title: Development and application of a novel reverse transcription real-time PCR method for miR-499 quantification.
abstract: OBJECTIVE
  MicroRNAs (miRNAs) are endogenous small RNAs of 21-25 nucleotides that can pair with sites in 3' untranslated regions in mRNAs of protein-coding genes to downregulate their expression. Recently, miR-499 and other miRNAs released in circulating blood have been reported as promising biomarkers for acute myocardial infarction (AMI). In the present study, we developed a novel reverse-transcription real-time PCR assay for human miR-499 quantification.
  
  
  METHODS
  miR-499 was reverse-transcribed with a 3' portion-specific primer into cDNAs. The cDNAs were further extended with overlap PCR. The extended cDNAs were determined by quantitative, real-time PCR. Synthetic miR-499 was put into the RT reaction over an optimal range to generate standard curves for absolute quantification of miR-499.
  
  
  RESULTS
  In the presence of 0.0001 amol/μL to 1.0×10⁶ amol/μL of synthetic miR-499, we observed a linear correlation (R²=0.999) between the logarithm of the amount of input RNA and the CT value. The miR-499 was reliably measured at a detection limit of 0.0001 amol/μL. The miR-499 measurements in spiked plasma samples indicated excellent correlation between the novel qRT PCR and classic stem loop qRT PCR. The qRT PCR analysis demonstrated that miR-499 was detected with higher levels in plasma from the patient with AMI in acute phase (AMI) compared with those from the control groups (P<0.001).
  
  
  CONCLUSIONS
  We developed a novel reverse-transcription real-time PCR assay for human miR-499 quantification. The good reproducibility and wide linearity range may permit more use of it in the quantification of other human miRNAs in future.
authors.affiliation: Department of Pathology and Laboratory Medicine, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China. Electronic address: zwdin@126.com.
authors.forename: Weidong
authors.initials: W
authors.lastname: Zheng
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Di
authors.forename: Yinghong
authors.initials: Y
authors.lastname: Liu
authors.forename: Ge
authors.initials: G
authors.lastname: Huang
authors.forename: Youwei
authors.initials: Y
authors.lastname: Zheng
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Fang
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry; D002138:Calibration; D018076:DNA, Complementary / Q000032:analysis* / Q000096:biosynthesis; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000097:blood*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
chemical_list: D018076:DNA, Complementary; C548683:MIRN499 microRNA, human; D035683:MicroRNAs
keywords: AMI; Real-time reverse-transcription PCR; miR-499