Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs
© Jensen et al; licensee BioMed Central Ltd. 2011
Received: 26 May 2011
Accepted: 26 August 2011
Published: 26 August 2011
microRNAs (miRNA) are short, endogenous transcripts that negatively regulate the expression of specific mRNA targets. miRNAs are found both in tissues and body fluids such as plasma. A major perspective for the use of miRNAs in the clinical setting is as diagnostic plasma markers for neoplasia. While miRNAs are abundant in tissues, they are often scarce in plasma. For quantification of miRNA in plasma it is therefore of importance to use a platform with high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the performance of three commonly used commercial miRNA quantification platforms: GeneChip miRNA 2.0 Array, miRCURY Ready-to-Use PCR, Human panel I+II V1.M, and TaqMan Human MicroRNA Array v3.0.
Using synthetic miRNA samples and plasma RNA samples spiked with different ratios of 174 synthetic miRNAs we assessed the performance characteristics reproducibility, recovery, specificity, sensitivity and linearity. It was found that while the qRT-PCR based platforms were sufficiently sensitive to reproducibly detect miRNAs at the abundance levels found in human plasma, the array based platform was not. At high miRNA levels both qRT-PCR based platforms performed well in terms of specificity, reproducibility and recovery. At low miRNA levels, as in plasma, the miRCURY platform showed better sensitivity and linearity than the TaqMan platform.
For profiling clinical samples with low miRNA abundance, such as plasma samples, the miRCURY platform with its better sensitivity and linearity would probably be superior.
microRNAs (miRNAs) are short 20-23 nucleotide long non-coding RNAs that are widely distributed in almost all eukaryotic organisms. They have multiple functions however the main function is believed to be post transcriptional regulation of protein levels [1, 2]. While miRNAs are often abundant in tissues, the amount found circulating in body fluids such as plasma and serum is often limited. It has been reported that the total RNA level in plasma is in the range 6-300 ng/ml [3, 4] and that the miRNA fraction constitutes only a few percent of this . The mechanisms regulating secretion of miRNA into circulation is still unclear. Reports have shown that while endogenous miRNAs appear stable in plasma/serum exogenous miRNAs are not, and as a result of this it has been suggested that endogenous circulating miRNAs are either encapsulated in microvesicles or bound to RNA-binding proteins in complexes, e.g. Ago2 and NPM1, protecting them from degradation [6–8]. Detailed knowledge of the biological function of circulating miRNA does not exist, however it has been shown that vesicular miRNAs can be transferred from cell to cell and influence the behavior of the recipient cells .
MicroRNAs have been reported deregulated in various diseases. Independent studies on different tissue materials have shown that miRNA expression profiles differ between healthy and diseased tissue, and various lines of evidence indicate that they have great potential as diagnostic, prognostic, and predictive biomarkers . It is technically demanding to quantify mature miRNAs based on the often low-abundance, short length of mature miRNA, homology between miRNA species, and the inclusion of the mature miRNA sequence in the primary miRNA (pri-miRNA) and precursor miRNA (pre-miRNA) transcripts. The latter makes it difficult to construct assays that are specific for the mature form. Nevertheless, multiple platforms for quantifying mature miRNAs exist, which are most commonly based on either quantitative real-time PCR (qRT-PCR) or microarrays, although alternatives exist . Common to all platforms are that a reverse transcription step is required to convert the RNA into cDNA prior to quantification. To date two different approaches have been utilized for this step. In the first approach, miRNAs are reverse transcribed individually using miRNA-specific reverse transcription primers e.g. stemloop primers. In the second approach, miRNAs are first tailed with a common sequence and then reverse transcribed using a universal primer. Subsequently, the cDNA levels of specific miRNAs are quantified by qPCR or microarray .
