Establishment Of RT-qPCR Assay For Hsa-miR-124a-3p In Plasma And Its Efficiency Evaluation In Prenatal Screening For Down Syndrome

Down syndrome(DS),which is the most common cause of neonatal mental and developmental defect in hunman,is a kind of genetic disease with the incidence rate about 1/700 in neonate.Ds children not only have mental deficiency but aslo are associated with heart disease,mental and physical retardation,and abnormality in a variety of organs and tissues.Unfortunately,there is no effective treatment for DS at present.Therefore,to screen the fetus with DS and avoid born of the fetus with DS is the most effectual prevention for DS.Prenatal diagnosis for DS is mainly classified into noninvasive and invasive testing,in which noninvasive testing includes DS screening and noninvasive DNA detection,and noninvasive inspection mainly includes amniocentesis,chorionic villus sampling(CVS)and cordcentesis.At present,Down’s screening is the most widely used method of DS prenatal screening.Its safety is high,but the specificity is low.Noninvasive DNA detection is costly,and the period of the test is long,at present which is mainly applied in the third-party medical organizations.Amniocentesis,CVS and cordcentesis obtain the fetal cells with invasive manipulation for Karyotype Analysis of Chromosomes.Although its specificity is high,but the invasive manipulation mainly rely on operators’ experience,and there is a 1% possibility risk of leading to the miscarriage after the invasive manipulation.Therefore,the establishment of a security,price-reasonable and specific prenatal detection mean is still the groping direction in the area of prenatal diagnosis,and microRNA(miRNA),which is a kind of common blood molecule as like cffDNA,begins to attract people’s attention.The miRNA is a kind of noncoded RNA molecular,consisting of 20 to 25 base pairs,with main function of regulating the expression of genes.miRNAs could bear the degradation of the RNA enzyme in blood and stay in circle blood stably for a long term,meanwhile miRNA could resist the etching of strong acids and strong alkali,so that it’s convenient for miRNA test.At same time,miRNA has high specificity of tissues and organs,which make miRNA an ideal circle blood markers of diseases.In former research,we found 135 kinds of moleculars which express different between the plasma of the second trimester of women who conceive healthy babies and Down’s babies,in which the expressive deviation of hsa-miR-124a-3p is the most.Therefore,hsa-miR-124a-3p is selected as the aim molecular to establish a new DS prenatal screening method.This study includes two parts,the first part is to establishment a RT-qPCR assay to detect hsa-miR-124a-3p of plasma,and the second one is a efficiency evaluation of RT-qPCR assay in prenatal screening for DS.Objectives:1.To construct and optimize the hsa-miR-124a-3pRT-qPCR assay.2.To approch the clinical value of hsa-miR-124a-3p assay in prenatal screening for Down syndrome.Methods:The hsa-miR-124a-3p was selected as the aim molecular to design and synthetise its reverse transcription primer and standards.Meanwhile,TaqMan? Small RNA assays and Taq Man? master mix with UNG were used to construct the RT-qPCR testing for hsa-miR-124a-3p assay.Then,the reaction system and conditions of RT-qPCR were optimized and evaluated.36 samples of healthy fetus maternal plasma and 11 samples of DS fetus maternal plasma were collected to detect the expression level of hsa-miR-124a-3p in plasma by hsa-miR-124a-3pRT-qPCR assay.All test results were statistically analyzed to evaluate the clinical values between RT-qPCR assay and Down’s screening。Results:1.The reverse transcription primer of hsa-miR-124a-3p for RT-qPCR testing was designed and synthetized and 100pmol/μL was used as its best working concentration.2.The standard substance of hsa-miR-124a-3p was synthetized and its quality was mearsured.3.The standard curve of RT-qPCR testing for hsa-miR-124a-3p was established with hsa-miR-124a-3p standards,which was evaluated to accord with requirement.4.The hsa-miR-124a-3pRT-qPCR assay was constructed,optimized and evaluated,1×10-2 to 1×10-6pmol/μL,1×10-8pmol/μL and 35 were identified as its linear range,lower limit of detection,and the cycles in which the assay is specific separately,lastly the reproducibility of the assay was proved well.5.Expression levels of hsa-miR-124a-3p in the maternal plasma of DS fetus and healthy fetus were mearsure and compared separately,and the early study result was verified.6.10-5 to10-3pmol/μL was identified as the concentration to predict the high risk of DS.7.A comparision between hsa-miR-124a-3pRT-qPCR assay and Down’s screening was carried on to find that the value of hsa-miR-124a-3pRT-qPCR assay to predict the high risk of DS was better.Conclusions:1.RT-qPCR assay was successfuly constructed to detect plasma hsa-miR-124a-3p quantitatively.2.Up-regulated expression of hsa-miR-124a-3p in the maternal plasma of DS fetus abnormaly,strongly suggested that hsa-miR-124a-3p may be a new specific blood markers for DS.3.10-5 to10-3pmol/μL was successfuly identified as the concentration to predict the high risk of DS,which laid the groundwork for application of hsa-miR-124a-3p in DS prenatal screening.4.Clinical value of hsa-miR-124a-3pRT-qPCR assay to predict the high risk of DS was proved better than Down’s screening.