. The results of this study showed that after surgery, the extubation time and the time of hospital discharge of patients in the observation group were all earlier than those in the control group, while the score of pain assessment and rate of complication was much lower in the observation group, and the SF-36 score of patients was much higher (all P < 0.05). The results of 5-HTT gene expression showed that there was no significant difference between the control and observation groups before the intervention. But one month after the intervention, gene expression in the observation group was significantly reduced compared to the control group (p <0.01). This significant decrease was also seen two months after the intervention (p <0.05). As mentioned before, the expression of the 5-HTT gene increases during the depression, therefore improvement of the patient's condition and quality of life decreased the expression of this gene. Hence, PRECEDE-PROCEED-Model-Based Health Education plays an influential role in reducing the expression of this gene. However, the passage of time has not been ineffective in lowering 5-HTT expression. In general, PRECEDE-PROCEED-Model-based health education could help patients establish a good system of health knowledge, which could encourage the patients to avoid the negative mood, optimize the rehabilitation process, improve the post-surgery rehabilitation and, finally, ameliorate the life quality of patients after surgery.

AURKA Regulates the Mechanism of ATR Mediated DNA Damage Response about Drug Resistance in Multiple Myeloma

Objective: Multiple myeloma (MM) is a cancer formed by malignant plasma cells. So far, drug resistance and relapse are major clinical problems in MM. Our previous studies showed that some chromosomal instability genes are involved in drug resistance, which AURKA expression is closely related survival. But the correlation between AURKA expression and clinical and how to participate in the mechanism of proliferation and drug resistance are unknown.Methods: Analyzing the Gene expression profiling (GEP) data and the relationship between AURKA expression and prognosis in MM. Bone marrow plasma cells from MM patients and health donor were collected by CD138. Detecting oncogene AURKA in presence of deletion and amplification by FISH. Detecting expression of mRNA by real-time qPCR. Immunofluorescence (IF) was used to detect the expression of protein. Comet assay was used to evaluate DNA repair. Then using the AURKA inhibitors and targeting AURKA, CCK-8 is used to detect the proliferation, WB detection of apoptosis, DNA Damage protein, IF detection of spindle formation and analyzing whether the AURKA is correlated with chromosome instability and drug resistance. In vivo and in vitro, combined with clinical medication bortezomib, Melphalan, lenalidomide. We detect apoptosis and DNA damage related protein and tumor volume in NOD/SCID mice. Changes in the culture supernatant of DNA damage response factor is used by real-time qPCR technique and ELASA detection of cell mass.Results: 1: Analyzing 350 patients with GEP data, the prognosis of patients with the AURKA high expression are better than the lowers (P=0). Patients with high expression of AURKA are mainly distributed in the high proliferation group. 2: Detecting the expression of mRNA by Real time qPCR technique, AURKA expression is significantly higher in newly diagnosed patients with 27.5% (n =11/n=40). The expression of AURKA protein is detected by IF assay. The results show that AURKA expression was higher in newly diagnosed (36.84%, n=14/n=38) and relapsed (50%, n=20/n=40) than that in normal donors. Patients with high expression of AURKA are closely related to the clinical relevance of anemia, bone destruction, high CR and high LDH. To further detect the relationship between AURKA expression and chromosome DNA copy number, the results show that AURKA copy number is normal in newly diagnosed patients (n=20). But there are 10% patients (n=2/n=20) with AURKA amplified in relapsed patients. 3: In vivo and in vitro, AURKA inhibitor combined with Bomtzomib, lenalidomide and Melphalan could enhance the sensitivity of BTZ and xenograft tumor biopsy showed reduced group AURKA, Ki67 expression. After over expression of AURKA, the proportion of cells with a single nucleus was decreased and the formation of multiple nuclei increased. Knocking down the AURKA, spindle formation is disorder. The expression of gamma-H2AX molecules and 53BP1 related DNA damage is abnormal. WB suggests that DNA damage is inhibited, phosphorylation of ATR, ATM, CHK2 reduction, phosphorylation of gamma-H2AX and BRCA increased. Further, real time qPCR technique and ELASA detection of DNA damage response factor in cell culture supernatant is mainly changed, such as IL-1 and IL6.Conclusion: High expression of AURKA is associated with poor prognosis in MM; Inhibition of AURKA can promote apoptosis, DNA damage and reduce the secretion of IL-6 that could enhance the sensitivity to bortezomib, lenalidomide and Melphalan. The abnormal expression of AURKA related spindle formation disorder and cell nuclear phenomena that cause chromosomal instability. AURKA is mainly involved in the drug resistance through the DNA damage response ATR-CHK1-gammaH2AX pathway. DisclosuresNo relevant conflicts of interest to declare.

