Target labelling for the detection and profiling of microRNAs expressed in CNS tissue using microarrays
© Saba and Booth; licensee BioMed Central Ltd. 2006
Received: 07 May 2006
Accepted: 12 December 2006
Published: 12 December 2006
MicroRNAs (miRNA) are a novel class of small, non-coding, gene regulatory RNA molecules that have diverse roles in a variety of eukaryotic biological processes. High-throughput detection and differential expression analysis of these molecules, by microarray technology, may contribute to a greater understanding of the many biological events regulated by these molecules. In this investigation we compared two different methodologies for the preparation of labelled miRNAs from mouse CNS tissue for microarray analysis. Labelled miRNAs were prepared either by a procedure involving linear amplification of miRNAs (labelled-aRNA) or using a direct labelling strategy (labelled-cDNA) and analysed using a custom miRNA microarray platform. Our aim was to develop a rapid, sensitive methodology to profile miRNAs that could be adapted for use on limited amounts of tissue.
We demonstrate the detection of an equivalent set of miRNAs from mouse CNS tissues using both amplified and non-amplified labelled miRNAs. Validation of the expression of these miRNAs in the CNS by multiplex real-time PCR confirmed the reliability of our microarray platform. We found that although the amplification step increased the sensitivity of detection of miRNAs, we observed a concomitant decrease in specificity for closely related probes, as well as increased variation introduced by dye bias.
The data presented in this investigation identifies several important sources of systematic bias that must be considered upon linear amplification of miRNA for microarray analysis in comparison to directly labelled miRNA.
MicroRNAs (miRNA) are an evolutionarily conserved, large new class of ~22 nucleotide (nt) long, gene regulatory RNA molecules that are involved in silencing mRNA transcripts through sequence-specific hybridization to 3' UTRs of mRNA molecules . In plants, gene silencing is mediated primarily through RNA interference where the miRNAs are fully complementary to their mRNA targets. In contrast, animal miRNAs are only partially complementary to their targets, and silence gene expression by mechanisms that involve the co-localization of miRNAs and miRNA targets to cytoplasmic foci known as P-bodies as well as degradation of target mRNA [2–8]. Concurrently, a role for miRNAs in proliferative diseases has also been suggested, specifically during cancers, where a large number of miRNAs appear to be de-regulated in primary human tumours [9–13]. The current paradigm that miRNAs represent a new layer of gene regulation has generated much interest in this field. Thus, detection of miRNAs, their expression analysis, and identification of potential regulatory targets (cognate mRNA) are burgeoning areas of research.
The most commonly used technique to detect miRNAs is a Northern blot. A Northern blot can reliably profile the transcription of miRNAs and has often been used in the analysis of developmental and tissue-specific expression patterns [18–22]. However, this method is also limited because it cannot be used for the simultaneous monitoring of hundreds of miRNAs and requires substantial amounts of sample. As such, microarray technology provides a promising alternative to the Northern blot as numerous miRNAs can be analyzed at once with relatively minimal amount of initial RNA investment .
A number of recent reports have outlined ways in which microarray technology can be used to detect and profile the expression of miRNAs isolated from cells or tissues [15, 16, 24–35]. These reports can be classified into several categories based on variations in the methodologies employed to prepare labelled-targets for hybridization. Firstly, there are reports in which the mature ~22 nt long miRNAs have been directly labelled and used for hybridization [24, 28, 30, 33–35]. Secondly, reports in which cDNA synthesized from the reverse transcription of adaptor-ligated miRNAs have served as the labelled miRNA targets for hybridization [15, 26, 29, 31]. In this category, either modified bases capable of binding or already containing a label have served as nucleotides for cDNA synthesis during reverse transcription, or the adaptor-specific primers used for reverse transcription were the source of the label for the miRNA. Thirdly, there are reports that are similar to the second category except that the cDNA is PCR amplified prior to serving as the targets for hybridization [16, 25, 27, 32]. In this category, the miRNAs were initially ligated to specific adaptors at both the 3' and 5' ends. Additionally, there are other novel methods that are presently being developed to measure miRNA expression [16, 36].
