Study On The Detection Of Salivary Exosomes Tumor Markers By Digital PCR Chip

Background:Tumor is one of the most threatening diseases to human life.Early detection and early diagnosis are of great significance for timely early treatment of tumors,reduction of the incidence of tumor death,and improvement of tumor prognosis.Exosomes,as nano-vesicles with lipid bilayer structure secreted by cells,exist in various body fluids such as blood,saliva,urine,cerebrospinal fluid,etc.It is an important pathway for intercellular communication.It participates in the body’s immune response,organ development,reproductive function and other physiological activities,as well as the formation and progression of cancer,cardiovascular disease,viral transmission and other pathological activities.Salivary exosomes are derived from saliva and their contents are mostly similar to plasma and are a truly non-invasive and easily sampled source of liquid biopsy samples.Some studies have shown that tumor markers in salivary exosomes are not only closely related to maxillofacial tumors,but also play an important role in the detection and prognosis monitoring of some distal tumors.In addition to common coding RNAs,recent studies have shown that the expression of long non-coding RNA（lncRNA）is also closely related to the occurrence and development of tumors.However,the amount of lncRNA in salivary exosomes is small and difficult to detect by conventional methods.Therefore,this thesis designed and prepared a digital PCR chip based on microfluidic method.Through the screening of target gene primers and the specificity of probe specificity,the detection of lung cancer-specific tumor marker lncRNA in salivary exudates was detected.It provides a new idea for the establishment of a new highsensitivity,high-specific detection method for salivary exosomes tumor markers.Methods: 1)Fabrication of chip: multi-layer lithography technology is adopted to make templates based on silicon chips.PDMS are then poured onto silicon wafers and heated to solidify.The PDMS chip is tightly bonded to the glass slide by means of heat bonding.2)dPCR chip sensitivity test: The H1975 cell cDNA was gradient diluted to different concentrations for detection,Analyze the linear range of the chip and the sensitivity of the detection.3)Saliva sample collection and treatment: Saliva samples are collected into a 50 ml centrifuge tube and the collection process should be placed on ice throughout.Extraction of salivary exosomal RNA was performed using QIAGEN’s exoRNeasy Serum/Plasma Midi Kit kit,and the resulting exosomal RNA was stored at-80 ℃.4)Salivary exosomal RNA reverse transcription: According to the instructions provided by the SuperScript III First-Strand Synthesis System kit for reverse.The reverse transcribed cDNA was stored at-80℃.5)Primer efficiency and specificity verification: The specificity of the primers of the target gene was verified by LightCycler 480 II and Biorad gel electrophoresis.Screening of target gene primers and improvement of probe specificity were completed.6)Comparison of qPCR and dPCR detection results: The same samples were selected for qPCR and dPCR detection respectively,and the detection results of the two methods was compared.7)Quantitative analysis of lncRNA in salivary exosomes: The microfluidic chip was used to detect the difference in expression of target genes in salivary exosomes of tumor and control populations,and double-tailed t-test was used for analysis.Results: 1)We designed a digital PCR chip based on microfluidic method for the detection of lncRNA in salivary exosomes.The chip is mainly composed of three parts: the inlet port area,the micro-chamber area and the sample outlet area.The micro-chambers and the inlet and outlet ports are connected by micro-pipes to form a whole.The PDMS chip was fabricated by photolithography,and then the chip was bonded to the slide by thermal bonding.Through testing,the minimum detection limit of the chip is 10 copies/μL.To meet the needs of the detection of saliva exosome lncRNA.2)The microfluidic chip was used to detect the expression of lung cancer tumor markers in saliva exosomes.The specificity and efficiency of target gene primers and probes were tested by qPCR.Based on previous studies,two lung cancer biomarkers,lncRNA uc011 cly.2 and NR₀46326,were detected and the results were consistent with the outcomes of plasma exosomes in the same period,and the expression of target genes was significantly different in tumor and healthy control groups,P < 0.001.We also compared the dPCR results with qPCR,which showed that the microfluidic chip had higher sensitivity.Conclusion:There is an important link between tumor markers in salivary exosomes and the development and progression of distant tumors.We explored the correlation between lncRNA in salivary exosomes and lung cancer by exploring the digital PCR chip method based on microfluidic method.These results indicate that microfluidic chips can be used for the detection of salivary exosomes,and the detection of lung cancer-specific markers in salivary exosomes provides a new strategy for the detection of lung cancer biomarkers.