Measuring microRNAs: Comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods
© Ach et al; licensee BioMed Central Ltd. 2008
Received: 28 March 2008
Accepted: 11 September 2008
Published: 11 September 2008
Determining the expression levels of microRNAs (miRNAs) is of great interest to researchers in many areas of biology, given the significant roles these molecules play in cellular regulation. Two common methods for measuring miRNAs in a total RNA sample are microarrays and quantitative RT-PCR (qPCR). To understand the results of studies that use these two different techniques to measure miRNAs, it is important to understand how well the results of these two analysis methods correlate. Since both methods use total RNA as a starting material, it is also critical to understand how measurement of miRNAs might be affected by the particular method of total RNA preparation used.
We measured the expression of 470 human miRNAs in nine human tissues using Agilent microarrays, and compared these results to qPCR profiles of 61 miRNAs in the same tissues. Most expressed miRNAs (53/60) correlated well (R > 0.9) between the two methods. Using spiked-in synthetic miRNAs, we further examined the two miRNAs with the lowest correlations, and found the differences cannot be attributed to differential sensitivity of the two methods. We also tested three widely-used total RNA sample prep methods using miRNA microarrays. We found that while almost all miRNA levels correspond between the three methods, there were a few miRNAs whose levels consistently differed between the different prep techniques when measured by microarray analysis. These differences were corroborated by qPCR measurements.
The correlations between Agilent miRNA microarray results and qPCR results are generally excellent, as are the correlations between different total RNA prep methods. However, there are a few miRNAs whose levels do not correlate between the microarray and qPCR measurements, or between different sample prep methods. Researchers should therefore take care when comparing results obtained using different analysis or sample preparation methods.
MicroRNAs (miRNAs) are small (~18–24 nucleotides) non-coding RNAs which bind to mRNAs to regulate protein expression, either by blocking translation and/or by promoting degradation of the mRNA target (reviewed in [1–3]), or alternatively by increasing translation [4, 5]. They have been found to be involved in numerous functions such as cell fate determination, cell proliferation, cell differentiation, and cell death (reviewed in [6, 7]). Profiles of miRNAs in various types of tumors have been shown to contain potential diagnostic and prognostic information (reviewed in [8, 9]). The number of known miRNAs has rapidly increased in recent years, and currently there are 722 human miRNA sequences reported in the Sanger Institute's miRNA database release 10.0 (miRBase) [10–12], with potentially many more yet to be reported [13, 14].
Several methods for global miRNA profiling are currently in common use. These include quantitative RT-PCR (qPCR) involving stem-loop RT primers combined with TaqMan PCR (Applied Biosystems) analysis [15, 16], qPCR with locked nucleic acid primers (Exiqon) , qPCR using poly(A) tailing (QIAGEN, Stratagene) [18, 19], high-throughput sequencing of small RNA libraries , and microarray analysis (for examples, see [21–26]). Typical experimental workflows often involve using different methods of measuring miRNAs at different research stages. For this reason, it is important to know how well the different measurements agree with each other. Several groups have compared microarray profiling results with those obtained by quantitative PCR for either a small number of genes or a small set of samples [21, 22, 26–30]; however there has been no systematic comparison of larger numbers of miRNAs across a widely diverse range of human tissues using the two methods.
In this study, we compared the relative expression of 61 different miRNAs across nine different human tissues, measured using both Agilent miRNA microarrays and TaqMan qPCR. The Agilent microarray platform features the direct end-labeling and profiling of mature miRNAs from total RNA without any size fractionation or amplification to minimize experimental loss, bias, or variations [31, 32]. The labeling reaction is performed under denaturing conditions to provide high labeling yield, minimal sequence bias , and consistently reproducible efficiency for every miRNA sequence [31, 32]. By incorporating hairpin structures in the microarray probe, base-pairing with the additional nucleotide incorporated during labeling, and empirical melting point-determination, the platform is capable of single-nucleotide discrimination in the miRNA sequences while specifically distinguishing the mature miRNAs from longer RNAs in the total RNA sample [26, 31, 32]. We chose to compare this microarray system against the Taqman qPCR system in particular, since at the time this work was performed this was the most commonly utilized miRNA qPCR system. We found excellent correlation between the microarray and PCR results for most of the miRNAs. We further examined two of the miRNAs showing low correlations by using spiked-in synthetic RNAs, and found that differential sensitivity between the two techniques is not the cause of the discrepancy.
Another factor which could potentially affect the results of an miRNA profiling study is the method used to isolate RNA from the biological sample. Both the Agilent microarray system and the TaqMan qPCR systems use total RNA as the starting material; however, it is unclear whether different total RNA preparation methods will yield systematically different miRNA profiling results. In this report, we compared the results of miRNA microarray profiling obtained with three different commonly used total RNA prep methods. We found that the results for most miRNAs were equivalent among the different sample preparation methods, but that measured levels of a small number of miRNAs differed systematically.