From the survey of published reports it is apparent that miRNA microarray analysis is a growing field but it is also in its infancy and there is a need for detailed comparison of data obtained from different methodologies before a consensus is drawn on the ideal method(s) for labelled target preparation. Based on this premise and our ultimate goal to analyze very limited amounts of miRNA obtained from procedures such as laser capture microdissection, we evaluated two different methods for the preparation of labelled-targets; targets prepared from amplified and non-amplified miRNA. The amplification of limited amounts of RNA prior to microarray analysis is a common strategy for longer transcripts; however, less is known about the effects of amplification of miRNAs for microarray analysis.
The primary aim of this investigation was to optimize a simple and dependable labelling protocol for microarray analysis of miRNAs from enriched mixtures of low molecular weight (LMW) RNA without gel purification that would have potential for use when the starting quantity of RNA is low. Our objectives for this investigation were; to determine whether the sensitivity of detection of miRNAs can be increased by amplification as compared to a direct labelling methodology, and to determine whether an accurate representation of the original miRNA population is retained following amplification.
Construction and specificity of the miRNA microarray
A total of 557 unique oligonucleotide probes to the majority of metazoan miRNAs in the miRNA registry (Release 5.0)  as well as to mature miRNA sequences from published literature at the time the project was initiated, were spotted onto glass slides in duplicate grids (See Additional File 1). Probes are in the same orientation as the miRNAs themselves. An initial evaluation showed that modifications of the probes to covalently link them to the slide surface was not necessary in order to allow high signal intensities with our choice of surface chemistry, epoxide coated slides from Corning (data not shown). We did however find that the addition of a 15 nucleotide poly(T) tract at the 5' end of the probes increased signal intensities, possibly by reducing steric hindrance due to the proximity of the short probe sequences to the slide surface.
Sequences of the miRNA probes spotted on the specificity microarrays
Probe sequence 5'-3'
Number of mismatches
From this data we do not expect the microarray to consistently resolve miRNAs that have only one or two base differences, especially towards the 5' end. The majority of identified miRNAs, however, differ from each other by 5 or more mismatches and so in most of the cases we can confidently discriminate between different miRNAs . Closely related miRNAs may require further analysis by other means to accurately measure their levels of expression [38, 39].
Development of a linear amplification labeling method for preparation of antisense miRNA targets
Confirmation of the validity of miRNA expression profiles following amplification
We went on to determine whether amplification affects the proportional representation of miRNA species identified from a complex mixture, in comparison to another method that does not include an amplification step.
Expression profiles for miRNAs isolated from brain tissue and amplified using the T7 linear amplification method as previously described were compared with profiles generated using antisense, tagged-cDNA hybridized to the array and signal amplified by dendrimer technology (See Additional File 2). The starting population of LMW RNA for all experimental procedures was prepared from total mouse brain as a single batch to ensure reproducibility. Each labelling reaction used between 100 and 250 ng of this LMW RNA as outlined in the methods section. The only variable factor was the labelled miRNA preparation technique employed. Total RNA consists of ~0.1% miRNA and the miRNA isolation technique we used results in a 10 fold enrichment of small RNAs (less than 200 nt long) and so between 1 ng and 2.5 ng of miRNA was used in each labelling reaction.
Significant signal was detectable on negative control spots, as well as to miRNA probe sequences from species other than mice, and not present in the mouse genome. We adjusted the intensity values for the spots to take this into account; an average signal intensity value was calculated for each slide from these control spots, and this was then subtracted from all spots on the array to determine net signal intensities. In contrast, hybridizations of target LMW RNA prepared without an amplification step (labelled-cDNA) had lower signal intensities, but little detectable non-specific hybridization to control spots; in this case the background correction step was omitted. The detection limit for miRNA labelled by the direct labelling technique was approximately 1.5 fmoles and the detection range was approximately 2.5 orders of magnitude as determined by serial dilution, labelling and hybridization of the synthetic miRNA (data not shown).