The qRT-PCR based platforms promise to be more sensitive than array based miRNA quantification platforms , and their use for analyzing samples with low miRNA levels, such as human plasma, is increasing [13–18]. A few reports have assessed the performance of a number of miRNA quantifying platforms, however they mostly focused on array based platforms and analysis of miRNA rich samples [12, 19–21]. As one of the major perspectives for miRNAs in the clinical setting is the use as diagnostic markers in screening for neoplasia in body fluids, it is very important to evaluate platform performance at the low miRNA levels found in such samples.
This motivated us to evaluate the performance of three commonly used commercial miRNA quantification platforms: GeneChip miRNA 2.0 Array (Affymetrix), miRCURY Ready-to-Use PCR, Human panel I+II V1.M (Exiqon), and TaqMan® Human MicroRNA Array v3.0 (Applied Biosystems Inc. (ABI)). Using plasma RNAs spiked with varying concentrations of 174 synthetic miRNAs we evaluated the specificity, accuracy, linearity, and sensitivity of the platforms.
The research was conducted in accordance with the Helsinki Declaration. Informed written consent was obtained from all patients according to local ethical regulations, and research protocols were approved by the Central Denmark Region Committees on Biomedical Research Ethics (J. no. 1999/4678). One milliliter of plasma was isolated from each of seven blood samples drawn prior to surgery from patients diagnosed with Colorectal Cancer (CRC). The plasma was pooled and total RNA including small RNAs, were purified according to the protocol supplied with the miRNeasy Mini Kit (Qiagen) with the exception that 1 μg MS2 carrier RNA (Roche) was added to the QIAzol Lysis Reagent prior to RNA purification in order to maximize the yield and minimize purification efficiency variation.
Synthetic miRNA samples and plasma RNA samples spiked with synthetic miRNAs
Two pools of 88 (pool A) and 86 (pool B) synthetic miRNAs with sequences corresponding to miRBase v14 were generated (Additional file 1, Table S1). Each pool contained 108 copies of each miRNA/μl dissolved in RNA storage solution buffer (Ambion) supplemented with MS2 carrier in a final concentration 10 ng/μl. These pools were mixed in ratios 1:4 and 4:1 to formulate the two synthetic samples #1 and #2. Synthetic sample #1 contained 0.2*107 copies/μl of the pool A miRNAs and 0.8*107 copies/μl of the pool B miRNAs and vice versa for the synthetic sample #2. Hence, comparison of miRNA quantifications from the two synthetic samples should show four-fold differences for all miRNAs.
To enable evaluation of platform performance on complex samples the synthetic miRNAs were spiked into two RNA aliquots from the pooled plasma RNA preparation described above. Thereby, two novel spiked plasma RNA samples (spiked plasma RNA #1 and #2) having the same concentrations of the synthetic miRNAs as the synthetic samples were generated.
Mapping of platform assays to miRBase v14 and identification of assays targeting the synthetic miRNAs
Complete lists of assays (including the miRBase version used for assay design) on the GeneChip miRNA 2.0 Array (Affymetrix), the miRCURY Ready-to-Use PCR V1.M panels (Exiqon), and the TaqMan® Human MicroRNA Array v3.0 TLDA cards (ABI) were obtained from the manufacturers. All platforms included various assays targeting sequences that could not be mapped to miRBase v14 or targeted non-human sequences. These were excluded from further analysis. In order to identify assays targeting the synthetic miRNAs, the assay target sequences were matched to the list of synthetic miRNAs. This revealed that of the 174 synthetic miRNAs the GeneChip platform contained matching assays for all 174, the miRCURY platform for 143, and the TaqMan platform for 155. In total, 125 of the synthetic miRNAs were represented on all three platforms (Additional file 1, Table S1).
Identification of assays targeting miRNAs with sequence homology to one or more of the synthetic miRNAs
The assay target sequences were aligned to the sequences of the synthetic miRNAs using a Smith-Waterman based sequence alignment algorithm, allowing up to four mismatches and eight nucleotide overhangs. Consequently, we identified assays targeting miRNAs with sequence homology to one or more of the synthetic miRNAs. The alignments are supplied in Additional file 2, Table S2.
