Development Of A SYBR Green â…  FQ-PCR With Stem-loop RT Primer To Detect MiR-155 And Its Clinical Primary Application

ObjectiveTo establish a method of SYBR Green I FQ-PCR with stem-loop RT primer for detecting miR-155. Detect miR-155 in CD4+T cell, HepG 2 cell, LO2 cell and SP2/0 cell with SYBR Green I real time quantitative PCR to primarily validate the feasibility of this method. Make a collection of specimens scraped from the cervix and detect their HPV gene type respectively. Detect miR-155 in these specimens respectively with this method. Primarily analyse the relation of miR-155 and cervix cancer and HPV gene types.Methods1,Design stem-loop RT primer refer to Caifu Chen. Detect miR-155 in CD4+T cell, HepG 2 cell, LO2 cell and SP2/0 cell with SYBR Green I real time quantitative PCR to validate the feasibility of this method. Analyze the melting curve simultaneously to evaluate the specificity of the test.2,Detect miR-155 in 60 cell specimens scraped from the cervix using this method. Analyse the relation of miR-155 and cervix cancer and HPV gene types. The PCR production was analyzed by agarose gel electrophoresis.Results1,The results showed that the SYBR Green I FQ-PCR writh stem-loop RT primer was highly specific and had a broad linear detecting range (10-1nmol/L～10-6 nmol/L, R=0.99). The melting curve analysis showed a single peak. The results of CD4+T cell, HepG 2 cell, LO2 cell, SP2/0 cell and the 60 cell specimens scraped from the cervix in this assay showed positive. The melting curve analysis showed a single peak and the PCR production migrated as a pure species during electrophoresis on 2% agarose gel.2,The expression of miR-155 in cell specimens scraped from the cervix is not related to their HPV gene types(P>0.05) but related to cervix cancer(P<0.05)in this study. It was reported for the first time.Conclusions The method of SYBR Green I FQ-PCR with stem-loop RT primer can detect miR-155 rapid, sensitively and specifictly for cell lines and cell specimen scraped from the cervix. It lays a foundation to further study the relationship between miR-155 and the mechanism of cervical cancer development in order to serve miR-155 as an early gene maker.