P107 Monitoring inflammatory bowel disease in clinical practice using gut microbial markers in faecal samples. Prospective study in a clinical cohort

Abstract Background The gut microbiota(GM) plays a pivotal role in the pathogenesis of inflammatory bowel disease (IBD) which includes Crohn’s disease (CD) and ulcerative colitis(UC). The degree of IBD activity, and therefore of inflammation, modifies GM composition towards an altered pattern called dysbiosis This IBD-dysbiosis pattern is different from healthy individuals(HI). The degree of dysbiosis is a good indicator of IBD activity and could be useful in monitoring IBD. The AIM of this study is to quantify the degree of IBD dysbiosis of the GM, and determine the utility as a biomarker for monitoring IBD disease compared with HI. Methods 80 IBD patients from 2 tertiary hospitals have been included. We included patients with clinical Remission (R), defined as Faecal Calprotectin (FC) levels &amp;lt;250 µg/gr., and Activity (A) (FC &amp;gt;250 µg/gr.) with mild to moderate disease. A blood sample (to determinate RCP, albumin and hemoglobin (Hb) levels) and stool sample (to determinate Faecal calprotectin (FC) and GM analysis) has been collected. DNA was extracted from fecal samples, using real-time qPCR techniques and 3 bacterial groups (Faecalibacterium prausnitzii (FP), Escherichia coli (EC), and total eubacteria (TE) that are highly representative of GM dysbiosis degree were analyzed. In regards to these bacteria, an index called FEI has been created to quantify GM dysbiosis. GM and FEI data has been collected in order to compare with standards analytical markers (Hb, Alb, RCP, FC). The duration of this study is 6 months with samples every three months (T0, T1 and T2). Results 35 UC and 45 CD patients were included. Initial treatment in 76,25% of cases were biological drugs. 55 % of patients were in remission at first sample. The analysis of clinical data showed that only PCR and CP had significantly differences between R and A (p=0,0037) and type of disease (p=0,018) and temporal evolution (p=0,004). Higher levels of FP are been observed in UC (p=0,0013) and higher levels of EC in CD (p=0,003) in all 3 samples. FEI values are higher in UC (p=0,001) and different between CD and UC and HI (p=0,015) indicating FEI ability to differentiate between 2 disease subtypes and HI. FEI cut-off point has been set at 1,869 (S 0,44 and E0,82; AUC 0,658) in T0. The IBD-GM compared with HI-GM showed lower FP abundance (p=0,002) and higher TE abundance (p=0,0001) with increased non-significant EC abundance. The A and R abundances for FP,FEI and TE showed significant differences between CD, UC and HI, indicating capacity to differentiate between activity and remission in T0. Conclusion Microbiological markers and FEI allow to differentiate between IBD subtypes and HI, and between A and R. The combination if RCP, FC and FEI can become a potential biomarker of IBD activity.

Gene expression of two innate cytokines in a miscarriage toxoplasmosis woman.

