transcription PCR ( RT-PCR ): trends and problems. J Mol Endocrinol, 2001, 29, 23-39. 4. Bustin S.A.: Quantitative real-time RT-PCR - a perspective. J Mol Endocrinol 2005, 34, 597-601. 5. Carvalho D.M., de Sá P.H., Castro T.L.P., Pinto A., Gil D. J. P., Bagano P., Bastos B., Costa L. F. M., Meyer R., Silva A., Azevedo V., Ramos R. T. J., Pacheco L. G. C.: Reference genes for RT-qPCR studies in Corynebacterium pseudotuberculosis identified through analysis of RNA-seq data. Antonie Van Leeuwenhoek 2014, 106, 605-614. 6. Dheda K
Joanna Pławińska-Czarnak, Joanna Zarzyńska, Janusz Bogdan, Alicja Majewska, Marek Karwański, Magdalena Kizerwetter-Świda, Jarosław Kaba, Krzysztof Anusz and Emilia Bagnicka
.C., Radtke A., Gerken G., Beckebaum S. (2008). Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR. BMC Cancer 8: 350. Cieslak J., Mackowski M., Czyzak-Runowska G., Wojtowski J., Puppel K., Kuczynska B., Pawlak P. (2015). Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells. PLoS ONE 10(10): e0139688. Coombes B.K., Hardwidge P.R., Finlay B.B. (2004). Interpreting the Host-Pathogen Dialogue Through Microarrays. Adv. Appl
NT Popov, DS Minchev, MM Naydenov, IN Minkov and TI Vachev
used in this study are shown in Table 1 . MicroRNA specific cDNA (5 μL) were subjected to pre amplification with peqGOLD Taq DNA Polymerase (VWR, Radnor, PA, USA) prior to the reverse transcription-(RT-PCR) step in order to enhance sensitivity of the test. The qRT-PCR was carried out using the Maxima SYBR Green qPCR Master Mix (Thermo Scientific) and the ABI PRISM ® 7500 system (Applied Biosystems, Foster City, CA, USA). All the experiments were performed in duplicate. Each sample was normalized using spiked-in control and relative quantification of miRNAs were
İbrahim Halil Demirsoy, Duygu Yolal Ertural, Şenay Balci, Ümit Çınkır, Kerem Sezer, Lülüfer Tamer and Nurcan Aras
Background: Metformin, a widely used biguanide class of anti-diabetic drug, has potential to increase insulin sensitivity and reduce blood glucose to treat type 2 diabetes (T2D). It has been reported that metformin has an activity on regulation of miRNAs by targeting several downstream genes in metabolic pathways. However, molecular mechanism underlying the process is still not fully known. In this study, it was aimed to identify differential expression profiles of plasma derived miRNAs following 3 months metformin treatment in patients with T2D.
Methods: The plasma samples of 47 patients with T2D (received no anti-diabetic treatments) and plasma samples of same 47 patients received 3 months metformin treatment was recruited to the study. Total RNAs were isolated from plasma and reverse transcribed into cDNA. Profiles of differential expressions of miRNAs in plasma were assessed by using of micro-fluidic based multiplex quantitative real time -PCR (BioMarkTM 96.96 Dynamic Array).
Results: Our results showed that expression profiles of 13 candidate miRNAs; hsa-let-7e-5p, hsa-let-7f-5p, hsa-miR- 21-5p, hsa-miR-24-3p, hsa-miR-26b-5p, hsa-miR-126-5p, hsa-miR-129-5p, hsa-miR-130b-3p, hsa-miR-146a-5p, hsamiR- 148a-3p, hsa-miR-152-3p, hsa-miR-194-5p, hsa-miR- 99a-5p were found significantly downregulated following metformin treatments in patients with T2D (p<0.05).
Conclusions: In conclusion, our finding could provide development of better and more effective miRNAs based therapeutic strategies against T2D.
E. Villar-Luna, J. A. García-Espinoza, O. Goméz-Rodriguez, R. I. Rojas-martínez and E. Zavaleta-Mejía
Capsicum annuum L. CM334 is susceptible to Nacobbus aberrans but highly resistant to Phy-tophthora capsici. Resistance to P. capsici is associated with the over-expression of various defense genes such as those encoding pathogenesis-related proteins. The transcriptional alterations of defense-related genes were determined in galls induced by N. aberrans (Na) in CM334 chili roots. Transcripts accumulation of WRKY-a, WRKY1, POX (peroxidase), PR-1 (pathogenesis-related protein 1), and EAS (5-epiaristolochene synthase) was estimated by qRT-PCR, and they were compared with those recorded in the incompatible CM334- P. capsici (Pc) interaction. The levels of all studied genes were significantly (P s 0.05) lower (WRKY1, POX and PR-1) or down-regulated (WRKY-a and EAS) in the presence of N. aberrans; in contrast, in the incompatible interaction, all genes were significantly up-regulated. The alterations induced by N. aberrans could be necessary to ensure the successful completion of its life cycle in CM334 chili roots.
Tatyana M. Kichukova, Nikolay T. Popov, Ivan S. Ivanov and Tihomir I. Vachev
Background: Development of biomarkers for autism spectrum disorder (ASD) has still remained a challenge to date. Recently, alterations of the expression of microRNAs (miRNAs) in peripheral blood, serum and post-mortem brain tissue have been linked to ASD. miRNAs are known to be secreted by various cell types and can mediate transmission of information into recipient cells and to modulate their physiological functions. On this basis it is assumed that circulating miRNAs could be useful biomarkers for the diagnosis or prognosis of pathological conditions.