RNA input for cDNA synthesis
When cDNA for the individual platforms were generated from the synthetic and spiked plasma RNA samples, the sample input quantities were adjusted such that, unless stated otherwise, the same number of synthetic miRNA copies were added per cDNA reaction (1*106 and 0.25*106 copies for the high- and low-abundance synthetic miRNAs). Similarly, when cDNA was generated from the pure plasma sample (including the no-RT control) the same volume of plasma RNA as for the spiked-plasma RNA samples was used (corresponding to 1/6 of the RNA extracted from 250 μl plasma). By this approach we enabled direct comparison of the miRNA quantities estimated from the cDNAs.
Duplicate aliquots of all investigated RNA samples were used for two separate reverse transcription reactions and the products of each reaction was used in separate qPCR amplifications. This enabled evaluation of reproducibility, of the reverse transcription and subsequent qPCR amplifications.
GeneChip assay setup
RNA was labeled using the 3DNA Array Detection Flash Tag RNA Labeling Kit (Genishere), according to manufacturers recommendations. First, poly(A) tailing was carried out at 37°C for 15 min in a volume of 15 μl reaction mix, which contains 1× Reaction Buffer, 1.5 μl 25 mM MnCl2, 1 μl 1:500 diluted ATP Mix and 1 μl PAP enzyme. Second, FlashTag Ligation was performed at room temperature for 30 min by adding 4 μl of 5× FlashTag Ligation Mix Biotin and 2 μl T4 DNA Ligase into the 15 μl of reaction mix. 2.5 μl of Stop Solution was added to stop the reaction. Samples were hybridized, washed and scanned with an Affymetrix Scanner.
miRCURY LNA™ Universal RT microRNA PCR setup
cDNA synthesis and real-time qPCR was performed using the miRCURY LNA™ Universal RT microRNA PCR system (Exiqon, Denmark) according to the manufacturers instructions. In brief, the RNA were tailed with a poly(A) sequence at their 3'end and then reverse transcribed into cDNA using a universal poly(T) primer with a 3'end degenerate anchor and a 5'end universal tag. The cDNA products were subsequently diluted 125 fold and transferred to the Ready-to-use microRNA PCR Human Panels (I + II) and quantified using SYBR green based real time PCR and LNA enhanced miRNA specific primers. The qPCRs were run on a 7900HT thermocycler (ABI) using the thermal-cycling parameters recommended by Exiqon. Raw Ct values were calculated as recommended by Exiqon using the RQ manager software v1.2.1 (ABI) with manual settings for threshold and baseline, i.e. all miRCURY assays were analyzed using a ΔRn threshold of 60 and baseline subtraction using cycles 1-14.
TaqMan® Human MicroRNA Array Set v3.0 setup
cDNA synthesis, pre-amplification, and real-time qPCR was performed as described in the protocol associated with the TaqMan® Human MicroRNA Arrays Set v3.0 (ABI). In brief, RNA was reverse transcribed using Megaplex RT Stemloop primers (pool A or B) and the TaqMan miRNA reverse transcription kit. For optimal sensitivity ABI recommends inclusion of a pre-amplification step. In this step the product of the reverse transcription reaction was pre-amplified using Megaplex PreAmp primers (pool A or B) and TaqMan PreAmp Master Mix. Finally, the pre-amplification product was diluted as indicated by the manufacturer and loaded onto the TaqMan A or B Array. The arrays were run using a 7900HT thermocycler (ABI). Raw Ct values were calculated as recommended by ABI using the RQ manager software v1.2.1 (ABI) with automatic baseline and threshold settings.
Statistical analysis and sequence alignment
Variance comparison test, Poisson randomness test, Mann-Whitney U test or Fisher's exact test were applied to assess differences or proportions in the obtained data . P-values < 0.05 were considered significant. Reproducibility and linearity were evaluated using Pearson correlation coefficients. The statistical analyses were carried out using STATA v10.1 (Statacorp), Excel 2007 (Microsoft), or the open source R-software http://www.r-project.org/. Sequence alignments were performed using a Smith-Waterman based sequence alignment algorithm .
