Rolling Circle Amplification Conbined Real-time PCR For The Detection Of MircoRNA

OBJECTIVES: Combined with real-time PCR technology,we developed a novel detection method called rolling circle amplification(RCA)-PCR.Due to RCA's signal amplification,this method has good sensitivity and selectivity.We also do some applications of this method in our research.METHODS:(1)Develop the stem-loop RT-PCR method to detect miR-200a-3p,and optimize the reaction conditions.(2)Develop the RCA-PCR to detect miR-200a-3p,and optimize the reaction conditions.At the same time we validated the new method.(3)The RCA-PCR method will be compared with the conventional stem-loop RT-PCR method in the sensitivity,selectivity and quantitation range.(4)The miR-200a-3p in total RNA which extracted from Hep G2 ? Huh7 ?BEL7402 ? HT29 ? HL7702 cells will be detected by RCA-PCR and Stem-loop RT-PCR method,respectively.RESULTS:(1)The detection method for miR-200a-3p by stem-loop RT-PCR was established successfully.Under the optimal reaction conditions,the sensitivity has reached 8pmol/L for miR-200a-3p.(2)Under the optimal reaction conditions,the sensitivity of RCA-PCR has reached 1 fmol/L for miR-200a-3p.Then method validation was performed,the results show that the method was in accordance with the method validation requirements.(3)The comparison of stem-loop RT-PCR and RCA-PCR was carried out: ? The quantitative detection range of stem-loop RT-PCR was 10 nmol/L~10 pmol/L,the limit of detection was 8 pmol/L,the lower limit of quantification(LLOQ)was10 pmol/L.? The quantitative detection range of RCA-PCR was 1nmol/L~10 fmol/L,the detection limit of the method was 1 fmol/L,the LLOQ was 10 fmol/L.In conclusion,the new method RCA-PCR was superior to stem-loop RT-PCR in sensitivity,selectivity and quantitation range.CONCLUSIONS: We developed stem-loop RT-PCR and RCA-PCR successfully for the detection of miR-200a-3p.This two methods were also applied to detect the real samples.The results show that the RCA-PCR method can achieve absolute quantification and stem-loop RT-PCR method only can achieve relative quantification for miR-200a-3p.