When comparing the endogenous human and Arabidopsis synthetic miRNAs, there appeared to be no systematic differences in the performance of the Arabidopsis miRNAs compared with the endogenous human miRNAs. For example, the majority of the variability measurements for the synthetic miRNAs were comparable to those of the human miRNAs (Table 2), despite the samples being independently spiked in each of the three replicates. Furthermore, the percent yields of miRs-159a and -172a after short RNA enrichment were within the range of the human miRNAs; miR-394a showed the lowest yield after enrichment (15.41% when measured with the Life Technologies assay, 12.55% with the Exiqon assay, Table 2) showing that this miRNA is at the edge of the range in percent yield, but is not dramatically different from the average value (25%), when considering the spread in values for all of the miRNAs. These findings demonstrate that the Arabidopsis spike-ins represent effective experimental controls for the analysis of endogenous human miRNA expression when performing the short RNA enrichment procedure. Interestingly, Cui et al. (38) showed that miR-159a also serves as an effective spike-in control for RNA extraction of plasma samples, demonstrating its general suitability for RNA isolation procedures. Other previous studies have also assessed the use of other non-mammalian spike-in miRNAs to control for technical variability associated with the entire RNA extraction process (8, 32, 36, 37). For example, Kroh et al. (37) used a pool of three C.elegans miRNAs to control for technical variation in sample extraction by spiking into plasma or serum after protein denaturation treatment. As some researchers have expressed concerns over the suitability of endogenous serum and plasma miRNAs as internal controls (37), synthetic non-mammalian spike-in miRNAs may be a method of choice for future studies. Therefore, further work is required to optimize the usage of miRNA spike-ins to control for technical variability in total RNA isolation and short RNA enrichment procedures, including the establishment of the most effective methods of sample normalization and the most suitable number of spike-in miRNAs to use. For example, whereas Kroh et al. (37) and Mitchell et al. (8) used multiple spike-in miRNAs, other researchers have used single spikes (32, 38).
In order to ensure specific amplification of miRNA targets, we employed a range of negative controls with each miRNA assay: reverse transcription no template control (RT NTC) reactions containing the reverse transcriptase enzyme but no template to evaluate DNA and RNA contamination or non-specificity; PCR NTCs to evaluate DNA contamination during PCR set up; carrier only reactions to evaluate non-specificity; and reactions containing no reverse transcriptase enzyme (no RTs) to evaluate DNA contamination throughout the reverse transcription and PCR procedures. For three of the Exiqon assays (let-7a and –c and miR-394a), there was a low level of background signal in several of the negative control groups, typically Cq >35 (Supplementary Table S2). Derivative melt curve analysis of these assays showed that the peaks for the negative controls were generally distinct from the experimental samples (Supplementary Figure S2), suggesting non-specific amplification. The Life Technologies assays generally showed an absence of any consistent signal in the no RT controls and PCR NTCs for all of the miRNAs, but of note there was a presence of signal in the RT NTCs. One of the Life Technologies assays (miR-159a) consistently showed a signal in the RT NTCs (Supplementary Table S2), but not in the corresponding DNA controls, indicating the amplification of a potentially contaminating source at the RNA level or non-specificity by the reverse transcriptase. This finding is of note as RT NTCs are not routinely performed in addition to no RT and PCR NTCs in many laboratories. These findings indicate that RT NTCs are an important control that should be included for miRNA RT-qPCR reactions in order to determine background levels of signal associated with the reverse transcriptase step as well as the PCR.
Comparison of two prominent miRNA RT-qPCR technologies, Life Technologies’ Taqman miRNA Assay (20) and Exiqon's miRCURY LNA Universal RT microRNA PCR assay, revealed that both assays displayed similar efficiencies but that the latter generated more variable measurements. The two assay technologies yielded significantly different copy number estimations for some of the miRNAs despite using the same standard curve templates for copy number interpolation, indicating that RT-qPCR technology can have a significant impact on the miRNA measurement. The external Arabidopsis spike-ins provided a useful process control for assessing technical sensitivity as they can be spiked at a known copy number and do not display significant cross-reactivity with any human miRNAs. Furthermore, the performance characteristics of the Arabidopsis miRNAs were comparable to those of the endogenous human miRNAs, in terms of the effects of the enrichment procedure and RT-qPCR technology. As miRNA profiling typically involves a series of steps that are sensitive to technical manipulations, the Arabidopsis spike-in miRNAs provide a robust method for the standardization of procedures for within or cross-platform comparisons. We have also demonstrated that short RNA enrichment of human total RNA material can result in a significant 4-fold reduction in miRNA signal when comparing equivalent volumes of total RNA and enriched material. The enrichment procedure had a variable effect on the miRNAs that were analyzed, resulting in a change in relative miRNA levels compared with the non-enriched material. These findings suggest that miRNA data from total and short RNA preparations may not be directly comparable.
This work was funded by the UK National Measurement System. We thank Alison Devonshire for critical reading of the manuscript.
The authors declare no competing interests.
Address correspondence to Nicholas Redshaw, LGC Limited, Queens Road, Teddington, Middlesex, UK. E-mail: [email protected]
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