In a pilot study evaluating the amount of plasma RNA input necessary to detect miRNAs we found that the qRT-PCR based platforms (TaqMan from ABI and miRCURY from Exiqon) reproducibly detected miRNAs using inputs ranging from all the RNA extracted from 250 μl plasma down to as little as 1/100 of this input (data not shown). In order not to assess the platforms at the brink of their sensitivity it was decided that for the platform evaluation the input per cDNA synthesis should be 1/6 of the RNA from 250 μl plasma. The GeneChip miRNA 2.0 Array platform from Affymetrix repeatedly failed to produce reliable signals at this input level (data not shown). This was not surprising, as the total amount of RNA isolated from 250 μl plasma was well below the minimum requirement of 100 ng total RNA stated in the manual for the platform. Consequently, due to the limited sensitivity the GeneChip platform was excluded from further assessment.
The number of mismatches to homologous synthetic miRNAs impacts assay cross-reaction tendency¤.
Number of nucleotide mismatches to synthetic miRNA*
Number of assays§
Number of false positive assays (n)#
False positive rate (false positives/number of assays) (%)
Assessment of platform reproducibility, Pearson correlation of duplicate measurements§.
All assays detected
The 125 spike-in miRNAs for which both platforms have assays
r (n detected)
r (n detected)
r (n detected)
r (n detected)
Spiked plasma RNA #1
Spiked plasma RNA #2
Having revealed that both platforms have difficulties recovering the expected fold change for a small subset of miRNAs we speculated if these poorly recovered miRNAs were the same for both platforms. This was assessed by testing if the differences (observed - expected fold change) of the two platforms were correlated. The analysis found no correlations in neither the synthetic nor the spiked plasma samples (r~0, Pearson correlation coefficient) (Additional file 3, Figure S2), indicating that the problematic miRNAs are different for the two platforms. Altogether, the recovery assessment indicates that the majority of the assays on both platforms are capable of detecting four-fold copy number differences and that the miRCURY platform appears to perform slightly better than the TaqMan platform. Further experiments are needed to evaluate if recovery is equally good at fold changes less than four-fold, however this is beyond the scope of the present study.
Many processing steps, each potentially introducing variation, are required to quantify miRNA transcript levels in biological samples, including plasma. To enable evaluation of reproducibility duplicate aliquots of all investigated RNA samples were used for two separate reverse transcription reactions and the products of each reaction was used in separate qPCR amplifications. A total of five RNA samples were investigated: synthetic RNA samples #1 and #2, spiked plasma RNA samples #1 and #2 and pure plasma RNA. The comparison of every duplicate pairs demonstrated median Pearson correlation coefficients of 0.985 and 0.952 for the miRCURY and TaqMan platforms, respectively (Table 2). As already mentioned, a few outlier assays with low reproducibility were observed for the TaqMan platform in the analysis of the synthetic samples (Additional file 3, Figure S1). Since the assays performed otherwise successfully this may relate to the specific LDA cards used for the synthetic samples rather than to the assays themselves. To facilitate comparison of the reproducibility measures for the two platforms, the analysis was repeated and this time restricted to the 125 spike-in miRNAs for which assays were present on both platforms. The same pattern was observed indicating that data produced by the miRCURY platform was slightly more reproducible than the TaqMan platform (Table 2).
Sensitivity and linearity
To enable assessment of sensitivity and linearity of the platforms, a five point 10-fold dilution series of spiked plasma RNA sample #1 were generated. For the miRCURY platform this translates to 5000/20000 (pool A/pool B miRNAs), 500/2000, 50/200, 5/20, and 0.5/2 template copies added per final PCR amplification at each of the five dilution points assuming that all the pre-PCR steps applied were 100% efficient. Even though the same RNA inputs were also used for the TaqMan cDNA syntheses, similar estimates of the input copy numbers for the final PCRs cannot be calculated as the TaqMan cDNA was pre-amplified.
