High-throughput amplification of mature microRNAs in uncharacterized animal models using polyadenylated RNA and stem-loop reverse transcription polymerase chain reaction.

This study makes a significant advancement on a microRNA amplification technique previously used for expression analysis and sequencing in animal models without annotated mature microRNA sequences. As research progresses into the post-genomic era of microRNA prediction and analysis, the need for a rapid and cost-effective method for microRNA amplification is critical to facilitate wide-scale analysis of microRNA expression. To facilitate this requirement, we have reoptimized the design of amplification primers and introduced a polyadenylation step to allow amplification of all mature microRNAs from a single RNA sample. Importantly, this method retains the ability to sequence reverse transcription polymerase chain reaction (RT-PCR) products, validating microRNA-specific amplification.

[1]  Kyle K. Biggar,et al.  MicroRNA Regulation in Extreme Environments: Differential Expression of MicroRNAs in the Intertidal Snail Littorina littorea During Extended Periods of Freezing and Anoxia , 2012, Genom. Proteom. Bioinform..

[2]  K. Storey,et al.  Akt signaling and freezing survival in the wood frog, Rana sylvatica. , 2013, Biochimica et biophysica acta.

[3]  Kyle K. Biggar,et al.  HIF-1α regulation in mammalian hibernators: role of non-coding RNA in HIF-1α control during torpor in ground squirrels and bats , 2012, Journal of Comparative Physiology B.

[4]  Kyle K. Biggar,et al.  Evidence for cell cycle suppression and microRNA regulation of cyclin D1 during anoxia exposure in turtles , 2012, Cell cycle.

[5]  N. Rajewsky,et al.  Deep conservation of microRNA-target relationships and 3'UTR motifs in vertebrates, flies, and nematodes. , 2006, Cold Spring Harbor symposia on quantitative biology.

[6]  Kyle K. Biggar,et al.  Amplification and sequencing of mature microRNAs in uncharacterized animal models using stem-loop reverse transcription-polymerase chain reaction. , 2011, Analytical biochemistry.

[7]  Kyle K. Biggar,et al.  Differential Expression of Mature MicroRNAs Involved in Muscle Maintenance of Hibernating Little Brown Bats, Myotis lucifugus: A Model of Muscle Atrophy Resistance , 2012, Genom. Proteom. Bioinform..

[8]  Jing Zhang,et al.  Regulation of p53 by reversible post-transcriptional and post-translational mechanisms in liver and skeletal muscle of an anoxia tolerant turtle, Trachemys scripta elegans. , 2013, Gene.

[9]  P. Karlovsky,et al.  Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum , 2011, Analytical and bioanalytical chemistry.

[10]  Kyle K Biggar,et al.  The emerging roles of microRNAs in the molecular responses of metabolic rate depression. , 2011, Journal of molecular cell biology.