Toxoplasma gondii infections are endemic in Iraq and represent serious problems. Human toxoplasmosis can be associated with serious clinical manifestations, particularly in developing fetus. This study was aimed to determine the distribution of genotypes and alleles, residing within interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) polymorphisms, among fetuses and neonates, congenitally infected with Toxoplasma gondii, and among uninfected control cases. Blood samples were collected from 125 aborted women with a history of single or recurrent miscarriage, in addition to fifty normal healthy control women. Molecular identification of the parasite was performed by detecting Toxoplasma B1 gene using real-time qPCR technique. IL-6 and IL-1β gene expression was assayed in each case-study samples by using RT-PCR. T. gondii was detected in recurrent toxoplasmosis aborted women at percent (16%). IL-1β and IL-6 gene expression was significantly increased in toxoplasmosis women compared with healthy control women. Fold expression of IL-1β was 9.5 in toxoplasmosis patients compared with one fold in healthy control. IL-1β and IL-6 over-expression was correlated to the high-risk toxoplasmosis infection and could be a biomarker for prognosis of the disease.

THE STUDY OFLONG NON-CODING RNAs(NAALADL2 AND XIST) GENEEXPRESSION IN DIFFUSE LARGE B CELL LYMPHOMA

Diffuse large B cell lymphoma (DLBCL) is the most predominant type of Non Hodgkin- Lymphomas, representing more than one third of all recently diagnosed cases. Long non-coding RNAs (lncRNAs) are a class of RNA not coding for protein having more than 200 nucleotides and have significant roles in tumor formation and they have been investigated in different types of solid and hematologic tumors, including DLBCL.This work aims to study the level of lncRNAs(NAALADL2 and XIST)gene expression in DLBCL.NAALADL2 and XIST gene expression levels in blood were performed by real time qPCR technique. Forty five DLBCL patients and forty five age and gender matched healthy controls were included in this study.There was significant statistical increase in the expression of NAALADL2 and decrease of XIST expression in patients compared to control group (p value 10.3 and<1.57 for NAALADL2 and XISTrespectively.There was significant positive correlation between NAALADL2 and advanced stage and a significant negative correlation is found between XIST and advanced stage. It may be concluded that NAALADL2 and XISTexpression levels were obviously associated with DLBCL and might determine the disease progression.

Expression levels of ABCG2 and CD61 genes in breast cancer tissues of Iranian population.

Breast cancer as the most common female cancer is a malignancy with heterogeneous course. Dysregulation of several genes has been associated with development of this malignancy. Among these genes are the stem cell markers CD61 and breast cancer resistance protein (BCRP or ATP-binding cassette super-family G member 2 (ABCG2)). ABCG2 is one of the major efflux transporters implicated in multidrug resistance in cancer cells. In the present study, we compared expression of CD61 and ABCG2 transcripts between 30 breast cancer tissues and matched adjacent non-cancerous tissues (ANCTs) using real time qPCR technique. There was no significant difference in expression of CD61 or ABCG2 between tumoral tissues and ANCTs (Expression ratios = 1.21 and 0.98, P values = 0.55 and 0.96, respectively). There was a trend toward association between relative expression of CD61 (tumoral tissues versus ANCTs) and patients' age (P =0.05) in a way that older patients tended to over-express this marker in their tumoral tissues compared with the matched ANCTs. Moreover, there was a significant association between expression of this gene and tumor size (P =0.04) in a way that all tumors with sizes less than 2cm showed down-regulation of CD61 (as compared with the matched ANCTs). Expression of CD61 was significantly higher in tumor tissues with extracapsular nodal extension compared with confined lesions (P =0.007). Moreover, expression of ABCG2 was significantly higher in tumor tissues of patients aged less than 55 years compared with older patients (P =0.04). There was no significant correlation between expression of CD61 and ABCG2 either in tumoral tissues or in ANCTs. The current investigation shows association or trends toward association between expression of two cancer stem cell markers and some clinical data of breast cancer patients such as extracapsular nodal extension, age and tumor size which might imply their importance in the pathogenesis of breast cancer.

