High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 14 August 2013
Accepted: 7 October 2013
Published: 10 December 2013
Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.
MicroRNAs (miRNAs) are a class of small (approximately 20 to 22 nucleotides), non-coding RNA species that are broadly expressed in almost all eukaryotes. They regulate diverse biological processes by targeting a large number of protein-coding mRNA transcripts . The widespread regulation of mRNAs by miRNAs is supported by the evidence obtained from computational analysis [2, 3], transcriptome analysis [4, 5] and proteome profiling [6, 7]. With recent developments in quantitative mRNA profiling, it is estimated that the median copy number of an mRNA is 17 molecules, and the dynamic range for mRNA copy number is approximately 104 in a single mammalian cell [8, 9]. Because miRNAs regulate mRNA expression in a stoichiometric manner [10, 11] and a conserved miRNA is predicted to recognize approximately 400 target sites [1, 12], such broad variation in mRNA expression suggests miRNAs could differentially regulate highly or lowly expressed mRNAs. Indeed, it was recently demonstrated that miRNAs could function either as a switch or a fine-tuner, depending on the copy number of both miRNAs and their cognate mRNA targets . Adding to the complexity of miRNA-mediated regulation, other RNA species, including competing endogenous mRNAs or circular RNAs, were recently shown to function as a molecular decoy by titrating miRNAs from their functional mRNA targets [13–16]. Collectively, these studies suggest that a highly expressed miRNA is capable of regulating many more mRNA targets and/or repressing mRNA targets more potently than a lowly expressed miRNA within a cell. Therefore, these findings highlight the importance of accurately determining the relative expression levels of different miRNAs in a given cellular context.
Three major platforms, quantitative real-time reverse transcription PCR (qRT-PCR), microarray and small RNA cloning followed by deep sequencing (miRNA-Seq), have been developed for genome-wide miRNA profiling. However, both qRT-PCR and microarray platforms are hybridization-based techniques suitable only for known miRNAs and are unable to provide single-nucleotide resolution for miRNA detection. Furthermore, due to the limitation of probe design for such a short sequence of miRNAs, absolute quantification of individual miRNAs requires extensive optimization and individual internal controls . In contrast, with the rapid development of next-generation sequencing techniques, miRNA-Seq has the potential to provide single-nucleotide resolution of miRNA species, facilitate de novo miRNA discovery, and offer nearly unlimited dynamic range for miRNA quantification . The enthusiasm towards miRNA-Seq, however, has been severely dampened by the identification of significant biases introduced during small RNA cDNA library preparation, often more than three orders of magnitude for individual miRNAs [19, 20]. Although the cloning biases are likely caused by many factors, the RNA ligation has been identified as one of the major sources . Consistent with this view, recent studies with modifications to the ligation reactions have shown some promise to reduce the cloning bias [21–23]. However, a highly effective method for genome-wide miRNA quantification by deep sequencing has yet to be successfully developed.
In this study, we investigate the highly biased miRNA quantification by current miRNA-Seq methods with 29 representative miRNAs. We demonstrate that inefficient and biased RNA ligation for both 3′ and 5′ adapter ligation is the major contributing factor for the inaccurate quantification. To develop an unbiased RNA cloning protocol, we have performed extensive tests with multiple customized adapters, RNA ligases as well as ligation conditions. As a result, we have established an RNA cloning strategy that allows nearly 100% ligation efficiency for both 3′ and 5′ ligation steps. When applied to a pool of either equimolar or differentially mixed synthetic miRNAs, our method achieves less than two-fold deviation from the expected results for each miRNA. We further demonstrate that the unbiased cloning is applicable to complex RNA mixtures using Caenorhabditis elegans total RNA with the synthetic mix spiked in. Finally, we apply our method to biological samples isolated from multiple mouse tissues as well as HeLa cells. Our results provide a quantitative view of miRNA expression for each of these samples. These data also reveal that most cistronic miRNAs that are derived from a single transcript are expressed at similar levels, and that miRNAs repress their target expression in a dose-dependent manner. Taken together, we have established a highly efficient miRNA-Seq method that accurately quantifies individual miRNAs whose expression levels are across four orders of magnitude. Furthermore, the optimized RNA ligation can be readily applied to other RNA ligation-based RNA-Seq methods to provide quantitative insights for RNA transcripts.