Assessment of sensitivity and reproducibility at different miRNA input levels, spanning five orders of magnitude, Pearson correlation of duplicate measurements§.
The 125 spike-in miRNAs for which both platforms have assays
miRNA copies pr. PCR amplification
r (n detected)
r (n detected)
As noticed during the analysis of the synthetic samples, the TaqMan platform had a few outliers (n = 5) in the duplicate measurements in the second dilution point. Again, these appeared to be caused by a few individual wells in one of the LDA cards amplifying poorly (data not shown). When these assays were excluded the Pearson correlation increased from 0.85 to 0.99 (Table 3), indicating that the TaqMan platform at this input level generates highly reproducible measurements.
This study reports the results of an evaluation of the performance of three commonly used commercially available miRNA quantification platforms. The focus was particular on performance in relation to minute levels RNA input, i.e. in the range of what can be extracted from 250 μl of human plasma. Consistent with previous reports we found qRT-PCR based platforms to have higher sensitivity than microarray based platforms . In fact in our hands the GeneChip miRNA 2.0 platform was not sensitive enough to reliably produce signals with the plasma RNA input levels studied. We nevertheless continued assessing the two remaining qRT-PCR platforms.
Using samples with known miRNA contents we were able to show that at abundant miRNA levels the technical reproducibility and sensitivity of these platforms was good and comparable. However, at low miRNA levels, particularly at 50/200 copies or below, the sensitivity of the miRCURY platform was significantly higher than for the TaqMan platform. Consistent with this we found that the number of miRCURY assays with a high degree of linearity (r2 ≥ 0.9) across four log scales of miRNA copies was significantly higher than for the TaqMan platform. Our evaluation of the two platforms' ability to recover four-fold differences revealed that at the investigated miRNA concentrations both platforms have an acceptable recovery. However, based on the better sensitivity and linearity of the miRCURY platform it is likely that at lower miRNA concentrations the results of a similar recovery analysis would have been in favor of the miRCURY platform. We did not address recovery of fold-changes less than four-fold, but expect that the performance of both platforms will decline with decreasing fold-changes.
Considering the inclusion of the reported sensitivity improving pre-amplification step in the TaqMan protocol it was surprising to find that at low miRNA levels the sensitivity and linearity of the miRCURY platform was better than the TaqMan platform. This could indicate that the sensitivity boosting effect of the pre-amplification step is less prominent than reported . However, it should be noted that other differences between the platforms also exist, and alternatively, these could also play a role for the better sensitivity of the miRCURY platform. For example, for cDNA synthesis the miRCURY platform uses a universal approach with poly(A) end-tailing and oligo(dT) primed reverse transcription, while the TaqMan approach megaplexes > 300 miRNA specific stem-loop primers for initiation of reverse transcription. Potentially the universal approach may be more robust and sensitive than the megaplexed approach. Another possibility is the inclusion of LNAs in the miRCURY primer designs. LNAs make assay design nearly independent of miRNA GC content and compensates for many of the compromises one otherwise would have to make with a short miRNA target sequence of just ~22 nucleotides. Noticeably, It has previously been reported that inclusion of LNAs improves both PCR specificity and sensitivity .