Results and discussion
Quantitative RT-PCR and Agilent microarray miRNA profiles correlate strongly
We previously reported that a comparison of Agilent microarray profiling and SYBR green-based quantitative RT-PCR (qPCR) of ten miRNAs in seven different human tissues found the two measurements correlated quite well . To perform a more extensive comparison, we analyzed the expression of 61 human miRNAs in nine different tissues (brain, breast, heart, liver, placenta, testes, ovary, skeletal muscle, thymus), using both Agilent miRNA microarrays and TaqMan stem-loop qRT-PCR . Aliquots of the same RNA samples were used for both the microarray and qPCR measurements. We chose these particular 61 miRNAs for several reasons. First, they represent a wide range of expression levels, as determined in an initial array analysis of some of the tissues. Second, they have wide differences in GC content, ranging from 23% (miR-190) to 68% (miR-328). Third, we chose several miRNAs which had potentially problematic sequences or exhibited atypical behavior during the development of the Agilent microarray platform: two of these did not show as good a linear titration curve as other miRNAs tested in a previous study (miR-126*, miR-296) , and two other miRNAs were previously reported not to be labeled by enzymatic methods similar (but not identical) to that used with the Agilent microarray assay (miR-208, miR-219) .
Of the 61 miRNAs examined, only miR-637 was not detected by either method in any of the tissues. The rest of the miRNAs assayed were detected in most or all of the tissues by both methods, with two exceptions: miR-208, expressed only in the heart  and at very low levels in skeletal muscle, and miR-138, expressed in the brain, and at lower levels in placenta and thymus (all data is shown in Additional File 1).
Measurements of spike-ins of miRNAs which systematically differ between platforms show linear sensitivity
Three different total RNA preparation methods show similar yields and quality
A question which arises when comparing the miRNA profiling results reported in different studies is whether the methods used to isolate RNA from tissue or cell line samples systematically affect the miRNA profiles. To examine whether the miRNA profile of a sample is affected by the type of total RNA prep method used, we prepared one large frozen cell pellet from each of two different human cell lines, HeLa (a cervical carcinoma line) and ZR-75-1 (a breast carcinoma line). We then subdivided these pellets into equal aliquots, and performed total RNA isolation on the aliquots using three different techniques: phenol/guanidinium (TRIzol, Invitrogen) followed by isopropanol precipitation, and two column-based techniques, miRNeasy (QIAGEN) and mirVana (ABI). Four to eleven replicate preps were performed on each cell type with each method.
List and characteristics of sample preps from HeLa and ZR-75-1 cell pellets.
No. of Preps
RNA Yield Mean (SD)
260:280 Mean (SD)
260:230 Mean (SD)
RIN Mean (SD)
Variability of hybridization results is highest between different prep methods
For miRNA microarray profiling analysis we took three of the replicate total RNA preps of each different prep method in each cell type and hybridized them to Agilent miRNA microarrays. A total of 42 hybridizations were done, with all but two samples hybridized at least twice (Additional File 16).
A good measure of the reproducibility of replicate measurements is the Root Mean Square (RMS) deviation of the natural logs of all signals that are well above background levels. The RMS deviation is approximately equal to the coefficient of variation of signals, and is an estimate of the proportional error of the measurement. For example, if the RMS deviation is 0.15 (15%), then a measured fold change between samples of 1.15 is a difference of one standard deviation.
We also plotted the RMS deviations for hybridizations involving prep replicate pairs from each of the three different prep methods (regardless of the hybridization day), in order to examine whether the different prep methods showed different amounts of variation between prep replicates (Figure 7b). The TRIzol prep replicates showed less variability than the other two prep methods. A Student's t-test between pair-wise comparisons of the three prep methods confirmed that this difference is statistically significant (data not shown). There was no significant difference in variability between the mirVana and miRNeasy prep replicates.
A small subset of miRNAs differ between prep methods
Grand means of the mean total gene signal for all hybridizations of each prep type.
Mean TGS (SD)
In this study we compared the expression levels of 61 miRNAs in nine human tissues as measured by both Agilent microarrays and TaqMan qRT-PCR. We found that 53/60 expressed miRNAs had correlations (R) > 0.9 between the two methods. For the two miRNAs that differed most between the two methods, spike-in studies found the differences are not due to differential sensitivity of the two methods, but are more likely due to interference from other RNAs in the complex mixture.
We also examined microarray-based miRNA profiles using three different total RNA sample prep methods. We found that while almost all miRNA levels correspond between the three different prep methods, a small subset of 2–10 miRNAs consistently differ by greater than 2-fold between different techniques. These differences were corroborated using qPCR, and are most likely due to true differences in the miRNA content of the extracted RNA. Thus, while all three methods are suitable for use in profiling miRNAs from total RNA, it may be prudent to pick one method and use it for the entire course of any particular study, in order to avoid these small profile differences due to the RNA preparation method.