Results and discussion
Characterization of systematic bias in current miRNA-Seq method
We first prepared an equimolar mixture of these 29 synthetic miRNAs and performed miRNA-Seq with the existing method [18, 20, 31]. Consistent with previous reports [19, 20], the cloning frequency of individual miRNAs varied by more than 1,000-fold (Figure 1C). We noted that miR-143, the second highest cloned miRNA in our pool, shows a much higher cloning efficiency (249-fold higher) than miR-145, its clustered, cistronic partner. Remarkably, such bias favoring miR-143 has been observed in many miRNA-Seq experiments using total RNA, including in our results from mouse hair follicles (Figure 1D; Figure S1A in Additional file 1). In many independent studies, miR-143 is consistently reported as one of the most highly cloned miRNAs; in sharp contrast to this, miR-145 often falls outside of the top 50 most cloned miRNAs [32, 33] (Figure 1D). However, miR-143 and miR-145 show very similar expression levels when measured by northern blotting  or qPCR-based absolute quantification (Figure S1B in Additional file 1). Similar to the results for miR-143/145, when we examined other clustered, cistronic miRNAs from the datasets generated by the current method, we observed significant variation in cloning frequency (Figure 1D). Thus, significant and widespread biases in the current miRNA-Seq method severely compromise the ability to quantify individual miRNAs and hinder the application of miRNA-Seq for identifying highly expressed miRNAs in a given cellular context.
RNA folding contributes to the systematic bias
High-efficiency RNA ligation
Having established an effective ligation method for the 3′ ligation, we re-visited the 5′ ligation. Similar to the 3′ ligation, the 5′ ligation efficiency in the current method was also very low. We observed 9% efficiency collectively for the mixed synthetic miRNAs (Figure 3E). In addition, the ligation efficiency for the thermostable ligase and the 5′ splint adapter was also very low at 7% and 2%, respectively (Figure 3E). These results prompted us to optimize the 5′ ligation efficiency. We took a similar approach based on our success in the 3′ ligation. This included: 1) the use of 3′ end NN randomized adapter; 2) further increasing the PEG8000 concentration to 20% in the reaction buffer; and 3) elevating the reaction temperature to 37°C. As shown in Figure 3F, these modifications optimized the 5′ ligation to approximately 97% efficiency. Taken together, these results demonstrate that we have established a highly efficient method for attaching both 3′ and 5′ adapters to miRNA substrates.
High-efficiency RNA ligation allows quantitative measurement of synthetic miRNAs by miRNA-Seq
Quantitative miRNA-Seq provides insights into miRNA expression in biological samples
Differentially expressed miRNAs repress their targets in a dose-dependent manner
In this study, we have developed a highly efficient miRNA-Seq method to quantify individual miRNAs within a biological sample (Figure 7E). Although the RNA-ligase-introduced bias has been widely recognized [19–22], a systematic solution generally applicable for a broad spectrum of small RNA substrates has not previously been developed. Such a bias, often leading to more than 1,000-fold deviation, has significantly hindered the utilization of miRNA-Seq for quantitative miRNA profiling in a high-throughput manner. Although in some studies the sequencing depth has approached billions of reads , the power of accurate quantification of miRNAs based on the cloning frequency remains poor . Because intermolecular RNA hybridization contributes to bias, the application of a thermostable RNA ligase or a 5′ splint adapter significantly reduces such bias. However, the ligation efficiency is low and the improvement by each strategy appears to vary for different RNA substrates. By examining individual steps during the small RNA cDNA library preparation, we have defined optimal conditions to achieve high ligation efficiency for both 3′ and 5′ ligation steps. We determined that the nucleotide composition of the linkers, the concentration of linkers, molecular crowding through the use of PEG8000, and ligation temperature are the primary determinants for highly efficient RNA ligation. We demonstrated with a wide spectrum of small RNA substrates that the ligation efficiency can approach 100%, a drastic improvement compared with the existing method. As a result, the high-efficiency RNA cloning leads to quantitative measurement of miRNAs by miRNA-Seq. To achieve such quantitative results for a pool of approximately 700 miRNAs for a single sample, we usually need approximately 4 to 5 million reads. With the data output routinely over 200 million reads from a single lane of a sequencer, in combination with the multiplexing capacity of the Illumina Hi-Seq platform, our miRNA-Seq method provides an extremely cost-effective and accurate approach for large scale miRNA analyses from numerous samples.
