title: A novel real-time PCR assay of microRNAs using S-Poly(T), a specific oligo(dT) reverse transcription primer with excellent sensitivity and specificity.
abstract: BACKGROUND
  MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases.
  
  
  RESULTS
  A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)(11) sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518.
  
  
  CONCLUSIONS
  With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers.
authors.affiliation: College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, People's Republic of China.
authors.forename: Kang
authors.initials: K
authors.lastname: Kang
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Zhang
authors.forename: Hongtao
authors.initials: H
authors.lastname: Liu
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Wang
authors.forename: Jiasheng
authors.initials: J
authors.lastname: Zhong
authors.forename: Zhenting
authors.initials: Z
authors.lastname: Huang
authors.forename: Xiao
authors.initials: X
authors.lastname: Peng
authors.forename: Yan
authors.initials: Y
authors.lastname: Zeng
authors.forename: Yuna
authors.initials: Y
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Jun
authors.initials: J
authors.lastname: Luo
authors.forename: Deming
authors.initials: D
authors.lastname: Gou
mesh_terms: D015687:Cell Hypoxia; D002460:Cell Line; D017931:DNA Primers; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D035683:MicroRNAs / Q000097:blood / Q000235:genetics*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D011071:Poly T; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D035683:MicroRNAs; D011071:Poly T