DYE-FREE ANALYSIS OF MIRNA PROFILING BY SEQUENCE-TAGGED STEM-LOOP REVERSE TRANSCRIPTION PCR COUPLED WITH PYROSEQUENCING
We have developed a novel low-cost and dye-free method for microRNA (miRNA) profiling, using sequence-tagged stem-loop reverse-transcription (RT) followed by PCR and quantitative pyrosequencing analysis. This method includes three steps: reverse transcription, PCR and pyrosequencing. First, miRNA was used for the synthesis of cDNA with miRNA- and source-specific reverse transcription primers consisting of a tail near the 5-end for supplying a universal PCR priming site, a source-specific sequence in the middle, and miRNA specific bases in the 3-end for annealing the miRNA strand. Second, after pooling sequence-labeled cDNAs from different sources, PCR was performed to amplify the templates. Third, pyrosequencing was applied for decoding and quantifying the source-specific sequences tagged in the amplicons. The signal ratio in the pyrogram is proportional to the amounts of miRNA among different sources. This assay is sensitive to femtomolar concentration of miRNA (40 fM). Using this method, precise quantification of mir-16 among different tissues of a mouse is achieved. This method enables fast, accurate, and sensitive expression profiling of miRNA from different sources.
miRNA dye-free sequence-tag stem-loop reverse transcription
Jing Hua Song Qinxin Zhou Guohua
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China Huadon
国际会议
The 6th International Forum on Post-genome Technologies(6IFPT)(第六届国际后基因组生命科学技术学术论坛)
北京
英文
277-281
2009-09-17（万方平台首次上网日期，不代表论文的发表时间）