Development and application of a novel reverse transcription real-time PCR method for miR-499 quantification

Objectives: 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. Design and 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/mu L to 1.0 x 10(6) amol/mu L of synthetic miR-499, we observed a linear correlation (R-2 = 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/mu L. 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. (C) 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.