Abstract
Since the discovery of microRNAs (miRNAs), different approaches have been developed to label, amplify and quantify miRNAs. The TaqMan® technology, provided by Applied Biosystems (ABIs), uses a stem-loop reverse transcription primer system to reverse transcribe the RNA and amplify the cDNA. This method is widely used to identify global differences between the expression of 100s of miRNAs across comparative samples. This technique also allows the quantification of the expression of targeted miRNAs to validate observations determined by whole-genome screening or to analyze few specific miRNAs on a large number of samples. Here, we describe the validation of a method published by ABIs on their web site allowing to reverse transcribe and pre-amplify multiple miRNAs and snoRNAs simultaneously. The validation of this protocol was performed on human muscle and plasma samples. Fast and cost efficient, this method achieves an easy and convenient way to screen a relatively large number of miRNAs in parallel.
Highlights
MicroRNAs are recently discovered small non-coding RNAs (∼22 nucleotides) regulating protein expression in animals and plants (Bartel et al, 2004). miRNAs can alter cellular function by binding the 3 -UTR of target mRNA and inhibit the expression of the corresponding protein by either repressing protein translation or promoting mRNA degradation (Krol et al, 2010). miRNAs can be highly and enriched in specific tissues and each miRNA can target multiple mRNA species (Lim et al, 2005; Sood et al, 2006)
Skeletal muscle is a highly plastic tissue able to adapt its size, structure and function in response to various internal and external stimuli, such as acute exercise, hypoxia, and training. miRNAs have been recently identified as novel, essential regulators of skeletal muscle health (Zacharewicz et al, 2013) and may account for specific regulation of muscle growth and differentiation (Buckingham and Rigby, 2014). miRNAs localization is, not restricted to cells and some miRNAs produced in cells are secreted in the bloodstream (Chen et al, 2008). miRNAs are reported to be highly stable in both plasma and serum (Mitchell et al, 2008) and circulating miRNAs expression is altered in pathological conditions
The protocol described in this manuscript is based on a fixed volume of room temperature (RT) product to be used in the polymerase chain reaction (PCR) reaction
Summary
MicroRNAs (miRNAs) are recently discovered small non-coding RNAs (∼22 nucleotides) regulating protein expression in animals and plants (Bartel et al, 2004). miRNAs can alter cellular function by binding the 3 -UTR of target mRNA and inhibit the expression of the corresponding protein by either repressing protein translation or promoting mRNA degradation (Krol et al, 2010). miRNAs can be highly and enriched in specific tissues and each miRNA can target multiple mRNA species (Lim et al, 2005; Sood et al, 2006). MiRNAs are reported to be highly stable in both plasma and serum (Mitchell et al, 2008) and circulating miRNAs expression is altered in pathological conditions. Skeletal muscle is a highly plastic tissue able to adapt its size, structure and function in response to various internal and external stimuli, such as acute exercise, hypoxia, and training. Their role in circulation is not yet clear, miRNAs are promising biomarkers for the diagnostic of various pathologies, injuries and health conditions (Chen et al, 2008; Baggish et al, 2011; Zampetaki et al, 2012b)
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