tailieunhanh - Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

important regulators of biological processes in animals and plants. MiRNAs regulate gene expression at the posttranscriptional level by binding to mRNAs and either inhibit translation or modify the stability of the mRNA. Due to the important biological role of miRNAs it is of great interest to study their expression level in the cells. Furthermore, miRNAs have been associated with cancer and other diseases [1] and miRNA expression can help in the diagnosis and prognostic of human disease [2,3]. The discovery of miRNAs in blood and their surprisingly high stability holds great promise for diagnosis of human disease with miRNAs as biomarkers [4]. Several studies have shown that the amount of individual miRNAs in. | Balcells et al. BMC Biotechnology 2011 11 70 http 1472-6750 11 70 BMC Biotechnology METHODOLOGY ARTICLE Open Access Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers Ingrid Balcells1t Susanna Cirera2t and Peter K Busk 3 Abstract Background MicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings. Results We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94 . The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification. Conclusions MiR-specific quantitative RT-PCR with DNA primers is a highly specific sensitive and accurate method for microRNA quantification. Background MicroRNAs miRNAs are small non-coding RNAs that are important regulators of biological processes in animals and plants. MiRNAs regulate gene expression at the posttranscriptional level by binding to mRNAs and either inhibit translation or modify the stability of the mRNA. Due to the important biological role of miRNAs it is of great interest .

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• MicroRNAs
• BMC Biotechnology
• cDNA synthesis
• synthetic templates
• Design of PCR primers
• Quantitative PCR
• BMC Pediatrics
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