Title: Development and clinical primary application of SYBR Green I FQ-PCR with stem-loop RT primer to detect miR-155
Abstract: Objective To establish a method of SYBR GreenⅠreal-time fluorescence quantitative polymerase chain reaction(FQ-PCR) with stem-loop reverse transcription(RT) primer to detect miR-155 and apply it primarily.Methods miR-155 in CD4+T cell was determined by SYBR GreenⅠreal-time FQ-PCR.The feasibility of this method was primarily validated.miR-155 in 60 cell specimens scraped from the cervix was detected.The relation of miR-155 with cervix cancer and HPV genotypes was analyzed.The PCR production was analyzed by agarose gel electrophoresis.Results The results showed that the SYBR GreenⅠFQ-PCR with stem-loop RT primer was highly specific and had a broad linear detecting range(10-1nmol/L-10-6 nmol/L,the lowest detecting limit: 10-7 nmol/L,r=0.99).The melting curve analysis showed a single peak.The results from CD4+ T cell lines and 60 cell specimens scraped from the cervix in this assay showed positive.The PCR product migrated as a pure band during electrophoresis.The expression of miR-155 in cell specimens scraped from the cervix was not related to their HPV genotypes in this study(P0.05),but was related to cervix cancer(P0.05).Conclusions The method of SYBR GreenⅠFQ-PCR with stem-loop RT primer can detect miR-155 rapidly,sensitively and specifically in cell lines and cell specimens scraped from the cervix.It lays a foundation to further study on the miR-155 and its clinical application.
Publication Year: 2010
Publication Date: 2010-01-01
Language: en
Type: article
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Cited By Count: 2
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