Salt oversensitivity derived from mutation breeding improves salinity tolerance in barley via ion homeostasis

Salt stress imposes a major environmental threat to agriculture, therefore, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any breeding strategy. In the present study, the expression profile of genes involved in ion homeostasis including salt overly sensitive (HvSOS1, HvSOS2, HvSOS3), vacuolar Na+/H+ antiporter (HvNHX1), and H+-ATPase (HVA) along with ion content measurement were investigated in two genotypes of Hordeum vulgare under 300 mM NaCl. The gene expressions were measured in the roots and shoots of a salt-tolerant mutant genotype M4-73-30 and in its wild-type cv. Zarjou by real-time qPCR technique. The critical differences between the salt-tolerant mutant and its wild-type were observed in the expressions of HvSOS1 (105-fold), HvSOS2 (24-fold), HvSOS3 (31-fold), and HVA (202-fold) genes in roots after 6-h exposure to NaCl. The parallel early up-regulation of these genes in root samples of the salt-tolerant mutant genotype indicated induction of Na+/H+ antiporters activity and Na+ exclusion into apoplast and vacuole. The earlier up-regulation of HvSOS1, HVA, and HvNHX1 genes in shoot of the wild-type genotype corresponded to the relative accumulation of Na+ which was not observed in salt-tolerant mutant genotype because of efficient inhibitory role of the root in Na+ transport to the shoot. In conclusion, the lack of similarity in gene expression patterns between the two genotypes with similar genetic background may confirm the hypothesis that mutation breeding could change the ability of salt-tolerant mutant genotype for efficient ion homeostasis via salinity oversensitivity response.

An improved real-time qPCR technique for quantification of intestinal bacteria in human fecal samples

The easy, cost-effective and rapid techniques for investigating the role of gut microbiota have become imperative owing to their importance in various diseases such as colon cancer and inflammatory bowel disease. Here we developed an absolute real-time qPCR technique for quantification of intestinal bacteria in human fecal samples for further investigation of gut microbiota composition in patients. At first, regarding bacterial species present in human fecal samples, species were cultured in anaerobic culture media and incubated at 37Cͦ in an anaerobic chamber. The number of CFU was counted. Eight fold serial dilutions of the bacterial suspension were prepared. To generate a standard curve for bacterial species in real-time PCR, DNA was extracted from different serial dilutions. Subsequently, DNA from human fecal samples was extracted. Now bacterial strains were applied as standard curve in absolute q PCR procedure for quantification of bacterial population in test samples. Based on absolute real-time analysis, intestinal bacteria were precisely quantified. The specificity of the primer pairs was checked by conventional PCR and real-time PCR regarding dissociation curve analysis. The amplification was linear in the range of 101-108. Dissociation curves had correlation coefficient values between 0.98 and 101/888. The efficiencies were between 95/073 and 99/804%. The absolute real-time PCR technique described here may be used for evaluating intestinal bacteria regarding diseases prevention.

1α,25-Dihydroxyvitamin D3 promotes bone formation by promoting nuclear exclusion of the FoxO1 transcription factor in diabetic mice

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the active form of vitamin D, which is responsible for reducing the risk for diabetes mellitus (DM), decreasing insulin resistance, and improving insulin secretion. Previous studies have shown that 1,25(OH)2D3 inhibited the activity of FoxO1, which has been implicated in the regulation of glucose metabolism. However, its function and mechanism of action in DM-induced energy disorders and also in bone development remains unclear. Here, using in vitro and in vivo approaches including osteoblast-specific, conditional FoxO1-knock-out mice, we demonstrate that 1,25(OH)2D3 ameliorates abnormal osteoblast proliferation in DM-induced oxidative stress conditions and rescues the impaired glucose and bone metabolism through FoxO1 nuclear exclusion resulting from the activation of PI3K/Akt signaling. Using alizarin red staining, alkaline phosphatase assay, Western blot, and real-time qPCR techniques, we found that 1,25(OH)2D3 promotes osteoblast differentiation and expression of osteogenic phenotypic markers (i.e. alkaline phosphatase (1), collagen 1 (COL-1), osteocalcin (OCN), and osteopontin (OPN)) in a high-glucose environment. Moreover, 1,25(OH)2D3 increased both total OCN secretion and levels of uncarboxylated OCN (GluOC) by phosphorylating FoxO1 and promoting its nuclear exclusion, indicated by Western blot and cell immunofluorescence analyses. Taken together, our findings confirm that FoxO1 is a key mediator involved in glucose homeostasis and indicate that 1,25(OH)2D3 improves glucose metabolism and bone development via regulation of PI3K/Akt/FoxO1/OCN pathway.

