Methodological Study On Rolling Circle Amplification Detection Of MiR-93-5p

Objective:This study aimed to develop a rapid and simple method for the direct detection of miR-93-5p using rolling circle amplification technology.At the same time,compared with the results of RT-qPCR detection of miR-93-5p,the detection sensitivity of the two methods was investigated.Detection of specific differences.In order to obtain the expected test results,different factors such as the secondary structure of the circular template were investigated.The detection of miR-93-5p was taken as an example to investigate the effect of different secondary structure lock probes on the rolling circle amplification.An optimal lock probe strand was screened for detection of miR-93-5p.Methods:1?Preparation of related reagents.2?The RNA structure software is designed with different secondary structure lock probes.3?RT-qPCR.4?RCA.5?Agarose gel electrophoresis.6?Polyacrylamide gel electrophoresis.7?Cell culture.Results:1?The optimal lock-type probe used in this study is a lock-type probe with no secondary structure.1.2?The optimal concentration of the cyclization link and the ligation sequence is 500nmol/L.3?The optimal time for the cyclization connection is 4 h.4?The optimal digestion time of the cyclized product was 9 h.5?The sensitivity range of RCA detection of miR-93-5p is1pM~10~4pM.6?The sensitivity range of PCR detection of miR-93-5p is 10 pM~10~4 pM.7?RCA and PCR methods have good specificity for detection of miR-93-5p.8?Both RCA and PCR methods can detect miR-93-5p from actual samples.Both methods are suitable for actual detection.Conclusion:1?A constant temperature detection platform based on rolling circle amplification was established,and a new technique for detecting miR-93-5p was developed,which was quick,simple,sensitive,specific,semi-quantitative and cost-effective.2?We used two methods of RCA and PCR to detect miR-93-5p.The system compared the sensitivity and specificity of RCA and PCR to detect miR-93-5p.Among them,the detection range of the RCA method is1pM~10~4pM,and the detection range of the PCR method is 10pM~10~4pM,and the detection sensitivity of the RCA method is one order of magnitude higher than that of the PCR method.Both RCA and PCR methods have good specificity and can specifically detect miR-93-5p.