We also assessed the specificity of the platforms and strikingly noticed that while the TaqMan platform generally showed no false positives in the no-RT control nearly 10% of the miRCURY assays were positive (even though detected at late Ct's, > 38). This could indicate that the miRCURY platform is less specific than the TaqMan platform. However, our analyses of the synthetic samples revealed that within the operative range, i.e. from the first detection to the detection threshold, the two platforms produce nearly identical numbers and rates of false positives (Figure 1). Hence, for practical purposes the specificity of the two platforms appears equal. Importantly, the number of false positives produced by both platforms increased exponentially with increasing detection thresholds. Hence, for both platforms the detection threshold should be chosen with great care to obtain acceptable false discovery rates when profiling biological samples. Sequence similarity between miRNAs has been reported to be potential cause of false positive detections [11, 26]. Consistent with this our analyses indicated that a significant fraction of the false positives, for both platforms, in particular at low Ct's was related to sequence homology (Figure 2). The latter is critical as this is also the detection range of the true positives making it practically impossible to distinguish the false from the true positives. As expected we found an inverse relationship between the number of nucleotide mismatches and the likelihood of a false positive detection (Table 1). While this was observed for both platforms the relationship was more pronounced for the miRCURY than the TaqMan platform. Along the same line the fraction of non-homology related false positives was larger for the TaqMan than the miRCURY platform (Figure 2). We do not understand the basis of the non-homology related false positives, but we perceive non-homology related amplification as more unspecific than homology related. In summary, our specificity evaluations indicate that the LNA based miRCURY platform compared to the stem-loop based TaqMan platform performs poorer in no-RT controls and slightly better, in terms of sequence specificity, in template containing samples. With both platforms it seems prudent to keep homology in mind when interpreting data from clinical samples and it may be advisable to consider carefully the likelihood of assay cross-reaction before taking a particular miRNA further, e.g. to in vitro studies.
We do not find it likely that the specificity issues of qRT-PCR will ever be completely eliminated; however, considering the ongoing technological shift to small RNA sequencing the issue may also soon be alleviated. The advantage of sequencing is that it is not hindered by variability in melting temperatures, coexpression of nearly identical miRNA family members, or post-transcriptional modifications. Moreover, it enables identification of novel miRNAs and unlike qRT-PCR the validity of sequencing results are not sensitive to changes in the miRNA sequences registered in miRBase. Currently the minimal input requirements for most small RNA liberary preparations are limiting for the use of the technology for profiling low abundance miRNA samples, such as human plasma. However, multiple approaches are being pursued to bring input requirements down, and the results are promising . Other limitations include the RNA ligation and the PCR amplification steps in the library construction protocols both of which bear inherent biases . Furthermore the tools for computational analysis of next generation sequencing data are still in their infancy. However, the technology has the potential to replace qRT-PCR as the preferred tool for profiling low abundance miRNA samples.
For now though, our data indicate that of the three tested platforms, the miRCURY platform with its better sensitivity and linearity in the low miRNA concentration range should be the platform of choice for analysis of low abundance miRNA samples. For analysis of samples with high miRNA abundance both qRT-PCR-platforms can be used, and probably also the GeneChip platform. However, when choosing platform one should keep in mind the poorly amplifying well phenomena we observed for some samples with the TaqMan platform. It caused artificial outlier measurements in the affected wells and while these outliers relatively easy can be identified by replication this approach may be prohibited by the availability of sample material and/or funding.
For the analysis of samples with abundant miRNAs - as can often readily be obtained from most tissue samples - both the TaqMan and miRCURY platforms will most likely yield good results. However, for studying samples with low miRNA levels, such as plasma, our data indicate that it probably would be beneficial to use the miRCURY platform due to its better sensitivity and linearity in the low miRNA concentration range. Future application of the platforms on plasma from e.g. cancer patients will clarify whether miRNAs form a new reproducible family of molecules to be used for cancer diagnosis and follow-up.
List of abbreviations
Applied Biosystems Incorporated
complementary DeoxyriboNucleic Acid
- (GC content):
Guanine and Cytosine content
Inter quartile range
Locked Nucleic Acid
Low Density Array
precursor microRiboNucleic Acid
primary microRiboNucleic Acid
quantitative Polymerase Chain Reaction
quantitative Reverse Transcription-Polymerase Chain Reaction
Acknowledgements and funding
This work was supported by the Danish National Advanced Technology Foundation, the John and Birthe Meyer Foundation, the Danish Council for Independent Research Medical Sciences, the Danish Council for Strategic Research, and the Lundbeck Foundation.
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