The ability to measure relative expression levels of different miRNAs within a single sample also offers an important approach to identify functionally significant miRNAs. Conventional gene expression analysis by qRT-PCR and microarray usually characterizes differential expression levels of individual miRNAs and identifies miRNAs with the largest fold changes from two or more samples. With the detection sensitivity of qRT-PCR approaching single molecules, some very lowly expressed miRNAs could be identified as highly differentially expressed, though their biological importance may be limited due to low copy number. Since miRNAs function by guiding the RNA-induced silencing complex to their cognate mRNAs and sequestering the mRNAs from productive translation , the ratio between miRNAs and mRNA targets is a key factor in determining the potency of a miRNA . In support of this, we demonstrate that the most highly expressed miRNA in HeLa cells, miR-21, has the highest capacity to repress the reporter expression, and the intermediately and lowly expressed ones (for example, miR-130a and miR-502) show much weaker repression. Thus, functionally important miRNAs could be identified as the most highly expressed miRNAs within a cellular context without the knowledge of differential expression patterns. Interestingly, in our analysis of miRNAs in both brain and heart, we observed that a small number (approximately 10) of miRNAs count for a major portion of the miRNA pool. This raises a possibility that the function of the miRNA pathway could be mediated by a handful of highly expressed miRNAs in each cell population.
Finally, our approach to enhance RNA ligation efficiency offers a general guide to improve all RNA ligation-based RNA-Seq methods. The enhanced ligation not only reduces the required amount of RNA input but also, more importantly, improves the representation of different RNA species/fragments in the sequencing libraries. These improvements will allow quantitative analysis of gene expression by fully harnessing the power of deep sequencing.
Materials and methods
DNA oligonucleotides to serve as 3′ linkers for miRNA cloning were ordered from Integrated DNA Technologies Coralville, Iowa, USA(see Table S3 in Additional file 1 for sequences). All oligonucleotides were ordered with a 5′ phosphate group; those used in sequencing experiments also included a 3′ dideoxycytosine. Prior to ligation, 3′ linkers were adenylated at the 5′ end using the MTH adenylation kit (New England Biolabs #E2610L Ipswich, Massachusetts, USA). Oligonucleotides were phenol/chloroform extracted, precipitated with 2.5 volumes of ethanol, and quantified by A260. Adenlyation was confirmed by 18% PAGE (Figure S2 in Additional file 1).
3′ ligation reactions
Five picomoles of indicated synthetic miRNA were 5′ end labeled using T4 polynucleotide kinase (Fermentas Hanover, Maryland, USA) with 25 μCi of γ-(32P) ATP (3,000 Ci/mmol). Labeled RNA was phenol/chloroform extracted, ethanol precipitated, and resuspended in distilled H2O. Initial reactions (Figure 3A) were composed of 25 femtomoles of labeled RNA, 1 picomole of 5′ adenylated DNA cloning linker, 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 20 units truncated T4 RNA Ligase 2 (residues 1 to 249, K227Q point mutant) (New England Biolabs M0242S Waltham, Massachusetts, USA), and 20 units RNase Inhibitor (Invitrogen N2611 Carlsbad, California, USA). Ligations were permitted to proceed for 90 minutes at room temperature. Reactions were stopped by the addition of an equal volume of 2X loading dye (7 M urea, 1× TBE, 0.01% bromophenol blue and 0.005% xylene cyanol), briefly heated to 95°C, and snap cooled on ice. RNA species were resolved on 15% acrylamide gels containing 7 M urea, exposed to a phosphorimager screen, and visualized by scanning on a Storm Imager (GE Healthcare Piscataway, New Jersey, USA). Subsequent, high efficiency reactions were identical with the following modifications; a final concentration of 10% (w/v) PEG molecular weight 8000 Da (PEG 8000) was included. Reaction times were increased from 90 minutes to 4 hours, and 3′ linker concentration was increased to 10 μM.
5′ ligation reactions
Synthetic 5′ 32P labeled RNA was subject to 3′ ligation and resolved on 15% PAGE-urea gels as described previously. The ligated product was excised and eluted and served as substrate for 5′ ligation tests. Initial 5′ ligation reactions were composed of the following: 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM ATP, 20% (v/v) DMSO, 10 units T4 RNA Ligase 1 (New England Biolabs M0204S), 20 units RNase Inhibitor (Invitrogen N2611), 5 μM 5′ RNA cloning linker, and 25 femtomoles of eluted substrate RNA. Reactions were carried out at room temperature for 2 hours. Subsequent high efficiency ligations were identical with the following modifications: no DMSO, 20% (w/v) PEG8000, 5′ RNA cloning linker increased to 10 μM, and temperature increased to 37°C. Reactions were stopped, resolved, and visualized as described above. For the thermostable ligation, a thermostable RNA ligase (Epicentre TRL8101K Madison, Wisconsin, USA) was used following the manufacture’s instruction.
