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Major depressive disorder (MDD) is the most frequent type of mental illnesses. Current predictions show that depression will be the leading cause of disease burden by the next decade. MDD affects around 10% of population and almost 30% of individuals referring to health care centers demonstrate depression related symptoms. Diagnostic methods such as DSM-IV and ICD-10 are not able to precisely diagnose MDD from other psychiatric disorders due to overlapping symptoms. Therefore, use of biomarkers as an accurate diagnostic tool is of high importance in clinical diagnosis for depression.
Emerging evidence show that MDD as a complex disease is caused by both environmental and genetic factors. Several investigations have reported the causal pathological and biological mechanisms related to depression. However, the underpinning molecular mechanisms of MDD are still unclear. In the last decade, small non-coding RNAs have emerged as master regulators of gene expression exhibiting involvement in pathology of numerous diseases. MicroRNAs (miRNAs), as a subclass of small non-coding RNAs with a length of 18-24 nt, modulate the expression of ~ 90% of genes via different mechanisms. These molecules play a wide variety of regulatory roles in numerous processes, involving cell proliferation and differentiation, development, and programmed cell death. MiRNAs are highly abundant in the brain and engaged in neural functions and development. The involvement of miRNAs in almost every biological activity highlights the difficulties related to finding specific causative genes linked to the etiology of psychiatric disorders.
Dysregulation of miRNAs in the brain has been related to the pathology of neurological and psychiatric diseases. However, transcriptome analysis of the brain samples in psychiatric disorders is invasive and has ethical issues. As such, blood samples have been suggested as a good source for transcriptome profiling in many diseases. The presence of circulating miRNAs in body fluids such as serum and plasma offers a non-invasive diagnostic tool for brain illnesses. Some recent studies have explored the expression of circulating miRNAs in blood samples from patients with depression. However, these studies were conducted on a small number of subjects which could be a significant limitation for their results. Here, we aimed to investigate serum miRNAs as potential diagnostic biomarkers in a large cohort of MDD patients and controls.
This study consisted of 610 MDD patients and 528 age-and sex-matched controls. MDD subject were randomly recruited from multiple healthcare centers (aged from 20 to 60 years). All MDD subjects were diagnosed for major depression by two psychiatrists, according to DSM-IV (the diagnostic and statistical manual of mental disorders, fourth edition, American Psychiatric Association, 2000) diagnostic criteria with a structured interview and were assessed by the Hamilton Depression Rating Scale (HDRS). Control individuals were recruited voluntarily at the Emam Sajad Hospital and were checked for any sign of mental disorder and excluded if positive. Furthermore, patients with a history of severe head injury, other mental disorders, dysaudia, vision disorder, epilepsy, cardiovascular disorders, pregnancy and thyroid disease were excluded. Also, the patients under anti-depression treatment were excluded. All enrolled participants prepared a written signed consent before sample conducting the experiments. The study procedures involving human participation were conducted according to the Declaration of Helsinki and ethical standards and approved by ethics committee of Yasuj University of medical sciences.
Blood samples were collected from patients and controls by use of phlebotomizing and transferred to collection tubes equipped with Clot activator (Sigma Aldrich). To separate serum from cellular components, samples were rotated end-over end at 25 ˚C for 20 min, then were placed at 25 ˚C for 25 min and centrifuged for 15 min at 3000 rpm (640 ×g) at 25 ˚C to isolate serum. To remove remaining circulating cells or debris in the samples, supernatant was again centrifuged at 5000 rpm (1780 ×g). As the next step, the clear supernatant was isolated and carefully transferred into RNase-free microtubes in 300 μl aliquots for miRNA extraction and stored at −80°C. Previously, it has been shown that the presence of lysed red blood cells in serum samples is the major source of variability in the level of miRNAs. Accordingly, all samples were then checked by a microplate reader spectrometer (Thermo Fisher Scientific) at a wavelength of 414 nm to distinguish non-hemolyzed sera.
RNA extraction and cDNA synthesis
RNA was extracted from 300 μl serum samples using a miRNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. Briefly, serum samples were lysed using QIAzol lysis reagent and Caenorhabditis elegans miR-39 as a synthetic spike-in control was then added as an internal normalizer to the tubes containing lysed samples. Chloroform was equally added and the samples were then centrifuged at 13, 000 rpm for 12 min at 4°C. Next, the clear supernatant was transferred to a new tube and mixed with absolute ethanol, centrifuged, washed, and eluted in 30 μl elution buffer in the collection tube. The quantity of extracted RNAs was analyzed by spectrophotometry.
After RNA extraction, cDNA synthesis was performed using miScript ІІ RT kit (QIAGEN) according to the manufacturer`s protocol. The reaction mixture with a 20 μl final volume contained 2 μl RNA, RT mix, Hispec buffer, dNTPs, and RNase-free water. Next, the prepared mixture was incubated 30 min at 37 ˚C, 60 min at 42˚C, 5 min at 95˚C and then held at 4◦C. The synthesized cDNA samples were kept at −20°C for the next step. miRNA profilingInitially, miRNA screening was performed by use of the Human Neurological Development & Disease miScript miRNA PCR Array (QIAGEN GmbH, Hilden, Germany), including 84 miRNAs which have previously been reported to be involved in the development of the brain and different neurological disorders. The array contained C. elegans miR-39 as an internal normalizer, snoRNA/snRNA (SNORD48, SNORD61, SNORD68, SNORD72, SNORD95, SNORD96A and RNU6-2) as normalization controls for the array data, miRNA reverse transcription control (miRTC) primer assays and positive PCR controls (PPC). In this phase, 214 MDD patients and 205 healthy age-and sex-matched controls were randomly selected for primary miRNA profiling. Next, prepared cDNAs were used for the profiling assay. MiRNAs expression was quantified using SYBR Green qPCR on an ABI 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). C. elegans miR-39 primer assay was used as an internal calibrator for the data sets.
To perform an accurate miRNA profiling, the Ct values from the data sets were normalized to the whole array Ct mean. Relative quantification, as recommended by the manufacturer, was obtained using the 2-ΔCT method. Differential expression of miRNAs was determined between the ADHD and control groups by Student’s t-test; a corrected p-value of <0. 00059 was considered as statistical significant.
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