Validation of microarray data by qRT-PCR
A third method, qRT-PCR was used for independent validation of miRNA microarray data for the relative expression of miRNAs in mouse brain . This method has been determined to be quantitative and sensitive; it requires as little as 50–100 ng of total cellular RNA as starting material and is specific enough to allow for discrimination between miRNAs differing by a single nucleotide. We determined the dynamic range of the qRT-PCR assay was from 1.5 pmoles to 1.5 amoles for miRNAs using a dilution series of the synthetic miRNA previously described.
The qRT-PCR assay proved to be the most sensitive method for the detection of miRNAs with 88 being positively detected in comparison to the microarray; 75 miRNAs detected with the cDNA labelled targets and 67 for the amplified targets. Amplification of miRNAs, in this case, did not result in increased sensitivity for low abundance miRNAs; probably due to the high background when using the amplified target which masked low intensity signals. The high background is likely due to non-specific hybridization of incomplete reverse transcription, or template-independent, products produced during the amplification steps that bind with less specificity than their full-length counterparts [31, 40].
With the exception of miRNAs not scored as present by microarray analysis, the results obtained from each of the methods evaluated were remarkably similar. Only 2 miRNAs were scored as present by microarray analysis using targets labelled by either the amplification or direct labelling techniques but not by the qRT-PCR assay. In turn, these 2 miRNAs had intensities that were very close to the cut-off values for the scoring of miRNAs as being present based on array signals; in fact 61 miRNAs were similarly scored as present by each method showing a high degree of agreement between the three methods evaluated.
We concluded from these results that although the sensitivity of microarray detection was lower than qRT-PCR, the accuracy of miRNA detection, however, was comparable within the detection range of the arrays. In addition, we found that the amplification step did not significantly alter the proportional representation of miRNAs in vivo, especially for those miRNAs present in high abundance. It is readily apparent from the expression map that the most abundant miRNAs, including brain specific or enriched miRNAs, miR-124a-1, -9-1, -9*-1, -127, -136, -138-1, -149, -154, -218-1, -219-1, -222, -125a, -125b-1, -128a, -26a-1, -29a, -29b-1, -30c-1, and -34a were equally identified by both types of microarray labelling techniques. Amongst the abundant miRNAs identified, detection of miR-124a-1 was especially important as it is often cloned at very high frequencies in the brain [18, 25, 41]. Furthermore, miR-124a-1 has been known to down-regulate approximately 174 genes in transfected HeLa cells and in the process alters the entire transcriptome to a neuronal-like mRNA profile .
Dye bias associated with amplification and direct labelling methods
The dye bias in favour of amplified targets may be due to the variability of incorporation kinetics and quenching susceptibilities of modified dNTPs in the amplification reactions, thus altering fluorescent signals . Normalization by lowess smoothing did not effectively remove dye bias from these self-self hybridizations. In contrast, the significantly lower dye bias for labelled-cDNA may be due to the dendrimer geometry which permits equal spacing between adjacent dye molecules thus preventing energy transfer and other types of quenching interactions which often reduce fluorescent signal . Furthermore, each cDNA transcript binds a single dendrimer and each dendrimer has a predetermined number fluorescent molecules. Cy3-labelled dendrimers have on average of 250 fluorescent molecules per dendrimer whereas the Cy5-labelled dendrimers have 243 flours per dendrimer, with a standard error of ± 4 molecules. Recent improvements in denrimer technology have resulted in nearly 900 fluorescent molecules per dendrimer. It was concluded that labelling transcripts for detection via dendrimers is likely a more reproducible methodology for quantitative assessment of miRNAs.
Validation of microarray derived expression ratios using qRT-PCR
Comparison of miRNA fold changes in a number of brain derived cell lines by Microarray and RT-PCR
Fold Change by Microarray
Fold Change by qRT-PCR
The primary aim of this investigation was to examine target labelling methodologies that could be used for our custom miRNA microarray that did not require labour intensive steps such as gel purification, were sensitive and specific, useable at high-throughput and more affordable than multiplex RT-PCR assay. In particular, we were interested to determine whether the amplification methodology routinely in use for mRNA profiling could be directly applied to the study of miRNAs. It was clear from our evaluation that high-throughput microarray analysis of miRNA expression could be performed accurately using amplified targets.