Waist-to-Hip Ratio (WHR) is Conversely Correlated to LAMP-2 Gene Expression Level: A Predictor for Lysosomal Lipid Entrance System

Background: Nutritional surveys focused on anthropometric measurements and gene profiles may present early predictors for atherosclerosis. Since lysosomal lipid entrance system is effectively related to the lysosomal membrane LAMP-1/-2 proteins thus the aim of this study was to evaluate the associations between their gene expression levels and anthropometric measurements. Methods: A total of 126 healthy volunteers participated in the study. The LDL-C, HDL-C, and TC values were measured by routine laboratory techniques. The WC, HC, BAI, HC and WHR measures were calculated on the basis of current anthropometric methods. The LAMP-1 and LAMP-2 expression levels were measured by Real Time qPCR technique. Results: The results showed that the HC, WC, WHR and BAI measures are significantly related to BMI value (r 0.6, p 0.0001; r 0.6, p 0.0001; r 0.1, p 0.006; r 0.55, 0.0001, respectively). The LAMP- 2 expression level was conversely related to LDL-C value, based on normal upper range (>130 mg/dl, p<0.005). Furthermore, the LAMP-2 expression level was conversely correlated to WHR (r - 0.35, p 0.009). Conclusion: We proposed that the lysosomal lipid entrance system relates conversely to abdominal adiposity. Furthermore, we suggested that the increase of WHR may indicate the increase of intracellular cholesterol synthesis due to the decrease of cholesterol uptake.

PCSK9-related LDL-C value is correlated to Rab5 isoform expression level

Circulating LDL metabolism is mostly related to the LDL internalization and intracellular lysosome-related degradation events. In this way, the competitive biologic agents and vesicular membrane-integrated or assembled proteins are important functioning factors. The aim of this study was to investigate the relationships between Rab5, Rab7, Lamp1 and Lamp2 gene expression levels, circulating PCSK9 value and, serum LDL-C level. One hundred ten subjects were recruited from healthy adults. Lipid profile was measured by laboratory routine techniques. PCSK9 value was measured with ELISA Kit. The Rab5, Rab7, Lamp1 and Lamp2 gene expression levels of buffy coat samples were measured using Real-Time qPCR technique. Serum LDL-C level was correlated to Rab5 expression level (r 0.25, p 0.02) and circulating PCSK9 value (r 0.3, p 0.003). Although PCSK9 elevated positively with LDL-C value but the Rab5 expression level increased up to 75centiles. Furthermore, multiple regression analyses showed that the Rab5 expression level is significantly correlated to LDL-C value (p 0.025, β 0.003), age (p 0.037, β −0.006) and Lamp1 expression (p 0.001, β 0.079) while the Rab7 expression level did not relate to the study factors except for Lamp1 (p 0.011, β 0.053). The results proposed that the LDL-C-correlated Rab5 expression level may be a primary cellular response due to reduced PCSK9-associated LDL influx. Furthermore, the LDL-C value was not correlated to Lamp1 and Rab7 expression levels. Our data suggested that the vesicular structures may be reduced with age.

Multigenic Delineation of Lower Jaw Deformity in Triploid Atlantic Salmon (Salmo salar L.).