Synthetic RNA library preparation
RNA oligoribonucleotides were produced by Thermo Fisher Scientific (Lafayette, Colorado, USA)(for complete list and sequences see Table S1 in Additional file 1). Oligos were resuspended in distilled H2O and mixed at an equal volume to generate an equimolar mixture of the 29 synthetic RNAs.
Small RNA cloning
RNA samples (5 to 1.25 picomoles for synthetic samples and 1 to 4 μg of total RNA for biological samples) were subject to high efficiency 3′ ligation as described above. Products were resolved on 15% PAGE-urea gels and stained with SybrGold (Molecular Probes S-11494 Carlsbad, California, USA). The region corresponding to ligated miRNAs (46 nucleotides) was excised, gel slices were thoroughly minced, and eluted in HSCB (400 mM NaCl, 25 mM Tris-HCl pH 7.5, 0.1% SDS) overnight at 4°C. Nucleic acids were precipitated in the presence of glycogen carrier via 2.5 volumes ethanol and 0.1 volumes sodium acetate. Pellets were washed in 70% ethanol, and resuspended in the 5′ ligation mix without enzyme. Samples were briefly heated to 70°C, snap chilled on ice, and enzyme added. Following ligation reactions cDNA was prepared using Superscript III RT (Invitrogen 18080-085) according to the manufacturer′s recommendation using 3′ linker-specific RT primer. Subsequent cDNA libraries served as templates for PCR amplification; amplicons were resolved on 8% native acrylamide gels. Bands of the correct molecular weight were isolated and used for high-throughput sequencing on the Illumina HiSeq2000.
A 17-bp DNA primer reverse complementary with nucleotides 6 to 22 of the canonical sequences of miR-203 was used in the primer extension experiment to detect the 5′ length variants of miR-203. Each isoform was quantified using Image J software where the signal from the two bands was summed and each presented as a proportion of the total signal. The assay was performed following the manufacture’s instructions (Promega #E3030 Madison, Wisconsin, USA).
Total RNA (10 μg) was electrophoresed through 20% denaturing urea-PAGE gels and electrophoretically transferred to GeneScreen Plus® Nylon Membranes (Perkin-Elmer, Inc. Waltham, Massachusetts, USA). Radiolabeled oligonucleotides complementary to mature miR-203 sequence (miRbase accession MIMAT0000236) were hybridized to the membranes in ExpressHyb Hybridization Solution™ (Clontech Laboratories, Inc. Mountain View, California, USA). Synthetic miR-203 and miR-203iso were loaded as size standards. Bands were quantified as described in the primer extension experiment.
HeLa cell cultures were propagated according to ATCC guidelines. Cells were plated in 24-well trays at a density of 0.1 × 106 cells per well. Sixteen hours post-plating cells were transfected with LT1 (Mirrus Bio Madison, Wisconsin, USA) according to the manufacturer’s instructions with 20 ng of the following constructs: pGL3 control, pGL3-miR502, pGL3-miR130a, and pGL3-miR21 along with 2 ng of Renilla (pRL-CMV), and 378 ng of 'filler' DNA (pLL3.7). Twenty-four hours post-transfection cells were assayed for luciferase activity using the Promega Dual Luciferase Assay Kit according to the manufacturer’s instructions. Data are the ratio of firefly to renilla luciferase activity, normalized to the control construct. Shown is a representative result from an experiment done in triplicate; error bars represent standard deviations.
All miRNA-Seq data have been deposited to the National Center for Biotechnology Information Gene Expression Omnibus with accession number GSE47858.
Small RNA cloning followed by deep sequencing
Quantitative reverse-transcription polymerase chain reaction
We thank D Wang for discussion and providing total RNAs from heart and brain and other members of the Yi lab for suggestions; M Han and B Kudlow for discussion and providing total RNAs from worm; N Lau, S Bradley, W Regan, M Rosbash and J Huntley for reagents and Illumina sequencing; R Dowell for advice on bioinformatics analysis; T Blumenthal for reading the manuscript. This publication was made possible by a start-up fund provided by the University of Colorado and a grant from the NIH (AR059697) to RY and awards from the American Cancer Society to RY and JEL.
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