Investigation of the sensitivity of detection, using a synthetic miRNA and an array of matched and mismatched probes, we found there to be an advantage in terms of sensitivity of detection for low-abundance miRNAs after amplification, in comparison to a direct labelling technology. However, when specificity was examined on the same platform, we found that the ability to discriminate between closely related sequences was reduced after amplification.
Using total brain LMW RNA as starting material for both the direct labelling and amplification protocols we measured very similar sensitivity and specificity for both methods; a TaqMan® quantitative RT-PCR method was used as the 'gold standard' for validation. We concluded that the reduction in specificity after amplification resulted in high non-specific signal intensities across the array, which masked detection of low abundance miRNAs. It is possible that by using an amplification technique, in which only full length targets are selectively labelled, to reduce non-specific hybridization, the sensitivity of detection for methods including amplification could be increased; potentially this increase in sensitivity could bring the range of detection for microarrays to a value similar to the 10 amoles detection limit that was observed for the qRT-PCR assay, and a requirement for μg rather than mg quantities of starting material. In this case, PCR amplification of reverse transcription products prior to linear amplification to select for full-length transcripts may be the solution to these problems . Initially, this would require the ligation of specific adapters to both the 3' and 5' ends of the mature miRNAs and the use of adapter specific primers for the PCR reactions .
It is worth noting at this point, a new addition to microarray based analysis of miRNAs; the RNA-primed array-based Klenow enzyme (RAKE) assays . In this technique, antisense DNA oligonucleotide sequences are used to probe for miRNAs and upon binding miRNAs serve as primers for Klenow enzyme based extension. During the generation of the double stranded fragment, tagged nucleotides are incorporated allowing for easy detection. The advantage of this method is that it does not require the manipulation of the initial RNA investment and also does not require the generation of a cDNA library or amplification. Most laboratories, however, have custom microarray platforms with optimized target labelling protocols and hopefully our results will alert those researchers in the field to possible areas of misinterpretation.
Lastly, we anticipate and encourage discussion about all aspects of microarray analysis of miRNAs, not only in the area of labelling but also in other areas of the technology where there is possibility for the introduction of systematic bias. High-throughput techniques in the analysis of miRNAs, such as microarrays, will be helpful to further understand the role(s) of miRNAs in normal and diseased cells and tissues.
For high-throughput analysis of limited amounts of miRNAs by microarrays we show that linear amplification of LMW RNA targets does not significantly increase the sensitivity of low abundance miRNAs, in comparison to direct-labelled targets. Additionally, the amplified targets also showed decreased probe specificity and increased variation introduced by dye bias.
MiRNA microarray construction
A microarray containing oligonucleotide probes complimentary to 557 miRNAs was constructed. These probes consisted of all the non-redundant miRNA sequences from the miRNA registry (Release 5.0) . The arrays were spotted in-house on Corning® epoxide coated slides; Probes were spotted in duplicate and additionally two full arrays were printed per slide. The probes were in sense orientation relative to the mature miRNAs and thus complimentary to either the cDNA or aRNA that would be derived from the mature miRNAs. The probes also had a 15 nucleotide long poly(T) tract at the 5'end for steric distancing from the array surface. Each probe was printed in duplicate and the signal intensities for averaged.
Low molecular weight RNA enrichment
Low molecular weight (LMW) RNA enrichment from whole mouse brain was performed using mirVana™ miRNA Isolation Kit (Ambion), according to the manufacturer's protocol. LMW RNA was DNase-treated for microarray target preparation using TURBO DNA-free™ (Ambion). Chemically synthesized miRNAs were purchased from Dharmacon.