Aim: The aim of this study was to test whether circulating miRNAs display differential expression profile in serum of ASD patients.
Patients and methods: The relative expression levels of 42 miRNAs were analyzed by stem-loop qRT-PCR assay in the serum of ASD patients compared to healthy controls.
Results: The results indicated that 11 miRNAs in ASD patients were substantially higher expressed than these in control subjects, and 29 miRNAs were lower expressed, respectively. In addition, target gene analysis displayed that the altered serum miRNAs targeted some important genes like alpha 1C subunit of voltage-dependent calcium channel, L type, (CACNA1C), beta 1 subunit of voltage-dependent calcium channel (CACNB1) and other genes involved in epigenetic processes like dicer 1, coding ribonuclease type III (DICER).
Conclusion: Our results suggested that differentially expressed miRNAs in serum might be involved in ASD molecular pathways, and serum miR-424-5p, miR-197- 5p, miR-328-3p, miR-500a-5p, miR-619-5p, miR-3135a, miR-664a-3p, and miR- 365a-3p might be able to serve as potential biomarkers for ASD because they displayed significant alterations in the expression profile in children diagnosed with ASD.
M. Ghaffari Novin, Azra Allahveisi, M. Noruzinia, F. Farhadifar, E. Yousefian, A. Dehghani Fard and M. Salimi
In some cases of infertility in women, human oocytes fail to mature when they reach the metaphase II (MII) stage. Mitochondria plays an important role in oocyte maturation. A large number of mitochondrial DNA (mtDNA), copied in oocytes, is essential for providing adenosine triphosphate (ATP) during oocyte maturation. The purpose of this study was to identify the relationship between transcript expression levels of the mitochondrial encoded gene (MT-CO1) and two nuclear encoded genes, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) in various stages of human oocyte maturation. Nine consenting patients, age 21-35 years old, with male factors were selected for ovarian stimulation and intracytoplasmic sperm injection (ICSI) procedures. mRNA levels of mitochondrial- related genes were performed by singlecell TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR). There was no significant relationship between the relative expression levels in germinal vesicle (GV) stage oocytes (p = 0.62). On the contrary, a significant relationship was seen between the relative expression levels of TFAM and NRF1 and the MT-CO1 genes at the stages of metaphase I (MI) and MII (p = 0.03 and p = 0.002). A relationship exists between the transcript expression levels of TFAM and NRF1, and MT-CO1 genes in various stages of human oocyte maturation.
B. Karpaga Raja Sundari and M. Ghosh Dasgupta
(2004): Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol 4: 14. BUSTIN, S. A. (2002): Quantification of mRNA using realtime reverse transcription PCR (RT-PCR) trends and problems. J Mol Endocrinol 29: 23-39. BUSTIN, S. A. and T. NOLAN (2004): Pitfalls of quantitative real-time reverse transcription polymerase chain reaction. J Biomol Tech 15(3): 155-166. CHEN, C. Y., M. H. HSIEH, C. C. YANG, C. S LIN and A. WANG (2010): Analysis of the cellulose synthase genes associated with
T.I. Vachev, V.K. Stoyanova, H.Y. Ivanov, I.N. Minkov and N.T. Popov
Schizophrenia (SZ) is a chronic neuropsychiatric disorder characterized by affective, neuromorphological and cognitive impairment, deteriorated social functioning and psychosis with underlying molecular abnormalities, including gene expression changes. Observations have suggested that fasciculation and elongation protein ζ-1 (FEZ1) may be implicated in the pathogenesis of schizophrenia. Nevertheless, our current knowledge of the expression of FEZ1 in peripheral blood of schizophrenia patients remains unclear. The purpose of this study was to identify the characteristic gene expression patterns of FEZ1 in peripheral blood samples from schizophrenia patients. We performed quantitative reverse-transcriptase (qRT-PCR) analysis using peripheral blood from drug-free schizophrenia patients (n = 29) and age and gender-matched general population controls (n = 24). For the identification of FEZ1 gene expression patterns, we applied a comparative threshold cycle (CT) method. A statistically significant difference of FEZ1 mRNA level was revealed in schizophrenia subjects compared to healthy controls (p = 0.0034). To the best of our knowledge, this study is the first describing a down-regulation of FEZ1 gene expression in peripheral blood of patients with schizophrenia. Our results suggested a possible functional role of FEZ1 in the pathogenesis of schizophrenia and confirmed the utility of peripheral blood samples for molecular profiling of psychiatric disorders including schizophrenia. The current study describes FEZ1 gene expression changes in peripheral blood of patients with schizophrenia with significantly down-regulation of FEZ1 mRNA. Thus, our results provide support for a model of SZ pathogenesis that includes the effects of FEZ1 expression.
Yong-Jie Yang, Zeng-Shan Liu, Shi-Ying Lu, Pan Hu, Chuang Li, Waqas Ahmad, Yan-Song Li, Yun-Ming Xu, Feng Tang, Yu Zhou and Hong-Lin Ren
ends (RACE), and then analysed tissue distribution and differential expression levels of OaCdc42 mRNA by RT-qPCR between infected sheep challenged with B. melitensis and sheep vaccinated with B . suis S2. Our aims were to discover potential diagnostic biomarkers to discriminate vaccinated sheep from those infected with virulent Brucella . Material and Methods Bacteria B . melitensis (smooth virulent strain, Bm) was isolated from naturally infected sheep. B . suis S2 (live rough avirulent strain, S2) was purchased from the Harbin Pharmaceutical