Lower jaw deformity (LJD) is a skeletal anomaly affecting farmed triploid Atlantic salmon (Salmo salar L.) which leads to considerable economic losses for industry and has animal welfare implications. The present study employed transcriptome analysis in parallel with real-time qPCR techniques to characterise for the first time the LJD condition in triploid Atlantic salmon juveniles using two independent sample sets: experimentally-sourced salmon (60 g) and commercially produced salmon (100 g). A total of eleven genes, some detected/identified through the transcriptome analysis (fbn2, gal and gphb5) and others previously determined to be related to skeletal physiology (alp, bmp4, col1a1, col2a1, fgf23, igf1, mmp13, ocn), were tested in the two independent sample sets. Gphb5, a recently discovered hormone, was significantly (P < 0.05) down-regulated in LJD affected fish in both sample sets, suggesting a possible hormonal involvement. In-situ hybridization detected gphb5 expression in oral epithelium, teeth and skin of the lower jaw. Col2a1 showed the same consistent significant (P < 0.05) down-regulation in LJD suggesting a possible cartilaginous impairment as a distinctive feature of the condition. Significant (P < 0.05) differential expression of other genes found in either one or the other sample set highlighted the possible effect of stage of development or condition progression on transcription and showed that anomalous bone development, likely driven by cartilage impairment, is more evident at larger fish sizes. The present study improved our understanding of LJD suggesting that a cartilage impairment likely underlies the condition and col2a1 may be a marker. In addition, the involvement of gphb5 urges further investigation of a hormonal role in LJD and skeletal physiology in general.

Plasma PCSK9 level affects passively LAMP-2 expression; an evidence of transcription network

Free cholesterol accumulation in lysosomes is related to highly glycosylated membrane-protein functions, endogenous cholesterol synthesis and, LDL influx. The LAMP-1 and LAMP-2 are two lysosome proteins involved in cholesterol transport. The study aim was to investigate the associations between LAMP-2 expression level, LAMP-2/LAMP-1 expression ratio, blood PCSK9 protein and LDL-C values. One hundred twenty six healthy subjects were selected during a medical interview. The biochemical parameters were measured using routine laboratory techniques. The plasma PCSK9 level was identified by ELISA method. The LAMP-1/LAMP-2 expression levels were estimated by Real time qPCR technique. The PCSK9 and LAMP-2 expression networks were designed with Cytoscape software and, were enriched using transcription factor databases. Linear correlations were observed between PCSK9 and LDL-C levels (r=0.34; p=0.002) and, TC/HDL-C ratio (r=0.35; p=0.001). The LAMP-2 expression level and LAMP-2/LAMP-1 expression ratio were inversely related to LDL-C values (p=0.02). The transcription factors were compartmented on the merged network and were strictly controlled the PCSK9 expression. The results suggested that the decreased LDL influx due to the increase of plasma PCSK9 level attenuates the LAMP-2 gene expression level. Furthermore, the merged network supported our findings that the LAMP-2 expression is not in relation to transcription factors involved with PCSK9 expression.

Applicability of the real-time PCR assay in the amplification of cyanobacterial DNA from preserved samples

Applic ability of the real-time PCR assay in the amplification of cyanobacterial DNA from preserved samples The study and monitoring of cyanobacterial blooms often involves the use of preserved samples to avoid cellular degradation. However, preserved samples may not be suitable for molecular biology studies because preservation methods can interfere with DNA quality/quantity. Real-time quantitative PCR analysis (qPCR) has been widely applied in molecular analysis and is considered a promising method for monitoring purposes. This study intended to evaluate the applicability of the real-time qPCR technique in samples that were subjected to different methods of preservation: (1) 15% Lugol’s iodine solution (2) 4% formaldehyde and (3) 25% glutaraldehyde. The ability to amplify and quantify DNA extracted from Planktothrix agardhii was assessed using the rpoC1 gene as the target fragment in both raw water samples and in vitro cultures. No reliable DNA amplification was obtained from glutaraldehyde-preserved samples. Successful amplification was obtained from Lugol’s and formaldehyde-preserved samples. In this case, however, the quantification that was achieved by real-time PCR cannot be used to infer cell numbers, because the Ct values that were obtained from the Lugol’s and formaldehydepreserved samples were significantly higher than the Ct values that were obtained from the unpreserved samples. Therefore real-time PCR can be used for the detection and identification of cyanobacteria in preserved samples but no reliable cell quantification can be performed using this method.