Preparation of labelled-cDNA targets
Array 900 miRNA RT Kit (Genisphere) was used to prepare labelled cDNA targets for microarray hybridization according to the manufacturer's protocol. Briefly, 250 ng of LMW enriched RNA was used as a template in a 100 μl Poly(A) Tailing and RT reaction containing 1× Reaction Mix, 2.5 mM MnCl2, 1 mM ATP, and 4 μL poly A polymerase (E-PAP Enzyme). After incubation at 37°C for 15 minutes the reaction was placed on ice and 2 μL of Cy3 or Cy5 reverse transcriptase primer was added. The reaction mix was incubated at 65°C for 10 minutes prior to addition of 23 μL of a second master mix containing per reaction: 10 μL of 5× First Strand Buffer, 5 μL of 0.1 M DTT, 2.5 μL of dNTP Mix, 1 μL of Superase-in RNase Inhibitor, 2 μL of SuperScript II Reverse Transcriptase (200 U) (Invitrogen), and 2.5 μL of nuclease-free water. Subsequently, the reaction was incubated at 42°C for one hour. Finally, 8.75 μL of 0.5 M NaOH/50 mM EDTA and 65°C for 15 minutes was used to inactivate Superscript II. Samples were concentrated to a volume of approximately 10–15 μL, using Microcon YM-10 Centrifugal Filter Devices (Fisher Scientific) according to the manufacturer's protocol.
Preparation of labelled-aRNA targets
LMW enriched RNA was polyadenylated using the Poly(A) Tailing Kit (Ambion); 250 ng of LMW enriched RNA was used as a template for a 25 μl Poly(A) Tailing reaction containing 1× Reaction Mix, 2.5 mM MnCl2, 1 mM ATP, and 1 μL poly(A) polymerase (PAP Enzyme). The reaction was placed on ice. For differential labelling of targets, approximately 100 ng of polyadenylated LMW RNA was mixed with 1 μL of T7 Oligo(dT) Primer, and nuclease-free water added to 12 μL. After incubation at 70°C for 10 minutes 8 μL of a reverse transcription mixture was added, 2 μL of 10 × First Strand Buffer, 1 μL Ribonuclease Inhibitor, 4 μL dNTP Mix, and 1 μL of Reverse Transcriptase, and incubation continued at 42°C for 2 hours. Following this 80 μL of second strand synthesis buffer was added, which included 1× Second Strand Buffer, 4 μL of dNTP Mix, 2 μL of DNA polymerase, and 1 μL of RNase H, and the sample incubated at 16°C for 2 hours. Newly synthesized cDNA was purified using cDNA Filter Cartridges and used for an in vitro transcription reaction to generate chemically modified aRNA. In vitro transcription was performed in a 40 μL reaction including the double stranded cDNA from the second strand synthesis, 3.75 mM aaUTP, 7.5 mM ATP, CTP, GTP Mix, 3.75 mM UTP, 1× T7 Reaction Buffer, and 4 μL of T7 Enzyme Mix. The mix was incubated for 14 hours at 37°C and then treated with DNase (2 μL per reaction) for 30 minutes at 37°C to remove template cDNA. The aRNA was purified using aRNA Filter Cartridges and the aRNA:dye coupling reaction was performed according to the manufacturer's protocol. Briefly, 5–20 μg of amino allyl aRNA was dried down to a thin film in a microfuge tube and 9 μL of coupling buffer, and 11 μL of either of the dyes Alexa Fluor Succinimidyl Ester 555 or 647 constituted in DMSO (one dye vial re-suspended in 88 μL of DMSO) (Invitrogen), were added. After incubating at room temperature for 30 minutes in the dark, 4.5 μL of 4 M hydroxylamine was added to quench the amine-reactive groups of the unreacted dye molecules. After incubation for 15 minutes at room temperature the labelled amino allyl aRNA was purified using aRNA filter cartridges.