Total RNA and cell samples
Total RNA samples from normal human tissues were from Ambion (Austin, TX). Frozen HeLa cell pellets were from Cell Trends (Middletown, MD), and frozen ZR-75-1 cell pellets were from BioProcessing Inc. (Portland, ME).
miRNA microarray analysis
miRNA microarrays were manufactured by Agilent Technologies (Santa Clara, CA)., and contain 20–40 features targeting each of 470 human miRNAs (Agilent design IDs 015508 (sample prep studies) and 016436 (nine tissue comparison studies)) . Sequences of the 470 miRNAs were obtained from the Sanger miRBase, release 9.1 [10–12]. Labeling and hybridization of total RNA samples were performed according to the manufacturer's protocol. 100 ng total RNA was used as input into the labeling reaction, and the entire reaction was hybridized to the array for 20 hours at 55°C. For the microarray versus qRT-PCR comparisons, the labeling and hybridizations of the nine human tissues were done 4–5 times, and the mean and standard deviation for each miRNA were calculated.
Microarray results were extracted using Agilent Feature Extraction software (v184.108.40.206) and analyzed using GeneSpring GX 7.3.1 software (Agilent Technologies) and Spotfire DecisionSite 8.1 software (TIBCO Software, Palo Alto, CA). Box plots were calculated using JMP 5.1 software (SAS, Cary, NC). Original microarray data is deposited in the Gene Expression Omnibus  (Series GSE11879).
All scatter plots of miRNA microarray data use the total gene signal, which is proportional to the total number of targets bound by the probes targeting each miRNA [31, 32]. For comparison of two hybridizations, the natural logs of the total gene signals for all genes expressing above 10x the background noise in both samples were regressed against each other, and the standard deviation of the residuals from the regression line were reported as the RMS deviation. For most pairs of samples prepared by the same method, residuals were normally distributed, so that the RMS deviation describes true random variation in the assay. In pairs of samples prepared by different methods, residuals of most of the miRNAs were also normally distributed, with systematic exceptions of some miRNAs as discussed in the text. No normalization was performed for either microarray or qPCR data, except for an overall intensity normalization applied to the average signals from different prep methods, as described in the text (Figures 8 and 9). For this comparison, the 75th percentile of the total gene signal for all the miRNAs on the array was calculated by sorting the total gene signals for 470 miRNAs on the array in ascending order, and the signals from the three methods were normalized to the signal from the 353rd miRNA.
miRNA qRT-PCR analysis
miRNA qRT-PCR analysis was performed using Taqman miRNA assays (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol. 5 ng total RNA was input into each reverse transcription reaction (RT) for each miRNA. Four replicates were done for each miRNA, consisting of two replicate PCR reactions from each of the two replicate RT reactions, and the results were averaged. PCR reactions were run on a 7500 Real Time PCR machine (Applied Biosystems) and analyzed using 7500 System SDS software (v1.4).
Synthetic miRNAs were manufactured by TriLink BioTechnologies (San Diego, CA) and spiked into human liver and placenta total RNA (Ambion). 100 ng of these RNA mixes were then used for labeling and hybridization onto the microarrays, while 5 ng were used as input into the reverse transcriptase reaction for qPCR. Two replicate microarray hybridizations and four replicate qPCR reactions were done for each dilution in each tissue.
Total RNA sample preps
Frozen cell pellets were resuspended in phosphate buffered saline and divided into equal aliquots of 5 × 106 (HeLa) or 1 × 107 (breast) cells and refrozen. Individual aliquots were subsequently thawed just before use.
TRIzol preps were performed according to the manufacturer's protocol (Invitrogen, Carlsbad, CA) using an isopropanol precipitation. Briefly, 1 ml of TRIzol reagent was added to the cell pellet and cells were lysed by repetitive pipetting, and then incubated at room temperature for 5 minutes. 200 μl of chloroform were added, followed by vigorous shaking and incubation for 2–3 minutes at room temperature. Samples were centrifuged 15 minutes at 12000 × g at 4°C. The aqueous layer was transferred to a new tube, and the RNA was precipitated by adding 0.5 ml isopropanol, incubating 10 minutes at room temperature, and spinning for 10 minutes (12000 × g at 4°C). Pellets were washed with 80% ethanol and resuspended in nuclease-free dH2O (Ambion).
miRNeasy total RNA preps (QIAGEN, Valencia, CA) were performed according to the manufacturer's protocol. The mirVana miRNA Isolation kit (Applied Biosystems) was used according to the manufacturer's protocol for total RNA isolation.
All total RNA preps were analyzed using the 2100 Bioanalyzer (Agilent Technologies), RNA 6000 Nano LabChip kits, and 2100 expert software (version B.02.05.SI360). Absorption spectra were measured on an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).
We thank Dr. Laurakay Bruhn for carefully reading and reviewing the manuscript. This work was supported by Agilent Technologies.
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