Some epidemiological features of BCoVs infection in Al-Qadisiyah Province by using real time-qPCR technique

This study was carried out to evaluate some epidemiological features of Bovine Coronavirus infection by using one-step real-time fluorogenic quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay based on SYBR Green I dye in detection. Coronaviruses detected by the same nucleocapsid (N) gene primers under 98% similarity with HECV-4408 (human enteric Coronavirus) in children according to NCBI with product size 124bp. 285 fecal samples have been examined by routine methods against pathogenic bacteria in the intestines (E.coli, Salmonella Spp.) and Cryptosporidium parvum, the samples positive for the parasite submitted to molecular testing because they may be mixed with coronavirus infections. 100 samples were screened for the presence of BCoVs antigens by using an immunochromatographic rapid test as a field fast test. Where 44% of samples showed positivity to BCoVs, out of 50 samples submitted to quantitative reverse transcription (RT-qPCR) assay. Out of 50 – 31 samples had been positive. We found that distribution of BCoVs was significantly higher in rural areas 33.3%-87.5% as compare to cities 71.4%-75%, the infection in males reach to75% vas 53.5% in females, high infection rate 62.9% in &lt; 1- 4 months age as compared with &gt; 1 month age 62.9%. The results of infection rate showed high percentage during February 77.7% while the percentage on (January, December, March, and April) was (66.6%, 66.1%, 54.5%, and 50%) respectively.

Analysis of Immune-Relevant Genes Expressed in Spleen of Capra hircus Kids Fed with Trivalent Chromium

Chromium is a biologically important element for humans and laboratory animals. Although the favorable effects of trivalent chromiumon immune responses of studied animals have been well documented, the precise mechanisms by which the chromium acts on immune system is relatively poor studied. In this study, real-time qPCR technique was employed to evaluate the expression profiles of four immune-related genes (B2M, MHCA, MHCB, and Rap2A) in spleens of the domestic goats, Capra hircus, feeding on four different levels of supplemental chromium (0, 0.5, 1, and 1.5 mg/day) as chromium– methionine. The results showed that 1.5 mg/day of supplemental chromium significantly increased the expression of the four studied genes (P <0.01). Since the studied genes play important roles in development, activation, and migration of lymphocytes, their increased expression seems to be an unknown mechanism by which chromium impose reinforcing effects on immune system. Therefore, supplemental chromium can be potentially used to improve immune responses especially in animals experiencing any type of stress such as invasion by a pathogen.

Microarray and miRNA analyses of brain lesions in African- American and Caucasians with multiple sclerosis

Objective: To study if differences in gene expression in brain tissue among African-Americans (AA) and Caucasian Americans (CA) with multiple sclerosis (MS) exist. Understanding any genetic differences is critical for better understanding of MS and its outcomes.Methods: Microarray and microRNA methods were used in chronic brain lesions of AA and CA patients with MS.Results: We found marked downregulation in GM2A (5.2 vs. 2.07), GALC (4.48 vs. 2.66), EIF1AY (4.54 vs. 1.57) and carboxypeptidase D (3.72 vs. 1.79), genes among chronic lesions taken from AA and CA brains and validated using real-time qPCR techniques. A total of 1108 genes were down regulated, compared to 467 genes that were upregulated in chronic MS lesions, compared to normal appearing brain matter (ratio of 2:1); a similar comparison between AA and CA brains revealed a total of 611 down regulated vs. 192 upregulated genes (ratio of 3:1). Interpretation: Significant downregulation of GM2A, GALC, EIF1AY and carboxypeptidase D in the AA lesions as compared to CA cohort could have implications for MS.

G.P.19.07 Gene and protein expression of muscle specific genes in children

TaqMan® low density arrays (LDA) is a real-time qPCR technique by which it is possible to quantitatively assess expression of various genes simultaneously on the same sample. In evaluation of muscle samples the assayed expression profiles will include genes encoding structural and functional proteins in skeletal muscle, proteins involved in activation, proliferation, maturation, and fusion of satellite cells as well as inflammatory mechanisms, thus covering broadly the field of muscle regeneration.