The Applications Of Capillary Electrophoresis In The Determination Of MiRNAs From Some Plants' And Animals' Cells

Capillary electrophoresis(CE),also known as High-performance capillary electrophoresis(HPCE),is a widely used separation technique.It is widely used in the separation of samples of biology and medicine.Due to its high efficiency,rapidity,and low sample consumption,it is very suitable for the separation of easily decomposable compounds such as microRNAs(miRNAs).MiRNA is a series of endogenous non-coding small RNAs and it plays a key role in gene expression.The occurrence of many diseases including cancers has close relationship with the abnormal expression of specific miRNAs.At present,miRNA has gradually become an important biomarker for disease diagnosis.MiRNA analysis technique plays a key role in the miRNA research and drug development process.Therefore,research on miRNA separation and high-sensitivity detection technology has a very important significance in clinical diagnosis in the future.This paper is mainly composed of the following chapters:In the first chapter,the research history,separation modes,detection methods,sample injection methods and on-line enrichment techniques of CE are introduced and reviewed.In addition,miRNAs and the analysis methods were also introduced.In the second chapter,three miRNAs,miRNA-156,miRNA-159 and miRNA-166 were separated and detected by non-gel sieving high-speed capillary electrophoresis(HSCE)with laser-induced fluorescence detection(LIF).Three strategies,namely disloading of laser light feedback,field amplification and high-voltage electrokinetic injection,were proposed to improve the detection sensitivity of the HSCE-LIF system.In order to obtain a sufficient resolution,DNA probes are appended with additional nucleotides as tails,giving them different sequence lengths.In a buffer solution of 1 × TBE(pH8.7)containing2.0%PVP and 0.4%HEC,the complexs of the three miRNAs could be separated within 3.2 min when applied with 3000V ×3 s injection voltage and 3000V separation voltage.The detection limits for the three miRNAs were 0.051,0.011,and 0.25 nmol/L respectively.Finally,banana leaves sipked with standard miRNA were used as samples to be tested.The results showed that HSCE-LIF with no-gel sieving mode is a very effective tool for the separation and detection of miRNAs.In the third chapter,we combined nucleic acid isothermal hybridization chain reaction(HCR)with HSCE-LIF to determine two low-content miRNAs,miRNA-21 and miRNA-31,in lung cancer cells(A549).The unreacted monomers(hairpin structure)were centrifuged off by using an ultrafiltration tube and the reaction product was preserved and analyzed by HSCE-LIF.Under the optimal experimental condition:a short capillary with an effective length of 4 cm,buffer solution containing 1 × TBE(pH 9.0),1.5%PVP and 0.3%HEC,injection voltage 3000 V,injection time 3 s,separation voltage 2500 V,the product peaks could be completely combined and separated within 1 min.SPSC(pH7.4)was used as the reaction buffer for HCR and the hybridization time was 4 h.The detection limits of miRNA-21 and miRNA-31 were 1.6×10-14mol/L and 1.1×1014mol/L respectively.This method was successfully applied to determine miRNAs in the cell A549.In the fourth chapter,a miniature sample reservoir on a piece of copper was developed.The sample reservoir could be used to preserve sample solution with volume 1-10 microliter.HSCE-LIF was used to separate and detect miRNAs in papaya leaves under no-gel sieving mode.The sample reservoir and the buffer reservoir were integrated on the same copper because the two reservoirs were grounded during the separation under no-gel sieving CE mode.The detection sensitivity was improved by the methods of field amplification and high voltage electrokinetic injection.Using the optimized buffer solution,namely 1 × TBE(pH8.8)containing 2.0%PVP and 0.6%HEC,the two miRNAs,miRNA-156 and c2 could be completely separated within 4 min under 3400 V separation voltage.The detection limits for them were 0.25 nmol/L and 0.11 nmol/L respectively.Finally,the method was applied to determine miRNAs spiked into the sample of papaya leaves.The recoveries were in the range of 82.3%-91.8%.The results showed the method was reliable.