Quantification of Circulating miRNAs
Quantitative Real-Time PCR
Quantitative real-time PCR (qPCR) remains the gold standard for validating the data derived from miRNA profiling experiments. Several characteristics of the mature miRNAs required the establishment of novel protocols for reverse transcription. The mature miRNAs are small in size, lack a poly(A) tail and encompass sequences that are present in the precursor transcripts and in the genomic DNA. Additionally, the reverse transcription system should be highly sensitive and able to discriminate among related miRNAs that only differ in a single nucleotide.
The use of stem-loop primers and the TaqMan technology led to the development of a very sensitive and accurate platform for miRNA validation. The stem-loop structure forms an RT primer/mature miRNA chimaera that extends the 3' end of the miRNA. The derived cDNA is the template for a TaqMan qPCR reaction that includes an miRNA-specific forward primer, a reverse primer and a dye-labelled TaqMan probe (Chen et al. 2005; Mestdagh et al. 2008). Compared to conventional linear primers, stem-loop RT primers show better specificity and sensitivity, probably due to the base stacking and spatial constraint of the stem-loop structure. This may also prevent binding to double-stranded genomic DNA molecules (Chen et al. 2005). Multiplexing assays that include pools of stem-loop primers are now available and can be applied to facilitate the simultaneous reverse transcription of multiple mature miRNAs. An additional step of preamplification of the RT product consisting of 10-14 cycles of PCR amplification can be included to increase the sensitivity of detection. A forward primer specifically designed for the target miRNA and a reverse universal primer are used in the preamplification reaction. Although there is an excellent correlation in the expression levels for the most abundant miRNAs obtained with or without preamplification, some variation is observed for the low-abundant miRNAs (Mestdagh et al. 2008). The directionality of the differential gene expression though is consistent, indicating that preamplification products are suitable for the analysis of relative miRNA expression. Further studies confirmed that the systematic bias does not prohibit the comparison of relative miRNA levels between samples (Linsen et al. 2009).
Besides stem-loop primers, polyadenylation of the mature miRNAs by poly(A) polymerase has been successfully applied for the reverse transcription of mature miRNAs. Oligo-dT primers that have a 3' degenerate anchor and a universal tag sequence on the 5' end are subsequently used to obtain the cDNA. This system is used in combination with SYBR Green to enable sensitive and specific quantification of mature miRNAs by qPCR. The combination of polyadenylation and the addition of a universal tag eliminate the risk of detecting genomic DNA (Tijsen et al. 2010; Wang et al. 2009, 2010). In a similar manner, LNA analogues can be introduced to the primers to increase specificity and detection efficiency. In this case, cDNA is synthesised using an miRNA-specific RT primer and a sensitive stable reverse transcriptase. Subsequently, the cDNA is amplified by qPCR using SYBR Green. LNA be incorporated in miRNA-specific primers in both the RT and qPCR reaction (Arroyo et al. 2011; Bryant et al. 2012; Jensen et al. 2011). This can increase PCR sensitivity and amplification efficiency and is particularly useful in multiplexing to specifically target the mature miRNAs of interest (Ballantyne et al.
2008). However, care must be taken with the design of LNA primers, as the number and position of LNA bases can substantially influence the outcome of the amplification and result in false positives (Latorra et al. 2003; Levin et al. 2006).