| Objective: Epilepsy is a chronic recurrent transient brain dysfunction syndrome,WHO is reported that the average prevalence rate of global active is about 4-10 ‰,and in developing countries, the proportion reached 6-10 ‰.At present,th there are about 50 million [1] epileptic patient all over the world, 9 million people in China,progressive increased four hundred thousand patients every year.The burden of Epilepsy for the patients and their family is heavy. Diagnosis of epilepsy at present is mainly on the basis of detailed clinical history, neurological examination,electroencephalogram(EEG), neuroimaging, family history, etc., but epilepsy is a kind of a seizure disorder, in attack period patients can be completely normal, and the history given by the patients and their family is often not enough detailed, so there are still some difficulties in diagnosis, so looking for early diagnosis specific biomarkers for epilepsy diagnosis of objective, accurate and convenient detection is one of the important problems needed to be resolved. Micro RNA(mi RNA) is a small single-stranded, non-coding RNAs. in recent years, the main researches in mi RNA were involved in tumor and other diseases, even in diseases of CNS, In epilepsy, mi RNAs have almost been studied in brain tissues and in animals’ circulation, but not in circulation of human. while the mi RNA has remarkable stability in the blood, and will change along with the fluctuation of the body’s physiological and pathological conditions, so our team speculated that micrornas in peripheral blood of epilepsy patients, there may be different expression which will become a potential biomarkers in early diagnosis of epilepsy and have an important application prospects.Methods:1. Initial screen: extract two groups of objects(50 cases each with case group and control group) early in the morning on an empty stomach peripheral Blood 2ml, applicate kit PAX- gene Blood micrornas kit(Qiagen) for the extraction of mi RNA, extraction process carried out in accordance with the kit instructions completely.Each sample to return 20 u L RNA solution, and mix respectively by the case group and the control group of mixed RNA 1 ml. Mix two groups of RNA withHiseq2000 high throughput sequencing to analysis case group and control group in whole blood all mi RNAs expression profiles.2. Training phase: Extracting a single sample of mi RNA 50 cases of case group and control group each, extraction method with initial screening.Using reverse transcription Kit One Step Prime Script mi RNA c DNA short Kit(Takara, Japan)reverse transcription of c DNA.Further more amplificate mi RNAs for early to screen out the differences of expression.3. Validation: Take the difference in complex screen of mi RNA of a single sample in large sample cases(100 cases control group, 100 cases) for further verification.Methods are same as the secondary screen.4. Data StatisticsResult: 1.the clinical features of the study population: Whole blood micrornas high-throughput sequencing showed a total of 200 kinds of micrornas in the two groups of whole blood expression differences statistically significant.2,Discovery phase using high-throughput sequencing :we found that compared with the control group, cases group exist 10 kinds of differentially expressed mi RNA:including the levels of expression of let- 7 f- 5 p, mi R- 106-5 p, b- 130- a- 3 p-146- a- 5 p, 126-5 p, novel- mi R- 164 rise significantly, mi R- a- 5 p, 15-194-5 p- 144-5 p- 181- c- 5 p lowered significantly.3,Secondary screening by q RT- PCR: The Results show that the expression of let- 7 f- 5 p, p- 130- a- 3, 5 p- 146- a-, 126-5 p, novel- mi R- 164 levels rise significantly, mi R- 194-5 p, the expression of 5 p- 181- c- level significantly lowered. Consider the simplicity and economy of clinical operation, we think the diagnostic value of mi R- 126-5 p is highest, which expected to be potential biomarkers in the diagnosis of epilepsy.Conclusion :This study first analysed mi RNAs genome-wide express spectrum of whole blood in patients with epilepsy, and selected 5 kinds of mi RNAs changeable expression, including let- 7-5 p, f- 130- a- 3 p- 146- a- 5 p- 126-5 p and mi R-181- c- 5 p,particularly, mi R- 126-5 p, with its high sensitivity and specificity, is expected to become the biological markers of epilepsy diagnosis.
|
PDF Full Text Request