Target hybridization to microarray
Tagged-cDNA hybridization followed the protocol outlined in the 900 miRNA RT Kit. A hybridization mixture consisting of the differentially tagged cDNA (10 μL of Cy3-labelled and 10 μL of Cy5-labelled targets) and 2 × SDS-based Hybridization Buffer pre-heated to 70°C (20 μL) was mixed and incubated at 75–80°C for 10 minutes, cooled to 50°C until loading and added to the microarray; specifically a 22 × 40 mm cover slip (mSeries Lifterslip™) (Erie Scientific) was centered over the grids and the preheated hybridization mixture was loaded under the cover slip. Microarrays were incubated overnight (16–20 hours) at 50°C in a dark humidified chamber (Genetix). Following hybridisation the cover-slips were removed and the arrays were washed in 2 × SSC, 0.2% SDS wash buffer preheated to 42°C for 15 minutes, 2 × SSC wash buffer at room temperature for 10–15 minutes, and 0.2 × SSC wash buffer at room temperature for 10–15 minutes. Arrays were dried by centrifugation at 1000 rpm for 2–3 minutes and the 3DNA system containing the fluorescent cyanine molecules were hybridized to the arrays; in this case the hybridization mixture contained Cy3 3DNA Capture Reagent (2.5 μL), Cy5 3DNA Capture Reagent (2.5 μL), Nuclease Free Water (15 μL), and 2 × SDS-based Hybridization Buffer. The mix was heated to 70°C for 10 minutes, cooled to 62–64°C and hybridized to the arrays for 4 hours at 62–64°C in a dark humidified chamber. Finally, the arrays were washed as previously described.
Differentially labelled aRNA targets were hybridized to the arrays according to the protocol outlined in the Amino Allyl MessageAmp™ Kit. Since the purified labelled aRNA was eluted in a final volume of 100 μL, it was necessary to firstly concentrate the samples to approximately 1–5 μL by vacuum drying in the dark. aRNA target was resuspended in Pronto!™ Short Oligo Hybridization Solution (Corning) to a final volume of 20 μL and differentially labelled aRNA targets combined and loaded onto arrays. Hybridized arrays were incubated for 16 hours at 50°C in a dark humidified chamber. Upon incubation, the slides were washed in 2 × SSC, 0.1% SDS pre-warmed to 42°C for 5 minutes, twice in 1 × SSC wash buffer at room temperature for 2 minutes, and two successive washes in 0.1 × SSC at room temperature for one minute each time. The arrays were dried by centrifugation at 1000 rpm for 2–3 minutes.
Microarray spot quantification and analysis
Microarrays were scanned using Agilent G2565AA and Agilent G2565BA Microarray Scanner System (Agilent). Feature extraction was performed using Array-Pro™ analyzer version 4.5 (Media Cybernetics). The intensities of duplicate spots on each array were averaged. Partek® software, version 6.2 Copyright© 2006 (Partek) was used to perform ANOVAs.
qRT-PCR (TaqMam®MiRNA Assays)
Reverse transcriptase reactions were performed using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). Each reaction contained 20 ng LMW enriched RNA, 1 mM dNTPs (with dTTP), 1 μL of 3.3 U MultiScribe™ Reverse Transcriptase, 1× Reverse Transcription Buffer, 3.75 U RNase Inhibitor, and 3 μL of RT primer. The reaction was carried out at 16°C for 30 minutes, 42°C for 30 minutes, and 85°C for 5 minutes. Semi-quantitative PCR reactions were performed according to the methodology outlined in the TaqMan® MiRNA Assay Kit (Applied Biosystems). Briefly, each reaction contained TaqMan 1× Universal PCR Master Mix (No AmpErase UNG), 1× TaqMan® MicroRNA Assay Mix, and 1.33 μL of the RT product in a total volume of 20 μL. Each reaction was incubated in an Applied Biosystems 7500 Real Time PCR System in a 96-well plate at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 minute. The threshold cycle (Ct) method was used to determine the relative quantities of each miRNA, and was defined as the fractional cycle number at which the fluorescence passes the fixed threshold.
To determine the fold changes in expression between miRNAs in a number of cell lines we used the following methodology. The expression of each miRNA relative to let-7a was determined using the ΔΔCt method. Average fold differences for each miRNA were calculated by comparing the relative expression (ΔΔCt values) in each of the cell lines tested in comparison to a standard (ΔΔCt values were compared for each cell line EOC 13.31, EOC 20, C8-B4, NB41A3, SK-N-FI, IMR-32 with the ΔΔCt value for Neuro-2a). All experiments were performed in triplicate.
The authors would like to thank Claude Ouellette and Shari Tyson of the NML DNA core for technical assistance in printing the custom miRNA oligonucleotide microarrays. The work was supported in part by the Canadian Biotechnology Strategy Fund: Genomics Initiative for Government Laboratories.
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