Amplification-Based Arrays

The microarray options listed above typically display lower sensitivity than quantitative PCR (qPCR). This observation led to the development of amplification-based arrays, whereby RNA was reverse-transcribed and quantification of miRNA content was performed using quantitative PCR. Three different methods of reverse transcription have been applied. The use of an miRNA-specific stem-loop reverse transcription (RT) primer and a pool of RT primers in a multiplex reaction is particularly popular. The RT primer/ miRNA complexes that are formed display extremely high specificity due to the presence of the stem-loop structure. The cDNA that is generated following extension at the 3' end of the miRNA is then loaded on a 384-well fluidic card that contains a panel of qPCR primers and TaqMan probes for the miR- NAs of interest. A qPCR reaction is run on a thermocycler, and the amplification values are calculated for the entire set of miRNAs. To increase the sensitivity of the assay, a preamplification step can be included. This platform has been widely applied in screening circulating miRNAs, and its sensitivity was further demonstrated in assessing the miRNA pools in the HDL fraction that harbour very low miRNA content (Mitchell et al. 2008; Vickers et al. 2011; Zampetaki et al. 2010). Additionally, it has higher sensitivity than hybridization-based assays as demonstrated by the lower false-positive rate of differential miRNA expression in the qPCR arrays compared to microarrays (Chen et al. 2009).

Alternatively, stem-loop RT primers can be substituted with an LNA-based system of reverse transcription (Arroyo et al. 2011) that includes addition of a poly(A) tail and the use of a polyT primer with a 3' degenerate anchor and a 5' universal tag. Subsequently, the cDNA template can be amplified using miRNA-specific LNA forward and reverse primers in a SYBR Green-based qPCR reaction that can provide higher sensitivity (Jensen et al. 2011; Mestdagh et al. 2014). Similar approaches are also available in other platforms with the omission of LNA analogues and the use of specifically formulated buffers to enhance specificity of the reaction or reliance on adapter ligation at the 5' end and universal RT primer at the 3' end to ensure accuracy and specificity in a single reaction.

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